The Golgi-associated protein p115 is required for the...

Molecular Mechanisms of the

Unconventional Secretion of Macrophage

Migration Inhibitory Factor (MIF)

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften

der RWTH Aachen University zur Erlangung des akademischen Grades

einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologin

Melanie Merk

aus

Freiburg im Breisgau

Berichter: Universitätsprofessor Dr. Jürgen Bernhagen

Universitätsprofessor Dr. Werner Baumgartner

Tag der mündlichen Prüfung: 19. Dezember 2008

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek

online verfügbar.

II

TABLE OF CONTENTS

A. Abbreviations ..................................................................................... VI

B. Acknowledgments .............................................................................. IX

C. Publications........................................................................................ XI

1 INTRODUCTION ................................................................ 1

1.1 Macrophage migration inhibitory factor.................................... 1

1.1.1 MIF – An Overview ...................................................................... 1

1.1.2 MIF in Inflammatory Disease ...................................................... 2

1.1.2.1 Endotoxemia and Sepsis ........................................................... 3

1.1.2.2 Rheumatoid Arthritis ............................................................... 4

1.1.2.3 Atherosclerosis .......................................................................... 4

1.1.2.4 Asthma ...................................................................................... 5

1.1.3 Molecular Mechanisms of MIF Action ......................................... 6

1.1.3.1 ERK 1/2 Activation ................................................................... 6

1.1.3.2 MIF Inhibits JAB1 Activity ...................................................... 7

1.1.3.3 MIF in Apoptosis ....................................................................... 7

1.1.3.4 MIF Upregulates TLR4 Expression ......................................... 8

1.1.3.5 MIF Receptors ........................................................................... 8

1.1.4 Structure, Enzymatic Activities and Inhibitors .......................... 9

1.1.4.1 Structure ................................................................................... 9

1.1.4.2 Enzymatic Activities ............................................................... 10

1.1.4.3 Small Molecule Inhibitors ...................................................... 11

1.1.5 The Structural Homolog D-Dopachrome Tautomerase ............ 12

1.2 Classical Secretion in Eukaryotes.............................................. 14

1.2.1 ER/Golgi Transport and the Golgi Apparatus ........................... 15

1.2.1.1 ER to Golgi Transport ............................................................. 15

1.2.1.2 The Golgi Apparatus ............................................................... 16

1.2.2 The Tethering Protein p115 ....................................................... 17

1.3 Non-Classical Secretion ................................................................ 19

1.3.1 Vesicle-Independent Translocation ........................................... 20

1.3.1.1 Membrane Blebbing/Exovesicles ............................................ 20

1.3.1.2 Plasma Membrane-Resident Transporters ............................ 20

1.3.2 Vesicle-dependent secretion ....................................................... 21

1.3.2.1 Lysosomal Secretion ............................................................... 21

1.3.2.2 Exosomes/Multivesicular Bodies ............................................ 22

1.3.3 Role of ABC transporters in unconventional secretion ............. 23

1.3.4 Unconventional Secretion of MIF .............................................. 24

2 MATERIAL AND METHODS .......................................... 25

2.1 Cell lines, Bacteria and Plasmids ............................................... 25

2.1.1 Cell lines ..................................................................................... 25

III

2.1.2 Bacteria ....................................................................................... 25

2.1.3 Plasmids ...................................................................................... 25

2.2 Equipment, Consumables and Chemicals ................................ 26

2.2.1 Equipment .................................................................................. 26

2.2.2 Consumables ............................................................................... 26

2.2.3 Chemicals .................................................................................... 27

2.2.4 Multi-Component Systems ......................................................... 28

2.3 Primary and Secondary Antibodies ........................................... 29

2.3.1 Primary Antibodies .................................................................... 29

2.3.2 Secondary Antibodies ................................................................. 29

2.4 RNAi-targeting Sequences ........................................................... 30

2.5 Quantitative PCR Primers ........................................................... 30

2.6 Media, Buffer and Solutions ........................................................ 30

2.6.1 Media ........................................................................................... 30

2.6.2 Buffers and Solutions ................................................................. 31

2.6.2.1 General Buffers ....................................................................... 31

2.6.2.2 Flow Cytometry ....................................................................... 31

2.6.2.3 Human MIF ELISA ................................................................ 32

2.6.2.4 Murine MIF ELISA ................................................................. 32

2.6.2.5 Murine DDT ELISA ................................................................ 33

2.6.2.6 Ni2+-Column Purification ........................................................ 33

2.6.2.7 MIF Purification ..................................................................... 34

2.6.2.8 DDT Purification ..................................................................... 34

2.6.2.9 SDS-PAGE and Western Blot ................................................. 35

2.6.2.10 Coomassie Staining ............................................................. 35

2.6.2.11 Immunofluorescent Staining .............................................. 35

2.7 Molecular Biology Techniques .................................................... 36

2.7.1 Measurement of DNA/RNA Concentration ............................... 36

2.7.2 Polymerase Chain Reaction (PCR) ............................................ 36

2.7.3 Enzymatic DNA Digestion ......................................................... 37

2.7.4 Agarose Gel Electrophoresis ...................................................... 37

2.7.5 Isolation and Purification of DNA from Agarose Gels .............. 37

2.7.6 DNA Ligation .............................................................................. 37

2.7.7 Heat shock Transformation of E. coli ........................................ 37

2.7.8 Plasmid Isolation ........................................................................ 38

2.7.9 Isolation of RNA and cDNA Synthesis ...................................... 38

2.7.10 Quantitative PCR ....................................................................... 38

2.7.11 Design and Production of short hairpin RNA ........................... 38

2.7.12 Preparation of Adenoviruses ...................................................... 39

2.8 Cell Culture Techniques ............................................................... 39

2.8.1 Cultivation and Treatment ........................................................ 39

2.8.2 Isolation of Macrophages from Blood......................................... 40

IV

2.8.3 Transient Transfection ............................................................... 40

2.8.4 Preparation of Cell Lysates ........................................................ 41

2.8.5 Immunofluorescent staining for microscopy ............................. 41

2.8.6 Fluorescence-based Quantification of FGF Export ................... 41

2.8.7 Chlamydia trachomatis (L1) Infection ...................................... 42

2.9 Proteinchemistry and Immunology Techniques .................... 42

2.9.1 Determination of Protein Concentration ................................... 42

2.9.2 SDS-PAGE .................................................................................. 42

2.9.3 Coomassie Staining .................................................................... 43

2.9.4 Western Blot ............................................................................... 43

2.9.5 Sandwich ELISA ........................................................................ 44

2.9.5.1 Human MIF ELISA ................................................................ 44

2.9.5.2 Murine MIF ELISA ................................................................. 44

2.9.5.3 Murine DDT ELISA ................................................................ 44

2.9.5.4 TNF-α, IL-1β and IL-6 ELISA ................................................ 45

2.9.6 Protein Purification .................................................................... 45

2.9.6.1 Overexpression of Proteins ..................................................... 45

2.9.6.2 Purification of Histidine-tagged Proteins .............................. 45

2.9.6.3 Purification of MIF ................................................................. 46

2.9.6.4 Purification of DDT ................................................................. 46

2.9.7 In vitro MIF/p115 Binding Studies ............................................ 46

2.10 In vivo Mouse Experiments .......................................................... 47

2.10.1 LPS Shock ................................................................................... 47

2.10.1.1 Serum Collection ................................................................. 47

2.10.2 Isolation of Bone Marrow-Derived Macrophages ...................... 48

3 RESULTS ........................................................................... 49

3.1 p115 Mediates the Secretion of MIF........................................... 49

3.1.1 p115 is a Novel Interaction Partner of MIF .............................. 49

3.1.2 The MIF/p115 Interaction is Direct and Specific ...................... 50

3.1.3 MIF Release from Human Monocytes ....................................... 51

3.1.4 Macrophage Activation Leads to a Redistribution of MIF ....... 53

3.1.5 p115 is Required for MIF Release in Monocytes ....................... 54

3.1.5.1 RNAi-Mediated Depletion of p115 ......................................... 54

3.1.5.2 Depletion of p115 Inhibits the Release of MIF ...................... 55

3.1.6 FGF-2 and FGF-4 Export in p115-Depleted HeLa cells ........... 57

3.1.7 Role of p115 in MIF Secretion in Macrophages ........................ 58

3.1.7.1 MIF and p115 are Co-Secreted in Activated Macrophages ... 58

3.1.7.2 Adenovirus-Mediated Depletion of p115 ................................ 62

3.1.7.3 p115 is Required for the Secretion of MIF in Macrophages .. 64

3.1.8 p115 Mediates the Release of MIF from Infected Cells ............ 67

3.1.9 Small Molecule Inhibitor Blocks MIF Secretion ....................... 70

3.1.9.1 Immunorecognition of 4-IPP-Modified MIF ........................... 70

3.1.9.2 4-IPP Targets the MIF/p115 Interaction ............................... 71

3.1.9.3 Effect of 4-IPP in LPS-Shock Model ....................................... 74

V

3.2 D-Dopachrome Tautomerase (DDT) ........................................... 76

3.2.1 Purification of D-Dopachrome Tautomerase ............................. 76

3.2.1.1 Purification of Histidine-tagged DDT .................................... 76

3.2.1.2 Purification of Native DDT Protein ....................................... 77

3.2.2 Establishment of a DDT-ELISA ................................................ 79

3.2.3 DDT is released from macrophages ........................................... 81

3.2.4 Effect of mif-gene deletion on DDT production ......................... 82

4 DISCUSSION ..................................................................... 84

4.1 Role of p115 in MIF Secretion ..................................................... 84

4.1.1 MIF is Released from a Pre-Formed Pool .................................. 85

4.1.2 MIF and p115 Associate at the Perinuclear Site ...................... 85

4.1.3 MIF and p115 Interact Directly ................................................. 86

4.1.4 p115 Is Required for the Stimulated Release of MIF ............... 87

4.1.4.1 p115 Overexpression does not Affect MIF Secretion ............. 89

4.1.4.2 MIF and p115 are Co-Secreted ............................................... 89

4.1.5 4-IPP Inhibits the Release of MIF and p115 ............................. 90

4.2 The Structural Homologue DDT ................................................. 92

5 SUMMARY AND OUTLOOK ........................................... 94

6 REFERENCES ................................................................... 97

VI

A. Abbreviations

Amino acids are abbreviated in the three letter code.

4-IPP 4-iodo-6-phenylpyrimidine

4-OT 4-oxalocrotonate tautomerase

ABC ATP binding cassette

ATP Adenosine triphosphate

CC Coiled-coiled

CD74 major histocompatibility complex, class II

invariant chain (Ii)

CHMI 5-carboxymethyl-2-hydroxymuconate

isomerase

CLP Cegal ligation and puncture

COP Coat protein complex

CSN5 Subunit 5 of the COP9 signalosome

Ct Chlamydia trachomatis

CXXC Cys-Xaa-Xaa-Cys

DDT D-Dopachrome tautomerase

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DTT Dithiothreitol

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

ER Endoplasmatic reticulum

ERK Extracellular signal-regulated kinase

FCS Fetal calf serum

FGF Fibroblast growth factor

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GFP Green fluorescent protein

HASPB Hydrophilic acylated surface protein B

VII

HSP Heat shock protein

HSPG Heparan sulfate proteoglycan

IB Immunoblot

IFN Interferon

IgG Immune globulin G

IL Interleukin

IP Immunoprecipitation

IPTG Isopropyl β-D-thiogalactopyranoside

ISO-1 (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-

isoxazole acetic acid methyl ester

JAB1 c-JUN activation domain-binding protein 1

JNK c-JUN N-terminal kinase

Kd Dissociation constant

kd kilo-Dalton (1 kDa = 1.6605 • 10-21 g)

LPS Lipopolysaccharide

MDR Multi-drug resistance

MIF Macrophage migration inhibitory factor

MVB Multivesicular body

NAPQI N-acetyl-p-benzoquinone imine

NMR Nuclear magnetic resonance

OD Optical density

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate-buffered saline

PCR Polymerase chain reaction

PDB Protein data bank

qPCR quantitative PCR

PI(4,5)P2 Phosphatidylinositol 4,5 bisphosphate

RA Rheumatoid arthritis

RNA Ribonucleic acid

RNAi RNA interference

RT Room temperature

VIII

SDS Sodium dodecyl sulfate

siRNA short interfering RNA

SNARE soluble NSF attachment receptor

SOD1 Cu, Zn superoxide dismutase

shRNA short hairpin RNA

TBS Tris-buffered saline

tER transitional endoplasmatic reticulum

TH T helper

TLR Toll-like receptor

TMB 3,3’,5,5’-Tetramethylbenzidine

TNF Tumor necrosis factor

TPOR Thiol protein oxidoreductase

Tris 2-amino-2-hydroxymethylpropane-1,3-diol

VTC Vesicular tubular cluster

IX

B. Acknowledgments

In the course of my dissertation, I was very fortunate to have the guidance

of Prof. Jürgen Bernhagen of the Department of Biochemistry and

Molecular Cell Biology at the RWTH Aachen University Hospital and Prof.

Rick Bucala of the Department of Internal Medicine at the Yale University

School of Medicine.

I want to thank Prof. Jürgen Bernhagen for providing the topic of my

thesis and making it possible for me to spend major parts of my thesis

abroad at Yale in the collaborative lab of Prof. Bucala. His scientific advice

and support were invaluable for the successful completion of my thesis.

I thank Prof. Rick Bucala for his continuous willingness to always discuss

the progress of my project and to help me not to lose track of the bigger

picture.

I thank Prof. Baumgartner for reviewing my thesis and accepting the Co-

Referat; and Prof. Rink for his participation in my committee.

I thank Prof. Walter Nickel and Antje Ebert of the Biochemistry Center in

Heidelberg for their help with the FGF studies, and Prof. Paula Kavathas

and Dr. Seung-Joon Lee from Yale University for their support with the C.

trachomatis experiments.

My thanks to all my colleagues in the Bernhagen and Bucala labs for their

support; especially to Lin Leng and Hongqi Lue for their unceasing advice

in lab techniques, Juan Fan for her patience while training me in mouse

experiments, and my bench-mates Birgitt Lennartz, Jason Griffith, Utako

Kaneyuki, Tiffany Sun and Tarah Connolly who were always helpful with

discussions.

X

I thank my fiancé Swen Zierow, for his help with the purification of

proteins, and his emotional support throughout my studies. I’m fortunate

to have you in my life.

I thank my family for their constant encouragement throughout my

academic studies and my life.

Lastly, I thank the Studienstiftung des deutschen Volkes for their generous

financial support of my thesis work.

XI

C. Publications

Parts of my thesis were published in international peer-reviewed journals:

1) D. Kamir, S. Zierow, L. Leng, Y. Cho, Y. Diaz, J. Griffith, C. McDonald,

M. Merk, R.A. Mitchell, J. Trent, Y. Chen, Y.K. Kwong, H. Xiong, J.

Vermeire, M. Cappello, D. McMahon-Pratt, J. Walker, J. Bernhagen, E.

Lolis, and R. Bucala. 2008. A leishmania ortholog of macrophage

migration inhibitory factor modulates host macrophage responses. J

Immunol 180:8250-8261.

2) M. Merk, J. Baugh, S. Zierow, L. Leng, U. Pal, S. Lee, A. Ebert, Y.

Mizue, J. Trent, R. Mitchell, W. Nickel, P. Kavathas, J. Bernhagen, and R.

Bucala. 2008. The Golgi-associated Protein p115 Mediates the Secretion of

Macrophage Migration Inhibitory Factor (MIF). Mol Biol Cell (in revision)

Introduction – 1

1 Introduction

1.1 Macrophage migration inhibitory factor

1.1.1 MIF – An Overview

While developing an assay to measure the migration of peritoneal cells in

capillary tubes, macrophage migration inhibitory factor (MIF) was

discovered in 1962 (1). The first function contributed to MIF was its

inhibitory action on the random migration of macrophages (2), and in the

following years, MIF was shown to activate macrophages leading to their

increased cell surface adhesion and phagocytosis (3). The molecular

mechanisms underlying MIF’s functions nevertheless remained unknown.

In 1989, a human MIF cDNA was first isolated and cloned, and the

expressed polypeptide had the expected molecular weight of 12.5 kDa (4).

Bernhagen et al. first cloned and purified murine MIF as a protein

released from anterior pituitary cells in response to bacterial

lipopolysaccharide (LPS) (5, 6). Soon macrophages were also described as a

major source of MIF; they were observed to release MIF after stimulation

with pro-inflammatory stimuli such as LPS, tumor necrosis factor (TNF) α

and interferon (IFN) γ, and even after stimulation with low concentrations

of anti-inflammatory glucocorticoids (7, 8). In 1999, a MIF knockout mouse

was constructed, showing that the deletion of the mif gene reduces

inflammatory responses (9). The discovery of the MIF cell surface receptor

CD74 (10), and later of the chemokine receptors CXCR2 and CXCR4 (11),

helped to improve the understanding of the molecular mechanism of MIF,

and reports about an association of high expression MIF alleles and

severity of inflammatory diseases further emphasized its role in disease

pathology (12-14).

Introduction – 2

1.1.2 MIF in Inflammatory Disease

Inflammation is the characteristic immune response of tissue to harmful

stimuli. It is a protective attempt to remove the injurious stimulus and

start the healing process. Causes for inflammation can be of physical

(mechanical, thermal, radiation), chemical (toxins, acids, allergens) or

biological (bacteria, viruses, parasites, fungi) nature.

There are two types of inflammation: acute and chronic. Acute

inflammation displays the five classic signs of inflammation: swelling,

redness, pain, heat, and loss of function. In contrast, chronic inflammation

does not show these symptoms, but is characterized by concurrent

inflammation, tissue destruction, and attempts at repair (15). MIF is an

important mediator in both acute and chronic inflammation. It was shown

to be a key mediator in the pathogenesis of endotoxemia/sepsis, arthritis,

glomerulonephritis, atherosclerosis, inflammatory bowel disease and

several other inflammatory and immune conditions (16).

MIF is a ubiquitously expressed protein that is most notably present in

tissue that is in direct contact with the host’s environment including

endothelial cells, and fibroblasts, the gastrointestinal tract, pituitary

glands, kidney, liver, lung, pancreas, skin and eyes. Immune cells are the

major source of circulating MIF, with monocytes/macrophages as the main

contributor, followed by T cells and dendritic cell. B cells, neutrophils,

eosinophils, basophils, and mast cells also release MIF (17).

The release of MIF is a common occurrence in inflammatory diseases, for

example the level of MIF is 5 – 10 times higher in serum of patients with

rheumatoid arthritis compared to a healthy control group (18). Table 1 lists

known stimuli causing the release of MIF.

Table 1: Stimuli responsible for MIF release. Table adapted from (17).

Stimulus Cell type

Endotoxin Monocytes/macrophages,

neutrophils, dendritic cells

Exotoxin Macrophages, splenocytes

Peptidoglycan PBMCs

Heat killed E. coli PBMCs

Introduction – 3

Heat killed S. aureus PBMCs

M. tuberculosis Macrophages

Influenza virus Pulmonary epithelial cells

TNF-α, INFγ Macrophages

C5a Eosinophils, neutrophils

IL-5 Eosinophils

IL-9 Mast cells

Nitric oxide Fetal membranes

Anti-CD3 antibodies T cells

Low-dose glucocorticoids Monocytes/macrophages, T cells

1.1.2.1 Endotoxemia and Sepsis

MIF is rapidly released by specialized pathway from pre-formed pools in

immune and pituitary cells after stimulation with LPS, a virulence factor

in the cell wall of gram-negative bacteria (5, 8, 19).

In a murine model of endotoxemia, MIF was rapidly released after

intraperitoneal injection of LPS, and peaked 8 – 20 h after stimulation.

The concurrent administration of recombinant MIF protein and LPS

decreased the survival rate compared to the control group (5). Moreover,

neutralizing MIF via antibodies, the deletion of the mif gene, or the

administration of small molecule MIF inhibitor prior to LPS challenge

improved the chances for survival significantly (5, 9, 20). These results are

in accord with models of E. coli injection in the peritoneum or cecal

ligation and puncture (CLP). Peritonitis leads to an increased MIF level in

the serum and treatment with anti-MIF antibodies has a protective effect

(21).

Of note, if mice with sublethal peritonitis had a secondary infection with

S. typhimurium, P. aeruginosa, or L. monocytogenes 48 h after the first

infection, the administration of recombinant MIF had a protective effect,

whereas applying anti-MIF antibody further enhanced the negative effect

of bacterial superinfection. This indicates that MIF has an important

regulatory role in the inflammatory response, with likely a positive

function in immune suppressed animals by reenabling the organism to

react adequately to a secondary infection (22).

Introduction – 4

1.1.2.2 Rheumatoid Arthritis

MIF is closely associated with the development of rheumatoid arthritis

(RA) (16). RA is the most common inflammatory disease of the joints, it is

estimated that 1% of adults in the U.S. and Europe suffer from RA. Key

pathologic characteristics of RA are a marked expansion of the synovium

by infiltrating leukocytes, the activation of apoptosis of resident cells, and

the participation of cytokines such as TNF-α and IL-1β. MIF is a key

regulator for the migration of leukocytes into the joint in RA (23).

In 1997, the role of MIF in inflammatory arthritis was reported for the

first time (24). In a experimental model of collagen type-II induced

arthritis the treatment with neutralizing anti-MIF antibodies before

immunization with collagen led to a delayed onset of RA, lowered the

frequency of arthritis, and reduced the levels of IgG2a. Treatment with

neutralizing anti-MIF antibody also showed protective effects in the model

of adjuvant-induced arthritis and antigen-induced arthritis (25, 26).

Recent studies employing mif-/- mice confirmed the importance of MIF in

arthritis. The severity of the disease, both in collagen-induced arthritis

and antigen-induced arthritis, was less pronounced in mice with a deletion

of the mif gene (27, 28). It was shown that both Th1 and Th2 cells

expressed high quantities of MIF, and that MIF stimulates the expression

of MMP (matrix metalloproteinase) in FLS (29).

In RA patients, MIF has been detected in synovial fluid, macrophages,

endothelial cells, fibroblast-like synoviocytes (FLS), and CD3+ synovial

lymphoid aggregates (30, 31), and MIF production is associated with

disease activity.

1.1.2.3 Atherosclerosis

Atherosclerosis is characterized by the growth of atherosclerotic plaques in

the vascular wall over the period of many years, leading to hardening and

constriction of arteries. Inflammation and accumulation of large numbers

of white blood cells (monocyte-derived macrophages, macrophage-derived

Introduction – 5

foam cells, to a lesser degree Th1 cells) is now regarded as a crucial force in

the initiation and progression of atherosclerotic lesion formation (32). Both

cytokines (TNF-α, GM-CSF, IL-12) and chemokines (CXCL8, MCP1) are

pivotal for the recruitment of monocytes and the activation of intimal

macrophages/foam cells.

Recent studies indicate an important role for MIF in atherogenesis,

atheroma formation and vascular disease. In 2002, the strong

overexpression of MIF in human atheroma lesions was reported for the

first time. It was also reported that the increased expression of MIF is

accompanied by an increase in disease progression. MIF protein was

upregulated in HUVEC cells when the cells were incubated with oxidized

LDL. Furthermore, stimulation of macrophages with oxidized LDL led to

an enhanced secretion of MIF (33). Importantly, through interaction with

the chemokine receptors CXCR2 and CXCR4, MIF is instrumental in

inflammatory leukocyte recruitment in atherosclerosis, targeting

monocytes and neutrophils through CXCR2 and T cells through CXCR4

(11). MIF is also causally linked to the development of atherosclerosis

lesions. Mice lacking the low-density lipoprotein receptor (ldlr) are prone

to develop atheroma. In comparison, mice lacking both ldlr and mif exhibit

a reduction in atheroma lesions (34). Furthermore, it was reported that

the neutralization of MIF in apoE-/- (apolipoprotein E) mice results in a

marked reduction in inflammatory responses associated with

atherosclerosis (35). It is also proposed that MIF serves as a link between

rheumatoid arthritis and atherosclerosis (36).

1.1.2.4 Asthma

Asthma is a chronic, inflammatory disease of the respiratory system. In

response to allergens such as tobacco smoke or air pollution, the airways

constrict, become inflamed and produce excessive amounts of mucus.

Studies in both mice and humans have supported a role of T helper 2 (Th2)

cells in asthma which mediate allergic inflammation by producing

Introduction – 6

cytokines leading to the production of IgE, and the differentiation,

proliferation and activation of eosinophils (37).

Rossi and colleagues showed that human eosinophils stimulated with C5a

or IL-5 release high amounts of MIF in vitro. Furthermore the

bronchoalveolar lavage fluid obtained from asthmatic patients contains

significantly elevated levels of MIF as compared to nonatopic normal

volunteers (38). In 2005, genetic studies observing the prevalence of

different MIF polymorphisms in asthma patients were performed. The

MIF gene has a functional promoter polymorphism consisting of a

tetranucleotide sequence, CATT, which is repeated five to eight times. A

high number of repeats results in an increase in MIF promoter activity

(39). While there was no association between particular alleles and

asthma incidence, a significant association between a mild form of asthma

and low-expression, 5-CATT MIF allele, was observed. Furthermore the

study demonstrated that mif-deficient mice show less pulmonary

inflammation and lower airway hyperresponsiveness than wildtype mice.

This was in accord with the finding that mif-/- mice have lower titers of

IgE and TH2 cytokines (13).

1.1.3 Molecular Mechanisms of MIF Action

1.1.3.1 ERK 1/2 Activation

Different stimuli lead either to a transient (rapid, within minutes) or

sustained (prolonged, for hours) activation of extracellular signal-

regulated (ERK) mitogen-activated protein kinases. MIF promotes both

the transient and the sustained ERK 1/2 signaling pathway (40, 41).

The sustained phase of ERK activation was shown to occur through

autocrine and paracrine stimulation and depends on protein kinase A (41,

42). It has been established that the sustained ERK 1/2 activation is

associated with an activation of cytosolic phospholipase A2, overriding of

glucocorticoids and a promotion of cell proliferation.

Introduction – 7

Lue et al demonstrated that the stimulation of fibroblasts with MIF leads

rapidly to a transient activation of the ERK 1/2 signaling pathway. They

also established that transient ERK phosphorylation by MIF is dependent

on a Src-kinase and leads to the phosphorylation and activation of the

transcription factor Elk.

1.1.3.2 MIF Inhibits JAB1 Activity

In a yeast two-hybrid screen JAB1 (c-JUN-activation domain-binding

protein-1) was identified as an intracellular binding partner of MIF (43).

JAB1, also known as CSN5 (subunit 5 of the COP9 signalosome),

regulates the degradation of p27Kip1 and activation of JNK (c-JUN N-

terminal kinase) and is a co-activator of AP-1, a transcription factor

implicated in cell growth, transformation and cell death. MIF antagonizes

these effects via the inhibition of the JAB1-dependent AP-1 transcription.

Furthermore, MIF stabilizes the cell cycle inhibitor p27Kip1 by inhibiting

the JAB1-mediated degradation of p27Kip1.

Recently, it was reported that JAB1 traps MIF inside cells in order to

prevent MIF secretion. The depletion of JAB1 protein led to an increased

secretion of MIF (44), whereas the overexpression of JAB1 led to an

inhibition of sustained ERK activation (40). Although there is no evidence

for an active participation of JAB1 in the secretory pathway of MIF, JAB1

nevertheless seems to have the ability to block MIF secretion and its

autocrine and paracrine functions.

1.1.3.3 MIF in Apoptosis

p53 has a central role in the regulation of the cell cycle. In a screen to

identify proteins which by-pass p53-dependent cell cycle control, MIF was

identified as a negative regulator of p53 activity (45). MIF sustains

macrophage survival by suppressing activation-induced, p53-dependent

apoptosis (46). Mitchell and colleagues reported that LPS-challenge in mif-

deficient mice results in decreased macrophage viability, decreased

Introduction – 8

proinflammatory function, and increased apoptosis when compared to

wild-type macrophages. In 2008, it was reported that MIF and p53

interact physically, and that cysteine-81 in MIF is of critical importance

for the physical and functional interaction (47). It is important to note that

in comparable studies a direct, physical interaction between MIF and p53

could not be detected (personal communication, J. Bernhagen).

1.1.3.4 MIF Upregulates TLR4 Expression

MIF has been implicated in the host response to LPS challenge in

numerous studies. Toll like receptor (TLR) 4 forms a receptor complex

with LPS binding protein and CD14, with TLR4 being the essential

signaling component when LPS is bound to the receptor complex. It was

demonstrated that mif-/- macrophages have a reduced expression level of

TLR4, but not of LBP or CD14. The downregulation of TLR4 in mif-

deficient mice leads to a hyporesponsiveness towards LPS resulting in a

reduced production of TNF-α, IL-1β, IL-6 and IL-12. (48).

1.1.3.5 MIF Receptors

Cytokines bind to their cell surface receptors and trigger an intracellular

signaling cascade. CD74 was discovered as the cell surface receptor of MIF

in 2003 (10). Surface plasmon resonance experiments between MIF and

soluble CD7473-232 revealed an equilibrium Kd of ~9 nM. Leng et al

reported that the MIF-dependent phosphorylation of ERK 1/2, the

production of prostaglandin E2 and cell proliferation requires the cell

surface expression of CD74. CD74 does not possess any obvious signal

transduction domains; therefore, the molecular mechanism of signaling

remained unclear for some time. Recent data showed that MIF’s binding

to CD74 leads to the phosphorylation of CD74 at the intracytoplasmic

domain in a CD44-dependent manner (49). The co-receptor CD44 was

demonstrated to be the missing link of MIF/CD74-mediated sustained

ERK activation via Src-kinases.

Introduction – 9

In 2007, the chemokine receptors CXCR2 and CXCR4 were discovered as

non-cognate receptors for MIF in MIF-mediated migratory and

atherogenic functions (11). MIF competed with cognate ligands for CXCR2

and CXCR4 binding, and directly binds to CXCR2 and CXCR4 with a Kd of

1.4 and 20 nM, respectively. Monocyte arrest induced by MIF involved

both CXCR2 and CD74. Furthermore, it was described that CD74 and

CXCR2 co-localize, suggesting a MIF signaling pathway via a

CD74/CXCR2 complex.

1.1.4 Structure, Enzymatic Activities and Inhibitors

1.1.4.1 Structure

The MIF protein consists of 114 amino acids. In 1996, the structure of MIF

was solved independently by crystallography (50, 51) and NMR (52). The

secondary structure of the MIF monomer consists of two α-helices and six

β-strands, four of these strands form a β-sheet (Fig. 1).

The structural analysis by crystallography revealed MIF to be a trimer,

stabilized by hydrogen bonds and a hydrophobic core. The three

dimensional structure of MIF possesses a strong homology to D-dopa-

chrome tautomerase (DDT) and the bacterial enzymes 5-carbodymethyl-2-

hydroxymuconate isomerase (CHMI) and 4-oxalocrotonate tautomerase (4-

OT) (53). These proteins are members of the MIF/tautomerase super-

family.

Although crystallographic analysis revealed MIF to be a trimer, other

studies, such as NMR and size exclusion chromatography, support the

idea that MIF forms a monomer or dimer (52, 54). The question of the

physiological state of MIF is still not answered to satisfaction.

Introduction – 10

Fig. 1: The three-dimensional structure of MIF. Left: Human MIF monomer. Right:

Human MIF trimer. Structures are drawn from PDB entry 1MIF using PyMOL (55).

1.1.4.2 Enzymatic Activities

There are two catalytic activities reported for MIF: a thiol protein

oxidoreductase (TPOR) and a tautomerase/isomerase reaction.

The base of MIF’s TPOR activity is located between residues 57 and 60 of

MIF in its so-called CXXC-motif. Although not reconcilable with the

crystallographic analysis of the recombinant reduced MIF trimer, the

cysteines are reported to form reversible, intramolecular disulfide bonds

(56). The CXXC-motif lies within the JAB1 binding site (57), and is

structurally sensitive as mutations especially at C60 result in dramatic

changes in the folding of MIF.

The second enzymatic activity of MIF is a tautomerase function which it

shares with all members of the MIF/tautomerase superfamily (DDT,

CHMI, 4-OT). Proline-1 is the catalytic base in all proteins, and is highly

conserved. It is not only present in vertebrate MIF, but also in MIF

orthologs found in parasites such as Plasmodium falciparum and

Leishmania major (58). So far the physiological substrate for the

tautomerase activity is not known, but non-physiological substrates have

been identified: D-dopachrome and hydroxyphenylpyruvate (59, 60) (Fig.

2).

Introduction – 11

Fig. 2: The tautomerase reaction of MIF. A) MIF catalyzes D-Dopachrome to 5,6-

dihydroxyindole-2-carboxylic acid. B) Keto-enol isomerization of both p-hydroxyphenyl-

pyruvate and phenylpyruvate

1.1.4.3 Small Molecule Inhibitors

The active site of MIF around proline-1 was reported to be of importance

for the pro-inflammatory actions of MIF. Mutational analysis revealed

proline-1 to be involved in the counter-regulation of glucocorticoids,

neutrophil priming and the upregulation of metalloproteinase-1 and 3 in

rheumatoid arthritis synovial fibroblast (61-63). Prototypic small molecule

inhibitors of MIF have been described that bind to the protein’s amino-

terminal tautomerase region. Prominent inhibitors are N-acetyl-p-

benzoquinone imine (NAPQI), (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-

isoxazole acetic acid methyl ester (ISO-1), and 4-iodo-6-phenylpyrimidine

(4-IPP) (61, 64, 65) (Fig. 3).

ISO-1 binds reversibly to MIF and does not only inhibit the tautomerase

activity of MIF, but also inhibits MIF’s ability to counter-regulate

glucocorticoids (61), and protects mice from endotoxic shock (20). NAPQI

binds covalently to MIF and the modification of MIF with NAPQI results

in an inhibition of its tautomerase activity, decreased cell binding ability

and a decreased immunorecognition (64). The inhibitor 4-IPP was found

via computational virtual screening and also binds covalently to the amino

acid proline-1. In contrast to NAPQI, which only bound to one third of MIF

molecules, almost all MIF molecules were modified by 4-IPP. This is

Introduction – 12

reflected in the IC50 determined by D-dopachrome tautomerase assay; ISO-

1 has an IC50 of ~50 µM, NAPQI of ~40 µM, and 4-IPP of ~5 µM.

Functionally, 4-IPP was reported to inhibit MIF-dependent cell motility

and growth in vitro (65).

Fig. 3: Small molecule MIF inhibitors. Left: NAPQI, Middle: ISO-1, Right: 4-IPP

1.1.5 The Structural Homolog D-Dopachrome Tautomerase

D-dopachrome and p-hydroxyphenylpyruvate serve as substrate for the

tautomerase activity of both MIF and D-dopachrome tautomerase (60, 66).

But in contrast to MIF, which converts D-dopachrome to 5,6-

dihydroxyindole-2-carboxylic acid, DDT decarboxylases D-dopachrome to

5,6-dihydroxyindole. The enzymatic activity of DDT was first described in

melanocytes (67); higher activities later were reported in human liver and

blood (68).

Murine DDT protein shares only 28% identity and 45% homology with

murine MIF. Of note, proline-1, the catalytic base of the tautomerase

activity in MIF is conserved in DDT. Furthermore, the active side residues

lysine-32 and isoleucine-64 also are present in DDT (Fig. 4). Interestingly,

DDT possesses no CXXC-motif which is associated with the TPOR activity

in MIF (56).

Introduction – 13

Fig. 4: Sequence alignment of mouse MIF with mouse D-dopachrome

tautomerase. The secondary structure of mouse MIF is shown (Pdb: 1MFI). Elements of

the secondary structures are indicated by α for α-helix and β for a β-strand of β-sheet, η

for a short α-helix and T for turns. The sequence identity of mouse DDT and mouse MIF

is 28%. Invariant residues among DDT and MIF are marked by black background; if

residues are similar they are marked by a box.

Human DDT protein was crystallized in 1999. The overall folding and

subunit topology are almost identical to human MIF, with two αβα motifs

related by a pseudo 2-fold symmetry and similar trimeric β-sheet packing

(Fig. 5) (69).

Fig. 5: Human D-dopachrome tautomerase. Left: Human DDT monomer. Right:

Human DDT trimer. Structures are drawn from PDB entry 1DPT with PyMOL (55).

To date, little is known about the biological functions and mechanisms of

action of DDT. It is highly expressed in the liver, and also found in

kidneys, brain, spleen, lung and the heart (70). In 2003, it was reported

that DDT activity is increased in UVB-induced blister fluid.

Correspondingly, the activity of MIF was also found to be increased (71). A

Introduction – 14

more recent study suggests a role for DDT in cancer. Coleman et al

reported about the individual and cooperative action of MIF and DDT in

angiogenesis showing that the depletion of MIF and/or DDT reduces the

basal secretion level of CXCL8 and VEGF from non-small cell lung

carcinoma cells. The transcriptional regulation of CXCL8 by MIF and DDT

appears to involve JNK, c-JUN and AP-1. Furthermore, they showed that

the MIF receptor CD74 is necessary both for MIF and DDT induced JNK

activation (72).

1.2 Classical Secretion in Eukaryotes

Over the last two decades, considerable progress was achieved in

improving the understanding of protein export. The process requires

multiple components including the endoplasmatic reticulum (ER), the

Golgi, and secretory vesicles.

Proteins targeted for the conventional export into the extracellular milieu

possess a signal peptide. This sequence directs the ribosome to the rough

ER membrane and initiates the transport of the growing peptide chain

across it into the lumen of the ER (73, 74).

The ER is one of the largest intracellular compartments in cells and

consists of a large network of membrane cisternae (75). Proteins are

translated into the lumen of the ER, where they either fold into their

native structure by themselves or with the aid of chaperones. The proteins

are screened for their proper folding (76), before being transported to the

Golgi in cargo vesicles (77). Misfolded proteins are degraded by the 26S

proteasome (78).

The Golgi, a highly dynamic organelle, is polarized with a cis-side facing

the ER, and a trans-side, communicating with the plasma membrane. On

their way through the Golgi, proteins are further processed mainly by

“trimming” of N-glycosylations obtained in the ER (79). Over the trans-

Golgi network, cargo proteins exit the Golgi and are transported to the

Introduction – 15

plasma membrane in vesicles. Proteins are released into the extracellular

space via a fusion of the carrier vesicles with the plasma membrane (80)

(Fig. 6).

Fig. 6: Secretory pathway. Proteins destined for export are transported in COPII-

coated vesicles from the ER to the cis-Golgi; they are leaving the Golgi at the trans-side

and are transported in vesicles to the plasma membrane. Resident ER proteins are

returned from the Golgi to the ER in COPI-coated vesicles.

1.2.1 ER/Golgi Transport and the Golgi Apparatus

1.2.1.1 ER to Golgi Transport

Proteins leave the ER not uniformly across the ER, but at distinct exit

sides, also called transitional ER (tER) sides. At these sites, no ribosomes

are docked to the ER, and an increase in budding activity is evident by

electron microscopy (81, 82). At the tER, COPII-coated vesicles bud off

carrying cargo to the Golgi.

Introduction – 16

These vesicles uncoat and fuse to form tubular clusters, termed vesicular-

tubular clusters (VTCs) (83). VTCs become part of the cis-Golgi network,

and ER-resident proteins are recycled in COPI-coated vesicles in

retrograde vesicle transport (84).

The ability of transport vesicles to fuse and form tubes is conferred by

tethering factors which ensure specific binding interaction and SNAREs

(soluble NSF attachment receptor) which facilitate the fusion of

membranes (85). Tethering proteins act like ropes between a vesicle and

target membrane. In addition to multi-component tethers such as the

exocyst, which facilitates the binding of vesicles to the plasma membrane

(86) or conserved oligomeric Golgi complex which is of importance in the

intra-Golgi transport (87), there are also filamentous tethering proteins

such as p115.

SNAREs are subdivided into v- and t-SNAREs referring to their presence

on either vesicles or target membranes. When v- and t-SNAREs are

pairing, they form a SNAREpin and fusion occurs.

It was reported that the tethering protein p115 which shares some

sequence homology with SNAREs, is required (at least in vitro) for the

initial interaction of SNAREs (88).

After cargo proteins reach the Golgi from the ER by tethering and fusion

of their carrier vesicles, proteins traverse through the Golgi.

1.2.1.2 The Golgi Apparatus

Over the last 30 years, one of the most controversial discussions in protein

export concerned the question how cargo proteins cross the Golgi and are

modified by resident Golgi proteins.

Two models are mainly discussed: On the one hand, there is the idea that

cargo arrives at the cis-side of the Golgi and then traverse through the

Golgi to the trans-side, the resident Golgi proteins stationary in their

tubular structure. On the other hand, there is the hypothesis that as

vesicles arrive from the ER, they form cis-tubular structures and then

Introduction – 17

progress to mature into trans-tubular structures. This implies that cargo

proteins remain in their distinct structures and resident Golgi proteins are

shuttled between the cisternae (89). In recent years, more and more

evidence supports this second hypothesis of cisternal maturation.

Recently, live imaging in yeast has provided direct evidence in support of

this hypothesis (90, 91).

1.2.2 The Tethering Protein p115

The transport between ER and Golgi requires the tethering and fusion of

vesicles. p115, belonging to the family of golgin proteins, is one of the best

studied tethering proteins involved in this process (92).

Golgins were originally identified as a group of proteins found in the sera

of patients with a variety of autoimmune diseases (93). Members of the

golgin family possess a coiled-coiled region forming a rod-like structure

(94). Localization studies showed that p115 predominantly localizes to the

cis-Golgi, and it was also detected on COPII-coated vesicles (95, 96).

p115 has a globular head, a extended coiled-coiled (CC) region and a short

acidic tail (Fig. 9). It forms a parallel homodimer by dimerization in the

CC region (97). Distinct domains interact with different proteins: the

acidic carboxy-terminal tail interacts with the golgin proteins GM130 (98)

and giantin (95), the CC1 domain of p115, a region with SNARE homology,

interacts with SNARE family members such as syntaxin 5 (88, 99), the

guanosine triphosphatase Rab1 mediates the recruitment of p115 by

interacting with the globular head (96).

p115, located at the cis-Golgi, tethers COPI-coated vesicles due to its

interaction with giantin and GM130. Furthermore, p115, located to

COPII-coated vesicles, tethers these vesicles in a SNARE-dependent

process to form VTCs. These VTCs then dock with the cis-Golgi facilitated

by an interaction between p115 and GM130. All tethering functions of

Introduction – 18

p115 are mediated through the interaction of p115 with Rab1 (Fig. 7)

(100).

Fig. 7: Tethering functions of p115. A) COPII-coated vesicles are tethered by p115.

Rab1 recruits p115 on these vesicles by interaction with the globular head. Tethered

vesicles fuse in a SNARE-dependent manner and form VTCs. B) Docking of VTCs to the

cis-Golgi is facilitated by the interaction of p115 and GM130. The acidic carboxy-terminal

tail of p115 binds to a basic amino-terminal region of GM130. C) Role of p115 in the

tethering of COPI-coated vesicles. Giantin, located on the COPI-coated vesicles interacts

with GM130 and p115 on the cis-side of the Golgi. Figure taken from (100).

In various studies it was demonstrated that p115 is essential for the

integrity of the Golgi. Both a depletion of p115 below detectable levels and

the injection of antibodies targeting p115 led to a fragmentation of the

Golgi (92, 101-103). It was furthermore reported that the fragmentation of

the Golgi by targeting p115 led to a decreased protein transport from the

ER to the cell surface (92).

Introduction – 19

1.3 Non-Classical Secretion

In contrast to the classical secretion of proteins where proteins are

exported via the ER/Golgi route, the pathway of non-classical secretion is

not uniform. Unconventionally secreted proteins do not possess a

hydrophobic signal peptide and therefore are not translocated into the

lumen of the ER. In contrast to classically secreted proteins, proteins

lacking a signal peptide are translated on free ribosomes. Evidence for this

heterogeneous group of proteins that is secreted from cells without

entering the ER/Golgi pathway came from the observation that leaderless

proteins are not inhibited in their secretion by brefeldin A (104-107).

Brefeldin A is an anti-fungal drug that interferes with the anterograde

protein transport from the ER to the Golgi, and causes a rapid

fragmentation of the Golgi (108, 109).

Distinct mechanisms were suggested to describe non-conventional protein

secretion including vesicular transport via either lysosomes and exosomes,

and vesicle-independent export via plasma membrane shedding and direct

translocation mediated by transporters in the plasma membrane (Fig.

8)(110).

Fig. 8: Potential export routes of non-conventionally secreted proteins. 1: Export

by secretory lysosomes; 2: Export mediated by plasma membrane-resident transporters;

3: Export through the release of exosomes derived from multivesicular bodies; 4: Export

mediated by plasma membrane shedding of microvesicles. Figure taken from (110).

Introduction – 20

1.3.1 Vesicle-Independent Translocation

1.3.1.1 Membrane Blebbing/Exovesicles

The plasma membrane is highly dynamic, and its reorganization is

controlled by small Rho GTPases. Specific Rho GTPases destabilize the

actin filaments resulting in the formation of rounded cell protrusions,

called membrane blebs (111). There are multiple, distinct types of

membrane blebs including necrotic and apoptotic blebs.

The export of the Leishmania protein HASPB (hydrophilic acylated

surface protein B) belongs to the group of unconventionally secreted

proteins employing the process of membrane blebbing (112). HASPB is

only produced in the infectious stages of the parasite life cycle, and is then

synthesized on free ribosomes. The amino-terminus of the protein becomes

both myristolylated and palmitoylated which are necessary modifications

for a membrane anchorage of the protein (113). Denny et al observed that

myristylation and palmitoylation of HASPB at its amino-terminal SH-4

domain leads to its redistribution to the plasma membrane. Both the

deletion of the complete amino-terminus and the mutation of the

myristylation site led to a redistribution of HASPB in the cytoplasm.

Mutation of the palmitoylation site led to an accumulation of HASPB at

the Golgi. In the secretion model proposed based on these observations,

HASPB is first located to the Golgi and subsequently transported to the

plasma membrane, probably using the conventional vesicular transport

system. Once anchored to the plasma membrane, the SH4-domain of

HASPB induces membrane blebbing in a Rho-dependent manner.

1.3.1.2 Plasma Membrane-Resident Transporters

Secretion facilitated by transporters residing in the plasma membrane is

another possible way to export proteins independent of the ER/Golgi

pathways. Fibroblast growth factors are heparan-binding growth factors

that are activators of tumor-induced angiogenesis. Fibroblast growth

factor (FGF) 2 is believed to be exported by direct translocation.

Introduction – 21

In an in vitro system employing inside-out vesicles it was demonstrated

that FGF-2 is translocated across the plasma membrane and that the

membrane-derived vesicles contain the molecular machinery necessary for

an efficient export of FGF-2. The process is independent of ATP or a

membrane potential; other proteins, among these MIF, were not able to

translocate into the vesicles (114). Before FGF-2 is able to translocate

across the plasma membrane, it needs to be targeted to the plasma

membrane. This likely occurs through the interaction with

phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) (115). It was

demonstrated that the downregulation of PI(4,5)P2 leads to a decreased

export of FGF-2. So far, the actual transporter of FGF-2 in the plasma

membrane has not been elucidated, but it is believed that heparan sulfate

proteoglycans (HSPGs) probably serve as an extracellular trap to ensure

the transport of FGF-2 into the extracellular space (116).

1.3.2 Vesicle-dependent secretion

1.3.2.1 Lysosomal Secretion

Secretory lysosomes share many features with conventional lysosomes.

But in contrast to conventional lysosomes which are multi-vesicular in

structure, secretory lysosomes have a more diverse structure. Both

secretory and conventional lysosomes contain classical markers such as

LAMP-1 and Hsc70, but secretory lysosomes additionally contain a specific

cell-type specific set of components (117).

Among the proteins reported to be secreted via lysosomes are interleukin-

1 (IL-1) β, heat shock protein (HSP) 70 and HMGB1 (118-120).

Andrei et al demonstrated that IL-1β is co-fractioning with endolysosomes

and that an inhibition of endolysosomal proteases leads to an increased

IL-1β secretion. Via immunolocalization they revealed that IL-1β-positive

vesicles also stain for the lysosomal marker LAMP-1 (118). Upon

stimulation of cells, it is believed that these vesicles undergo fusion with

Introduction – 22

the plasma membrane and release IL-1β into the extracellular milieu

(121). Similarly HSP70, a protein released from prostate carcinoma cells

after heat shock, appears in the extracellular space at the same time that

LAMP-1 appears on the cell surface, suggesting that lysosomes facilitate

the unconventional secretion of HSP70.

1.3.2.2 Exosomes/Multivesicular Bodies

Exosomes represent another vesicle-based mechanism for export of

proteins independent of the ER/Golgi pathway. Exosomes are a distinct set

of membrane vesicles homogenous in form and size (122, 123). Exosomes

were first identified as a mechanism for externalization of obsolete

membrane proteins during reticulocyte maturation (124).

The characterization of exosomes is based on morphology and biochemical

criteria. The diameter of exosomes is between 50 and 90 nm, and they are

released when multivesicular bodies (MBVs) fuse with the plasma

membrane. Many cell types are known to release exosomes including

hematopoietic cells, reticulocytes, B- and T-lymphocytes, dendritic cells

and tumor cells (125).

It was recently reported that the unconventional secretion of Cu, Zn

superoxide dismutase (SOD1) is facilitated by exosomes (126). SOD1 was

detected on isolated exosomes, but not on membrane blebs. In this study,

the authors furthermore found p115 both on exosomes and membrane

blebs.

Another role for exosomes in the process of non-classical protein secretion

is the export of the heat shock protein 90α (HSP90α) (127). In human

keratinocytes, transforming growth factor α stimulates the rapid secretion

of HSP90α via an exosomal secretion pathway. The inhibitor dimethyl

amiloride inhibited the secretion of HSP90α in a concentration-dependent

manner by blocking exosome-mediated protein secretion, while Brefeldin

A did not show any inhibitory effects.

Introduction – 23

A further complication in the study of unconventional protein secretion

comes from the fact that for some unconventionally secreted proteins

several potential secretory pathways were described for one protein.

Potential secretory pathways that have been described for interleukin-1

(IL-1β) for instance, include cell surface blebbing and the formation of

microvesicles that lyse in the extracellular space (128), and the fusion of

endolysosomal vesicles with the cell surface plasma membrane (118).

1.3.3 Role of ABC transporters in unconventional secretion

The ATP binding cassette (ABC) transporter superfamily is one of the

largest protein families known, with members in almost all organisms

from bacteria to mammals and plants. These transmembrane proteins

actively transport specific substrates across extra- and intracellular

membranes. In eukaryotes, these proteins mainly transfer molecules to

the outside of the cell. Classified in seven groups, 48 ABC transporter

proteins are known in humans (129). Multiple drug resistance (MDR)-1

was the first ABC transporter characterized in humans and it is also the

best studied. MDR transporters are important in pharmacology, for

example, they pump tumor suppressor drugs out of cancer cells (130).

ABCA1 is a member of the subfamily ABCA and has been implicated as a

regulator in cellular cholesterol and phospholipid homeostasis.

Additionally, ABCA1 is reported to be involved in the unconventional

secretion of several proteins; among these are IL-1β, HSP70, and Annexin-

1 (119, 131, 132). It was demonstrated that the pharmacological inhibitor

of ABCA1, glyburide, inhibits the release of these proteins. In the case of

Annexin-1 the results obtained by using pharmacological inhibition were

confirmed in siRNA experiments targeting ABCA1. To date, no study was

undertaken in regard to Annexin-1 and vesicles. However, it remains

unclear whether ABC transporters are directly or indirectly involved in

the unconventional secretion pathway of these proteins.

Introduction – 24

1.3.4 Unconventional Secretion of MIF

As mentioned previously, MIF belongs to the heterogonous group of

proteins that do not possess a signal peptide and are secreted by a

specialized, unconventional pathway. MIF from bovine liver cell lysates or

monocytes/macrophages does not become post-translationally N-

glycosylated although two potential N-glycosylation sites are present in

the MIF cDNA sequence (6, 105). Only after the experimental fusion of

the leader-sequence of IL-2 to MIF, N-glycosylations of MIF were

observed, indicating that native MIF is excluded from the classical

secretory organelles ER and Golgi. Furthermore, brefeldin A, an inhibitor

of the ER/Golgi pathway did not affect the stimulated release of MIF from

monocytes/macrophages and neutrophils, whereas glyburide and

probenicide, inhibitors of the ABCA1 transporter, blocked the MIF

secretion (105, 133). Of note, there is evidence that MIF is localized to

vesicles in certain cell types (pituitary cells, β-cells of the pancreatic islets,

and epididymal cells) (134-136). Interestingly, other unconventionally

secreted proteins that are inhibited in their secretion by glyburide (IL-1β,

HSP70) are also associated with vesicles.

Material and Methods – 25

2 Material and Methods

2.1 Cell lines, Bacteria and Plasmids

2.1.1 Cell lines

Description Cell type Morphology

HeLa Human cervix carcinoma

cells

Epithelial, adherent

HEK 293 Human embryonal kidney

cells

Epithelial, adherent

RAW 264.7 Murine ascites leukemia

cells

Macrophage/monocyte,

adherent

THP-1 Human peripheral blood

leukemia cells

Monocyte, suspension

2.1.2 Bacteria

Strain Genotype Origin

E. coli

DH5α

F- mcrA Δ(mrr-hsdRMS-mcrBC)φ80lacZ

ΔM15 ΔlacX74 recA1 araD139 Δ(araleu)

7697 galU galK rpsL (StrR) endA1 nupG

Invitrogen,

Carlsbad, CA

E. coli

BL21 (DE3)

F- ompT hsdSB (rB-mB-) gal dcm rne131

(DE3)

Invitorgen,

Carlsbad, CA

2.1.3 Plasmids

Plasmid Insert Resistance/

Promotor

Origin

pET22b p115 Amp/ T7 this work

pET22b DDT Amp/ T7 this work

pET11b MIF Amp/ T7 Bucala +

Bernhagen

lab

pENTR/U6 shRNA targeting p115 or

lacZ

Kan/U6 this work

pAd/Block-iT

Dest

shRNA targeting p115 or

lacZ

Amp/U6 this work

pAd/CMV/V5-

DEST

p115 Amp/CMV this work

pAd/CMV/V5-

DEST

lacZ Amp/CMV this work


2.2 Equipment, Consumables and Chemicals

2.2.1 Equipment

Equipment Manufacturer

Äkta FPLC system GE Healthcare, Piscataway, NJ

Amaxa nucleofector Amaxa, Gaithersburg, MD

Amaxa nucleofector Amaxa, Cologne, Germany

Analytical balance Acculab, Columbia, MD

Beckman Alegra6 centrifuge Beckman, Fullerton, CA

Centrifuge 5417 R Eppendorf Eppendorf, Westbury, NY

Centrifuge 5417 R Eppendorf Eppendorf, Wesseling-Berzdorf,

Germany

CO2 incubator Heracell Thermo Scientific, Waltham, MA

CO2 incubator Heracell Heraeus Instruments, Hanau,

Germany

Electrophoresis Power Supply BioRad, Hercules, CA

Heat block thermo mixer comfort Eppendorf, Westbury, NY

HERA-Safe sterile bench Heraeus Instruments, Hanau,

Germany

HERA-Safe sterile bench Thermo Scientific, Waltham, MA

iCycler BioRad, Hercules, CA

HiTrap chelating HP column GE Healthcare, Piscataway, NJ

Light microscope Carl Zeiss, Thornwood, NY

Light microscope Olympus CK40 Olympus Co GmbH, Hamburg,

Germany

LSM510 confocal microscope Carl Zeiss, Thornwood, NY

NuPAGE/XCell II Blot Module Invitrogen, Carlsbad, CA

NuPAGE/XCell SureLock Invitrogen, Carlsbad, CA

ND-1000 Spectrophotometer NanoDrop, Wilmington, DE

PTC-100 PCR BioRad, Hercules, CA

Q Sepharose Anion exchange

chromatography column

GE Healthcare, Piscataway, NJ

Superdex 75 size exclusion column GE Healthcare, Piscataway, NJ

Sorvall Centrifuge Thermo Scientific, Waltham, MA

SpectraMax Plus Molecular Devices, Sunnyvale, CA

2.2.2 Consumables

Consumables Manufacturer

BioMax MS Film Kodak, Rochester, NY

BioMax MR Film Kodak, Rochester, NY

Blotting paper Whatman, Clifton, NJ

C8 Sep-Pak Waters, Milford, MA

Cell culture equipment BD Falcon, Bedford, MA


Cell culture equipment Greiner, Frickenhausen, Germany

Centricon Amicon Bioseparations

Culture Slides BD Falcon, Bedford, MA

Eppendorf tubes Eppendorf, Westbury, NY

Eppendorf tubes Eppendorf GmbH, Wesseling-

Berzdorf, Germany

ImmunoModule ELISA plate Nunc, Rochester, NY

ImmunoModule ELISA plate Nunc, Roskilde, Germany

NuPAGE 4-12% Bis-Tris-Gels Invitrogen, Carlsbad, CA

Polypropylene tubes BD Falcon, Bedford, MA

PVDF membrane Millipore, Bedford, MS

Thin wall PCR tubes BioRad, Hercules, CA

2.2.3 Chemicals

Chemicals Manufacturer

Acetonitrile J.T.Baker, Phillipsburg, NJ

Agarose Sigma, St. Louis, MO

Ampicillin Sigma, St. Louis, MO

Bacto Agar BD, Franklin Lakes, NJ

BSA American Bioanalytical, Natick, MA

Bradford Reagent BioRad, Hercules, CA

Dimethylsulfoxide Sigma, St. Louis, MO

DMEM Invitrogen, Carlsbad, CA

DMEM Invitrogen, Eggenstein, Germany

DNA ladder Invitrogen, Carlsbad, CA

Dry milk powder BioRad, Hercules, CA

DTT Sigma, St. Louis, MO

EDTA American Bioanalytical, Natick, MA

FBS Invitrogen, Carlsbad, CA

FBS Invitrogen, Eggenstein, Germany

Formaldehyde American Bioanalytical, Natick, MA

Glycerol Sigma, St. Louis, MO

Goat serum Sigma, St. Louis, MO

human Serum Cambrex, Walkersville, MD

Imidazole Sigma, St. Louis, MO

iQ Sybr Green BioRad, Hercules, CA

Kanamycin Sigma, St. Louis, MO

Lipofectamine 2000 Invitrogen, Carlsbad, CA

Lipofectamine 2000 Invitrogen, Eggenstein, Germany

Lipopolysaccharide 0111:B4 Sigma, St. Louis, MO

Lipopolysaccharide 0111:B4 Sigma, Munich, Germany

Luria Broth medium Invitrogen, Carlsbad, CA

Na3VO4 Sigma, St. Louis, MO

NaCl J.T.Baker, Phillipsburg, NJ

NaF Sigma, St. Louis, MO


β-Mercaptoethanol Sigma, St. Louis, MO

Methanol Fisher scientific, Pittsburgh, PA

Nonidet P-40 Roche, Indianapolis, IN

NuPAGE LDS sample buffer Invitrogen, Carlsbad, CA

NuPAGE SDS running buffer Invitrogen, Carlsbad, CA

NuPAGE transfer buffer Invitrogen, Carlsbad, CA

Phosphate buffered saline Invitrogen, Carlsbad, CA

Penicillin/Streptomycin Invitrogen, Carlsbad, CA

PIC Complete Roche, Indianapolis, IN

Protein Free Blocking buffer Thermo Scientific, Waltham, MA

Protein G Sepharose Sigma, St. Louis, MO

RPMI 1640 Invitrogen, Carlsbad, CA

RPMI 1640 Invitrogen, Eggenstein, Germany

RT² SYBR Green SuperArray, Frederick, MD

SeaBlue 2Plus Invitrogen, Carlsbad, CA

SOC medium Invitrogen, Carlsbad, CA

Sodiumdodecylsulfate Sigma, St. Louis, MO

Superblock Pierce, Rockford, IL

Streptavidin-HRP Promega, Madison, WI

Stripping buffer Thermo Scientific, Waltham, MA

TAE buffer Invitrogen, Carlsbad, CA

TMB substrate system Dako, Carpinteria, CA

Tris American Bioanalytical, Natick, MA

Triton-X-100 Sigma, St. Louis, MO

Trypsin/EDTA Invitrogen, Carlsbad, CA

Tween-20 Sigma, St. Louis, MO

2.2.4 Multi-Component Systems

Kits Manufacturer

AccuScript cDNA synthesis Stratagene, La Jolla, CA

Biotin protein labeling Roche, Indianapolis, IN

ELISA (TNF-α, IL-1β, IL-6) ebioscience, San Diego, CA

EZ-Link NHS-SS-Biotin Pierce, Rockford, IL

GeneClean Qbiogene, Carlsbad California

QIAprep MiniPrep Qiagen, Valencia, CA

QIAprep MiniPrep Qiagen, Hilden, Germany

RNeasy Mini Kit Qiagen, Valencia, CA

Qiashredder Qiagen, Valencia, CA

SlowFade Antifade Invitrogen, Carlsbad, CA

Super Signal West Dura (ECL) Pierce, Rockford, IL


2.3 Primary and Secondary Antibodies

2.3.1 Primary Antibodies

Antigen Application* Species/Type** Manufacturer

ß-Actin WB Mouse/M Sigma, St. Louis, MO

Ct-LPS FACS Mouse/M DAKO, Carpinteria, CA

DDT WB/ELISA Rabbit/P this work

GAPDH WB Goat/P Santa Cruz

Biotechnology, Santa

Cruz, CA

MIF (3H2F) ELISA Mouse/M Bucala lab

MIF (XIV14.3) ELISA Mouse/M Bucala lab

MIF (N20) WB/ELISA Goat/P Santa Cruz

Biotechnology, Santa

Cruz, CA

MIF (IIID9) IFA Mouse/M Bucala lab

MIF

(MAB289)

ELISA Mouse/M R&D Systems GmbH,

Wiesbaden, Germany

p115 WB/IFA Rabbit/P Calbiochem, San Diego,

CA *WB: Western blot, IP: immunoprecipitation, IFA: Immunofluorescent assay, FACS: fluorescent –

activated cell sorting, ELISA: Enzyme-Linked Immunosorbent Assay

**P: IgG polyclonal, M: monoclonal (IgG1)

2.3.2 Secondary Antibodies

Description Application* Species Manufacturer

Anti-goat HRP WB/ELISA Donkey Santa Cruz Biotechnology,

Santa Cruz, CA

Anti-mouse HRP WB Horse Cell signaling, Danvers, MA

Anti-mouse

Biotin

ELISA Goat R&D Systems GmbH,

Wiesbaden, Germany

Anti-mouse

Alexa568

IFA Rabbit Invitrogen, Carlsbad, CA

Anti-rabbit HRP WB Goat Cell signaling, Danvers, MA

Anti-rabbit

Alexa488

IFA Goat Invitrogen, Carlsbad, CA

*WB: Western blot, IFA: Immunofluorescent assay, ELISA: Enzyme-Linked Immunosorbent Assay


2.4 RNAi-targeting Sequences

Description Sequence

RNAi GM130 5'-ATGAGAACATGGAGATCACC-3'

RNAi p115-2 5'-GCAGCTGGATTCATCTAATAG-3'

RNAi p115-1 5'-GCAGTTGGTCCAAGGCTTAT-3'

RNAi lacZ provided by Invitrogen, Carlsbad, CA

RNAi PIP-K Validated siRNA, Qiagen, Hilden, Germany

2.5 Quantitative PCR Primers

Description Sequence

MIF-forward 5'-CGGACAGGGTCTACATCAA-3'

MIF-reverse 5'-CTTAGGCGAAGGTGGAGTT-3'

β-Actin-forward 5'-GGATGCAGAAGGAGATCACTG-3'

β-Actin-reverse 5'-CGATCCACACGGAGTACTTG-3'

For quantification of p115 mRNA levels, p115 and β-Actin primers were

designed and synthesized from SuperArray.

2.6 Media, Buffer and Solutions

2.6.1 Media

All media contained 1 U/ml penicillin/streptomycin

Complete growth medium for HEK293,

HeLa and RAW264.7

Concentration,

percent

DMEM 90

FBS 10

Complete growth medium for THP-1 Concentration,

percent

RPMI 1640 90

FBS 10

Complete growth medium for primary human

macrophages

Concentration,

percent

RPMI 80

human AB serum 20


Complete growth medium for bone marrow

derived macrophages

Concentration,

percent

DMEM 50

FBS 20

L929 supernatant 30

2.6.2 Buffers and Solutions

All buffers were prepared with distilled water, if not stated otherwise.

2.6.2.1 General Buffers

TBS, pH 7.4 Concentration

Tris-HCl, pH 7.4 20 mM

NaCl 150 mM

TBS-T, pH 7.4 Concentration

TBS (buffer)

Tween-20 0.05%

RIPA Concentration


NaCl 150 mM

EDTA 2 mM

Nonident P-40 1%

Sodium deoxycholate 0.5%

SDS 0.1%

Protease inhibitors complex 1x

2.6.2.2 Flow Cytometry

Staining buffer Concentration

PBS (buffer)

FBS 1%

Sodium azide 0.1%

Fixing buffer Concentration

PBS (buffer)

Formaldehyde 3.7%


2.6.2.3 Human MIF ELISA

Washing buffer Concentration

PBS (buffer)

Tween-20 0.05%

Blocking buffer Concentration

PBS (buffer)

Sucrose 1%

BSA 1%

Reaction buffer Concentration

PBS (buffer)

EDTA 1mM

BSA 0.1%

Tween-20 0.05%

Primary antibody Concentration

PBS (buffer)

Anti-MIF antibody clone 3H2F 10 µg/ml

HRP-coupled secondary

antibody

Concentration

Reaction buffer (buffer)

Peroxidase-conjugated anti-MIF

antibody

1x

2.6.2.4 Murine MIF ELISA

Washing and reaction buffer are the same as for human MIF ELISA. For

blocking, Protein Free T20 Blocking buffer is used.


PBS (buffer)

anti-MIF antibody clone XIV.14.3 15 µg/ml

Secondary antibody Concentration

reaction buffer (buffer)

Anti-MIF antibody N20 0.6 µg/ml


Tertiary antibody Concentration


Anti-goat antibody HRP conjugated 2 µg/ml

2.6.2.5 Murine DDT ELISA

Washing and blocking buffer are the same as for human MIF ELISA.


PBS (buffer)

Anti-DDT antibody 15 µg/ml

Reaction buffer Concentration

TBS (buffer)

BSA 0.1%

Tween-20 0.05%

Secondary antibody Concentration


anti-DDT antibody (biotinylated) 0.6 µg/ml

Tertiary antibody Concentration


Streptavidin-HRP conjugate 2 µg/ml

2.6.2.6 Ni2+-Column Purification

Binding buffer Concentration

Tris-HCl, pH 8 50 mM

NaCl 200 mM

Glycerol 10%

Imidazole 20 mM

β-mercaptoethanol 10 mM


Elution buffer Concentration

Tris-HCl, pH 8 50 mM

NaCl 200 mM

Glycerol 10%

Imidazole 500 mM

β-mercaptoethanol 10 mM

2.6.2.7 MIF Purification



NaCl 150 mM



NaCl 2000 mM

Denaturation buffer Concentration

TBS (buffer)

Urea 8 M

Renaturation buffer Concentration

TBS (buffer)

DTT 5 mM

2.6.2.8 DDT Purification



NaCl 50 mM



NaCl 1000 mM


2.6.2.9 SDS-PAGE and Western Blot

Running buffer Concentration

MES running buffer 20x (NuPAGE) 1x For separation of low molecular weight proteins

Running buffer Concentration

MOPS running buffer 20x

(NuPAGE)

1x

For separation of high molecular weight proteins

Transfer buffer Concentration

Transfer buffer 20x (NuPAGE) 1x

Methanol 10%

Blocking buffer Concentration

TBS-T (buffer)

Dry milk powder 5%

Washing buffer Concentration

TBS-T (buffer)

2.6.2.10 Coomassie Staining

Staining solution Concentration

Coomassie Brilliant Blue R-250 0.25%

Methanol 40%

Glacial acidic acid 10%

Destaining solution Concentration

Methanol 40%

Glacial acidic acid 10%

2.6.2.11 Immunofluorescent Staining

Fixing solution Concentration

PBS (buffer)

Formaldehyde 3.7%

Triton-X-100 0.1%


Blocking solution Concentration

PBS (buffer)

Goat serum 5%

2.7 Molecular Biology Techniques

2.7.1 Measurement of DNA/RNA Concentration

The concentration and purity of DNA/RNA was assessed by photometric

analysis. The absorption of the nucleotides was measured at OD260 and

OD280. One OD260 equals 50 µg/ml DNA and 40 µg/ml RNA. The ratio

between OD260 vs. OD280 assesses the purity of the sample with respect to

protein contamination. A quotient of 1.8 to 2.0 indicates an

uncontaminated sample.

2.7.2 Polymerase Chain Reaction (PCR)

DNA fragments were amplified using native Pfu DNA polymerase

(Stratagene). In contrast to the Taq polymerase, Pfu polymerase has a

proofreading activity, resulting in fewer mutations in the process of

amplification.

The reaction mixture was prepared according to the manufacturer’s

recommendations. The profile for amplification included:

Step Number of cycles Temperature Duration

1 1 95 ºC 45 sec

2 25 – 30 95 ºC

Tm – 5 ºC

72 ºC

45 sec

45 sec

1 min per kb of PCR target

3 1 72 ºC 10 min


2.7.3 Enzymatic DNA Digestion

For the hydrolytic restriction of DNA, vector/insert DNA was incubated

with the desired restrictionendonucleases according to the manufacturer’s

instructions.

2.7.4 Agarose Gel Electrophoresis

In order to identify and/or purify DNA after PCR or DNA restriction, DNA

was separated according to its mass in 1% agarose gels. The running

buffer contained ethidium bromide for visualization of DNA under UV

light.

2.7.5 Isolation and Purification of DNA from Agarose Gels

DNA from PCR reactions or restriction reactions were run on an agarose

gel and the desired bands cut from the gel with a razor blade. DNA was

eluted using GENECLEAN® III Kit according to their instructions.

2.7.6 DNA Ligation

For the ligation reactions, T4 DNA ligase was used. 50 – 100 ng vector

DNA were incubated with the insert at a molar ratio of 1:3 in the

recommended buffers with 5 U T4 Ligase at 4 ºC over night.

2.7.7 Heat shock Transformation of E. coli

After thawing an aliquot of competent cells on ice, bacteria were incubated

with plasmid DNA for 30 min on ice prior to transformation. Heatshock

transformation occurred via incubation for 30 sec at 42 ºC. Subsequently,

bacteria were kept on ice for two minutes and then incubated at 37 ºC for 1

h in SOC medium. Bacteria were then plated on LB-agar plates containing

the appropriate antibiotics.


2.7.8 Plasmid Isolation

Plasmids were isolated with the Plasmid Mini Kit from Qiagen according

to the manufacturer’s recommendations.

2.7.9 Isolation of RNA and cDNA Synthesis

For the isolation of RNA the RNeasy kit was used according to the

manufacturer’s instruction. RNA was converted to cDNA by reverse

transcriptase. Oligo-dT primers were used for priming polyadenylated

RNA. For an efficient cDNA synthesis, RNA together with the primers

were denatured at 65 ºC for 5 min, followed by a 5 min cooling period at

room temperature to allow the primers to anneal to the RNA. The

synthesis reaction was carried out at 42 ºC for 1 h. After heat inactivation

of the reverse transcriptase, cDNA was stored at –20 ºC.

2.7.10 Quantitative PCR

Quantitative PCR (qPCR) is a modification of the polymerase chain

reaction, and is used to analyze gene expression. After the synthesis of

cDNA, primers specifically amplify the gene of interest in the presence of

SYBR green which only emits fluorescence after intercalating into double

stranded DNA.

The qPCR was performed on the iCycler from BioRad. The amplification

profile included denaturation at 95 °C for 5 min, followed by one step

annealing and elongation for 1 min at 60 °C for a total of 40 cycles. For

verification of specific product amplification a melting curve was

performed.

2.7.11 Design and Production of short hairpin RNA

The Block-it RNAi designer from Invitrogen was used to design short

hairpin RNA molecules (shRNA) specific to human p115. Two constructs

were designed: p115-1 from position 2625 to 2645 (5'-


GCAGCTGGATTCATCTAATAG-3') and p115-2 from position 1601 to 1620

(5'-GCAGTTGGTCCAAGGCTTAT-3') in the human p115 mRNA sequence

(NM_003715). Both constructs share no significant sequence homology

with other genes as analyzed by BLAST search. As negative control,

shRNA constructs against lacZ (provided from Invitrogen) were generated.

Oligonucleotides were cloned into the RNA expression vector pENTR/U6

and confirmed by sequencing.

2.7.12 Preparation of Adenoviruses

shRNA constructs p115-2 and lacZ in the pENTR/U6 vector were

recombined with the adenoviral vector pAd/BLOCK-iT-DEST using

Gateway LR Clonase according to the manufacturer’s protocol. The

resulting p115/pAd plasmid was transfected into HEK293 and amplified

following the standard procedure described in the manual. The viral titer

was determined using AdenoX Rapid Titer Kit (Clontech, Palo Alto, CA).

2.8 Cell Culture Techniques

2.8.1 Cultivation and Treatment

All cell culture work was performed in BL2-approved laminar flow hoods.

For cultivation, cells were kept in a humidified atmosphere at 37 ºC with

5% CO2. All media and solution were sterile and endotoxin free. Adherent

cells were passaged at a confluence of 70 – 80%. Cells were washed twice

with PBS and then detached from culture flask using Trypsin/EDTA. Cells

were re-plated in fresh medium at a confluence of 10%. Suspension cell

were passaged every third day.

For differentiation of THP-1 monocytes to macrophages, cells were treated

with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 h.

Subsequently, cells were cultured for additional 48 h (137).


For MIF secretion assays, cells were incubated in RPMI + l% FBS medium

for 3 h prior to stimulation to quiescent cells. MIF secretion was

stimulated with lipopolysaccharide (LPS). THP-1 monocytes and primary

human macrophages were stimulated with 10 µg/ml LPS, PMA-

differentiated macrophages were stimulated with 0.1 µg/ml LPS.

2.8.2 Isolation of Macrophages from Blood

Human PBMCs were isolated by Ficoll-Hypaque gradient centrifugation.

The cells were resuspended in RPMI 1640 medium supplemented with

20% human AB serum and plated at 2.5x106 cells per well in 24-well cell

culture plates. After 2 h of culture, the adherent cells were washed

extensively with PBS and cultured for 1 week to allow differentiation into

monocyte-derived macrophages (MDMs).

2.8.3 Transient Transfection

Depending on the cell type, different methods were employed for

transfection. HeLa and HEK293 cells were transfected with Lipofectamine

2000, a cationic, lipid-based transfection reagent. THP-1 monocytes were

transfected by Amaxa electroporation utilizing program V-001 and the

nucleofector solution V.

Adenovirus-mediated transfection was performed to efficiently transfer

plasmids into differentiated THP-1 macrophages and primary human

macrophages. PMA-differentiated cells were incubated with a multiplicity

of infection (MOI) of 50 over night; primary macrophages were incubated

with 50 MOI of adenovirus for 2 h.

Depending on the cell type, transfection method and construct, cells were

cultured between 48 and 96 h after transfection.


2.8.4 Preparation of Cell Lysates

The preparation of cell lysates for Western blot analysis included the

following steps: washing of cells with ice cold PBS, lysis of cells by

incubation in RIPA buffer containing protease and phosphatase inhibitors

for 20 min at 4 ºC, removal of genomic DNA and cell debris by

centrifugation for 10 min at 14,000 rpm at 4 ºC.

2.8.5 Immunofluorescent staining for microscopy

Cells were plated at a confluency of 80% on culture slides. For fixation and

permeablization, cells were incubated with fixation solution for 20 min at

37 ºC. Cells were thoroughly washed with PBS, and then incubated with

blocking buffer for 30 min at RT. Cells were incubated with primary

antibody over night at 4 ºC, washed three times with PBS and then

incubated with secondary antibody for 1 h at RT. To reduce fading during

microscopy, the cells were mounted with Antifade. Primary and secondary

antibodies were diluted in blocking solution according to the

recommendations of the manufacturer.

2.8.6 Fluorescence-based Quantification of FGF Export

HeLa cells were genetically modified to express FGF-2-GFP and FGF-4-

GFP fusion proteins in a doxycycline-dependent manner (138). To quantify

the secretion of FGF fusion proteins, cells were transfected with

knockdown constructs targeting either p115, PIP-K (115), or a control

sequence. After 96 h, the expression of the fusion protein was induced by

doxycycline for 24 h. Cells were prepared for flow cytometry to detect cell

surface associated FGF-2-GFP and FGF-4-GFP, respectively. Primary

antibodies were decorated with APC-conjugated secondary antibodies and

GFP fluorescence (intracellular and cell surface) and APC fluorescence

(cell surface) were determined employing FACSCalibur flow cytometer


(BD Bioscience). Untransduced HeLa cells were used to determine

autofluorescence signals.

2.8.7 Chlamydia trachomatis (L1) Infection

THP-1 cells were infected with C. trachomatis at an MOI of 10 for 1 h at

RT by slow rotation in RPMI medium. Cells were washed with medium

and resuspended in RMPI containing 10% FBS without antibiotics and

cultivated at 1x106 cells per ml in 12 well plates. The infection rate was

analyzed by flow cytometry. For FACS analysis, cells were washed in

staining buffer and then fixed in 3.7% formaldehyde in PBS for 20 min at

RT. Subsequently cells were permeabilized with BD Perm/Wash by

incubating for 20 min on ice. Cells were stained with mouse anti-Ct LPS

FITC antibody.

2.9 Proteinchemistry and Immunology Techniques

2.9.1 Determination of Protein Concentration

The measurement of protein concentration was performed according to

Bradford (139). After diluting the Bradford reagent 1:5 in water, samples

were diluted 1:100 in Bradford solution. After incubation for 5 min at RT,

the absorbance was measured at 595 nm. The concentration of the

samples was calculated by interpolation from a BSA standard curve.

2.9.2 SDS-PAGE

Proteins were separated according to their size in the NuPAGE system

using Bis-Tris 4 – 12% polyacrylamide gradient gels. Samples were boiled

for 5 min in LDS sample loading buffer and separated at 180 V in

NuPAGE/XCell SureLock chambers. Depending on the size of the protein

analyzed, either MES running buffer for small molecular weight proteins


(3 – 50 kd) or MOPS running buffer for large molecular weight proteins

(50 – 200 kd) was used.

2.9.3 Coomassie Staining

Coomassie brilliant blue is a triphenylmethane dye binding to the basic

amino acids of proteins. To stain proteins after a SDS/PAGE, the gel is

incubated with the Coomassie staining solution for 30 min at RT. For

visualization of proteins, the gel was washed with destaining solution

until only the dye bound to the proteins remains. The destaining solution

was changed repeatedly.

2.9.4 Western Blot

Proteins separated in a SDS-PAGE were transferred to a methanol-

activated PVDF membrane using the XCell II Blot Module. The unspecific

binding sites on the membrane were blocked by incubating the membrane

for 2 h at RT in blocking buffer. The primary antibody was diluted 1:1000

or according the manufacturer’s recommendations in blocking buffer and

the membrane was incubated with primary antibody solution either for 2

h at RT or over night at 4 ºC. Before the incubation with the HRP-coupled

secondary antibody, the membrane was washed 3 times. The secondary

antibody was diluted in blocking solution at 1:5000 and incubated with the

membrane for 1 h. After washing the membrane, proteins were visualized

by detecting the chemiluminescence emitted after addition of the ECL

substrate. If the membrane was re-probed, the membrane was incubated

in stripping buffer for at least 30 min at RT. After a thorough washing the

membrane, immunoblotting procedure was repeated. For densitometric

analysis, the Scion Image program from NIH was used.


2.9.5 Sandwich ELISA

2.9.5.1 Human MIF ELISA

The human MIF ELISA is a one-step solid phase assay performed as

described previously (13). The samples were mixed with anti-MIF

antibody-HRP construct and added to an ELISA plate pre-coated with

anti-MIF antibody 3H2F. During the incubation, the MIF antigen binds

simultaneously to the capture antibody on one side and the HRP-coupled

anti-MIF antibody on the other side. After incubation and washing to

remove unbound enzyme, the amount of bound enzyme in each well was

detected by adding the substrate TMB. The reaction was stopped by

adding an acidic stop solution, and the resulting color read at 450 nm in a

microtiter plate spectrophotometer. The concentration of MIF in the

samples was calculated by interpolation from the MIF standard curve.

2.9.5.2 Murine MIF ELISA

MIF concentration was measured as described previously (21). Briefly, 96-

well microtiter plates were coated with the anti-MIF monoclonal antibody,

washed, and blocked. 50 µl aliquots of each sample were added to the plate

and incubated overnight at 4 °C. The plate was washed and incubated

with polyclonal anti-MIF antibody (N-20). This was followed by the

addition of anti-goat IgG antibody conjugated to HRP, and the antibody

complexes were quantified after the addition of the substrate TMB.

2.9.5.3 Murine DDT ELISA

96-well microtiter plates were coated with 15 µg/ml polyclonal anti-DDT

antibody over night at 4 ºC. Subsequently the plate was washed and

blocked with 1% BSA, 1% Sucrose in PBS for 2 h at RT. 50 µl aliquots of

each sample were added to the plate and incubated for 2 h at RT. After

washing, biotinylated anti-DDT antibody was added. This was followed by

washing and the addition of a streptavidin-HRP conjugate, which was


incubated for 1 h at RT. The amount of bound enzyme in each well was

detected by adding the substrate TMB. The DDT concentrations were

calculated by extrapolation from a sigmoidal quadratic standard curve

using mouse DDT.

2.9.5.4 TNF-α, IL-1β and IL-6 ELISA

To measure the concentration of TNF-α, IL-1β and IL-6 the commercially

available ELISA kits from ebioscience were used according to the

manufacturer’s instructions.

2.9.6 Protein Purification

2.9.6.1 Overexpression of Proteins

The DNA sequence of the protein of interest was cloned into the bacterial

expression vectors (pET from Novagen). After transforming the constructs

into competent E. coli BL21 (DE3), cells were cultured over night. The

following day, LB medium was inoculated with the over night culture to

obtain an OD600 of 0.1. Bacteria were grown until an OD600 of 0.6, and the

expression of the protein was induced by the addition of IPTG to a final

concentration of 1 mM. The expression lasted for 4 h; afterwards the

bacteria were pelleted by centrifugation for 10 min at 3,000 rpm at 4 ºC.

The supernatant was discarded and pellets stored at –20 ºC.

2.9.6.2 Purification of Histidine-tagged Proteins

After overexpression of histidine-tagged p115 or DDT, bacteria were

resuspended in binding buffer and lysed by French Press. The crude

bacterial extract was purified using a HiTrap chelating HP column with a

linear gradient of 20 – 400 mM imidazole in 50 mM Tris, pH 8.0 and 200

mM NaCl. The peak fractions, as determined by SDS-PAGE, were

subjected to gel filtration chromatography. Protein purity was verified by

SDS-PAGE/Coomassie staining and Western blotting.


2.9.6.3 Purification of MIF

MIF was purified as described previously (6). Briefly, after overexpression,

pelleted bacterial were resuspended in 50 mM Tris-HC1 pH 7.5, 150 mM

NaCl and lysed using a French Press. The lysates was sterile filtered and

then subjected to a MonoQ anion exchange chromatography. The MIF

containing fractions were applied to a C8-SepPak reverse phase column.

Weakly bound material was eluted with 20% acetonitrile. MIF then was

eluted with six column volumes of 60% acetonitrile, frozen at –80 ºC, and

lyophilized. For renaturation, MIF was dissolved in 20 mM sodium

phosphate buffer containing 8 M urea and 5 mM DTT and dialyzed

against 20 mM sodium phosphate buffer containing 5 mM DTT, followed

by 20 mM sodium phosphate buffer alone.

2.9.6.4 Purification of DDT

Native DDT was overexpressed in E. coli BL21 (DE3). The bacteria pellet

was resuspended in 50 mM Tris-HCl, pH 7.4, 50 mM NaC1 and lysed

using a French Press. The lysate was sterile-filtered and subjected to a Q

Sepharose anion exchange chromatography. The column was equilibrated

with Tris-buffered saline. DDT was eluted with a salt gradient (50 mM

Tris-HCl, pH 7.4, 1 M NaC1) and 7.5 ml fractions were collected. The

DDT-containing fractions were combined, concentrated and subjected to a

Superdex 75 size exclusion chromatography. One fraction contained pure

DDT protein. One liter of E. coli culture resulted in 10 mg DDT protein.

The protein was stored at –20 ºC. LPS content of the purified protein was

not analyzed.

2.9.7 In vitro MIF/p115 Binding Studies

96-well protein-absorbent plates were coated with 1 µM p115 per well.

After coating over night, the plate was washed with TBS-T and blocked

with Superblock. After pre-incubating MIF at concentrations ranging from


0 – 4 µM with 150 nM biotinylated MIF for 2 h, the samples were added to

the plate and incubated over night at 4°C. For detection of bound

biotinylated MIF, a streptavidin-alkaline phosphatase conjugate was

added after washing the plate. Using p-nitrophenylphosphate as a

substrate for the alkaline phosphatase, the amount of captured protein

was measured at 405 nm. For a reverse set-up of the assay, 1 µM MIF was

coated and 150 nM biotinylated p115 incubated with unlabeled p115 at

concentrations from 0 – 4 µM. Values are plotted as percent absorbance at

405 nm relative to wells containing biotinylated MIF or p115 alone.

2.10 In vivo Mouse Experiments

2.10.1 LPS Shock

LPS shock experiments were performed with 6 – 8 week old female

BALB/c mice.

4-IPP was diluted in DMSO to a concentration of 1 M. For the

administration of 4-IPP, the stock solution was diluted in corn oil to a final

concentration of 10 mg/ml. 100 µl of 4-IPP or vehicle control was injected

intraperitoneally 2 h prior to LPS injection. 17.5 mg/kg LPS (0111:B4) was

intraperitoneally injected for survival experiments (5). When serum

cytokines levels were monitored, shock was induced with 15 mg/kg LPS.

2.10.1.1 Serum Collection

Blood was collected from mice by cardiac puncture. The mice were

anaesthetized with isoflurane. After the mouse was under a deep surgical

anesthesia, blood samples were taken from the heart by a thoracotomy

through the diaphragm. Blood was withdrawn slowly to prevent the heart

from collapsing.

Blood samples were incubated at 37 ºC for 30 min and then at 4 ºC over

night. The next day, samples were centrifuged for 10 min at 1,600 rpm,

and serum supernatant was transferred to a fresh tube.


2.10.2 Isolation of Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages were isolated from C57BL/6 mice. To

obtain the tibia and femur for isolation of bone marrow, mice were

sacrificed and legs dissected. After the removal of tissue from the bone,

bone marrow was expelled by a jet of medium from both ends of the bone.

4x106 cells were plated per petri dish in 10 ml of conditioned medium.

After 4 days of cultivation, the medium was exchanged and cells were

cultured for at least another 24 h. For experiments the cells were scraped

off and plated at a concentration of 0.5x106/ml.

Results – 49

3 Results

3.1 p115 Mediates the Secretion of MIF

3.1.1 p115 is a Novel Interaction Partner of MIF

In a yeast two-hybrid screen in the laboratory of Prof. Bucala, p115 was

identified as a novel interaction partner of MIF. The interaction was

affirmed by co-immunoprecipitation in a mammalian cell system.

For the yeast two-hybrid screen, MIF cDNA served as “bait” and a human

pituitary cDNA library was the prey (5). After co-transformation of bait

and prey plasmids into Saccharomyces cereviciae cdc25H, 90 colonies were

identified in a primary screen, but secondary and tertiary screening ruled

out 75. The remaining 15 colonies carried proteins binding specifically to

MIF. By DNA sequencing, one of the positive clones contained the carboxy

terminal 780 bases of p115. p115 is a large protein of 962 amino acids, and

consists of a globular head, four conserved coiled-coiled (CC) domains and

a short acidic tail (97) with MIF binding at the CC region and/or the acidic

tail (Fig. 9).

Fig. 9: Schematic structure of p115. Top: Full length p115 with its globular head

(blue), four coiled-coiled domains (black), and an acidic tail (red). Bottom: Carboxy-

terminus of p115 (residues 702-962) that interacts with MIF.

The intracellular interaction between MIF and p115 was further

investigated by co-immunoprecipitation in mammalian cells. In the

prostate carcinoma cell line LNcap, an endogenous interaction between

MIF and p115 was observed by Western blot analysis for p115 after

immunoprecipitation with anti-MIF, but not an isotype control antibody

(Fig. 10).

Results – 50

Fig. 10. Co-Immunoprecipitation of MIF and p115. Endogenous MIF/p115

complexes were co-immunoprecipitated from LNCap cells using a monoclonal anti-MIF

antibody. p115 was detected by Western blot using a polyclonal anti-p115 antibody. IB:

immunoblot.

3.1.2 The MIF/p115 Interaction is Direct and Specific

The discovery of p115 as an intracellular interaction partner of MIF was a

new information that was pursued in this thesis for further validation and

to address the question whether p115 could be involved in the secretion of

MIF.

In order to biochemically confirm and further characterize the interaction

between MIF and p115, the carboxy-terminal region of p115 identified in

the yeast two-hybrid screen (p115702-962) was recombinantly produced and

purified. The purity and specificity of the product was verified by SDS-

PAGE followed by Coomassie staining or Western blot analysis visualizing

the p115702-962 protein with an anti-p115 antibody (Fig. 11).

Fig. 11: Purified recombinant p115702-962. Purity and specificity revealed by SDS-

PAGE followed by Coomassie staining (left panel) and Western blot (right panel).

Results – 51

A specific interaction between MIF and p115 was observed in an in vitro

competition binding assay employing recombinant, immobilized p115702-962

and biotinylated MIF. As shown in Fig. 12, increasing concentrations of

MIF, but not the addition of equimolar amounts of control protein

(lysozyme), inhibited the interaction of biotinylated MIF with p115.

Protein-protein interaction was verified by performing the reverse assay,

i.e. measuring the binding of biotinylated p115702-962 to immobilized MIF.

Fig. 12: MIF binding to p115 demonstrated by a competitive binding assay. Left

panel: MIF binds to immobilized p115702-962 in a concentration-dependent manner. Right

panel: p115702-962 binds to immobilized MIF in a concentration-dependent manner.

Lysozyme served as a negative control. Results are expressed as mean±S.D. of duplicate

measurements and are representative for three independent experiments.

3.1.3 MIF Release from Human Monocytes

Although MIF does not possess a leader sequence, it is released from cells

in both a regulated and a specific manner. Human THP-1 monocytes were

stimulated with 10 µg/ml LPS and an increase in the concentration of MIF

in the supernatant was observed (Fig. 13, A). MIF release was detectable 2

h after stimulation and reached its plateau after 4 h.

The observed increase in the supernatant content of MIF was not the

result of cell death, as the cell viability was verified by lactate

dehyrogenase (LDH) assay. Supernatants from both LPS-stimulated and

solvent control treated cells showed 3 – 5 mU/ml LDH activity. In

contrast, cells treated with 0.1% Triton-X-100, a detergent, showed 100

mU/ml LDH activity.

Results – 52

The increase in MIF protein concentration in the supernatant after LPS

stimulation was not accompanied by an increase in the level of MIF or

p115 mRNA in the monitored period of 6 h (Fig. 13, B). These data

indicate that the initial release of MIF from THP-1 monocytes occurs from

pre-formed pools and does not require the transcription of MIF mRNA.

The results are in accord with previous reports that were based on the

immunostaining and in situ hybridization of murine tissues after LPS

administration in vivo (19, 140).

Fig. 13: Cell stimulation increases MIF secretion without affecting MIF or p115

mRNA levels. A) Time course of MIF secretion from LPS-stimulated, THP-1 monocytes.

THP-1 cells (1×106/ml) were treated with LPS (10 µg/ml) or PBS (control) and

supernatants were collected at the indicated time for measurement of MIF by specific

ELISA. Results are expressed as mean±S.E. of duplicate measurements from two

independent experiments (n=4). P values were calculated by unpaired Student’s t-test for

all time points. *p<0.01 for stimulated vs. unstimulated cells. B) MIF and p115 mRNA

levels in LPS-stimulated THP-1 monocytes. RNA was isolated at indicated time points

and analyzed by quantitative PCR. Results are expressed as mean±S.E. of three

independent experiments.

Results – 53

3.1.4 Macrophage Activation Leads to a Redistribution of

MIF

The cellular distribution of MIF in macrophages in response to LPS

stimulation was studied. In unstimulated macrophages, MIF appears

present throughout the cytoplasm and p115 is localized predominantly in

the perinuclear area in a pattern that is consistent with the Golgi (Fig. 14,

A), which is in agreement with prior reports (95, 98). Upon activation of

primary macrophages with LPS, MIF redistributes from the cytoplasm to

a plasma membrane-proximal area, and a portion of p115 now appears

dispersed in the cytoplasm (Fig. 14, B).

Results – 54

Fig. 14: Immunolocalization of p115 and MIF in macrophages. A) Macrophages

were plated on a four-chamber culture slide (2.5×105 cells/ chamber). The cells were

cultivated overnight, fixed with formaldehyde, and permeabilized with Triton-X-100.

MIF (red) and p115 (green) were visualized by immunostaining with specific antibodies

(1:200). Yellow indicates an overlapping signal after merging. B) Macrophages were

stimulated with LPS (10 µg/ml) or PBS (unstimulated) for 4 h, fixed with formaldehyde,

and permeabilized with Triton-X-100. MIF and p115 were visualized by immunostaining

with specific antibodies. Images are representative for n=40 cells. Scale bar=10 µm.

3.1.5 p115 is Required for MIF Release in Monocytes

3.1.5.1 RNAi-Mediated Depletion of p115

p115 plays an essential role in vesicle transfer from the endoplasmatic

reticulum to the Golgi complex and in Golgi re-assembly after mitosis (92,

141). MIF secretion is not inhibited by brefeldin A, a pharmacological

Results – 55

inhibitor of the Golgi-retrograde transport, MIF does not enter the Golgi,

and it is not glycosylated (105). It was nevertheless of interest to examine

whether p115 was necessary for the release of MIF in response to

activating stimuli. RNA interference (RNAi) constructs were generated for

the gene-specific silencing of p115 and their efficacy tested in human

HeLa and THP-1 cells. As shown in Fig. 15, two RNAi constructs specific

for p115 (p115-1, p115-2) reduced the concentration of p115 protein by 50

– 70% in the two cell types tested.

Fig. 15: RNAi-mediated depletion of p115. HeLa or THP-1 cells were transfected with

RNAi constructs specific for p115 (p115-1, p115-2) or a control plasmid (mock) and

analyzed for cellular p115 content by Western blotting 48 h after transfection.

3.1.5.2 Depletion of p115 Inhibits the Release of MIF

The objective was to address, whether a p115 knockdown reduces the

amount of MIF produced by LPS-stimulated THP-1 monocytes.

Transfection of the p115-1 or p115-2 RNAi sequences resulted in a 30 –

40% decrease in MIF release in response to LPS, while basal levels of MIF

production were unaffected (Fig. 16). Of note, the LPS-stimulated

secretion of the two conventionally-secreted cytokines, TNF- and IL-6,

which both bear hydrophobic signal peptides, was not affected by a

reduction in p115 protein. These data point to a potential specificity of

p115 in mediating MIF secretion, and also indicate that the cellular

depletion of p115 by the RNAi approach used herein does not appreciably

affect the functioning of the ER/Golgi pathway.

Results – 56

Fig. 16: p115 depletion inhibits MIF secretion. THP-1 monocytes (1x106/ml) were

transfected with p115 RNAi or mock RNAi plasmids and stimulated with LPS for 4 h.

Supernatants were collected and analyzed for MIF, TNF and IL-6 by specific ELISA.

Results are expressed as the mean±S.E. of four independent experiments. Statistical

significance was determined for each p115 RNAi construct against mock RNAi by

unpaired Student’s t-test, *p<0.05, **p<0.01.

GM130 is a protein known to interact with the carboxy-terminal acidic tail

of p115 (95). With the purpose of studying the role of GM130 in MIF

secretion, GM130 was depleted in THP-1 cells by siRNA. The depletion of

GM130 protein in THP-1 cells had no effect on LPS-stimulated MIF

release (Fig. 17).

Results – 57

Fig. 17: Depletion of GM130 does not affect MIF secretion. A) THP-1 cells were

transfected with GM130 RNAi or mock control. 96 h after transfection, cells were lysed

and GM130 content analyzed by Western blot and densitometry. Results are expressed as

mean values ±S.D. of three independent experiments. B) GM130-depleted or control

THP-1 cells were stimulated with LPS for 4 h, supernatants were collected and assayed

for MIF content. Results are expressed as mean values ±S.E. of one experiment measured

in duplicates; results are representative for three independent experiments.

3.1.6 FGF-2 and FGF-4 Export in p115-Depleted HeLa cells

To gain a broader understanding of the role of p115 in unconventional

protein secretion, the export of the two cytokines, FGF-2 and FGF-4, was

studied. While both of these proteins are released from cells in response to

tissue injury (142-144), FGF-4 is secreted conventionally whereas FGF-2

is translocated directly across the plasma membrane (114).

In collaboration with the group of Prof. Walter Nickel, using a previously

established doxycycline-inducible expression system (138 113), both total

and cell surface-associated FGF-2 and FGF-4 protein levels were

measured. Cellular depletion of p115 did not inhibit the export of either

FGF-2 or FGF-4, while the secretion of FGF-2 was inhibited by depletion

of phosphatidylinositol kinase (PIP-K), as previously reported (115) (Fig.

18). The finding that the release of both conventionally secreted cytokines

(TNFa, IL-6 and FGF-4) and unconventionally secreted cytokines (FGF-4)

is not affected by a depletion of p115 suggests a specific role for p115 in

the stimulated export of MIF.

Results – 58

Fig. 18: FGF export does not depend on p115. HeLa cells were transfected with

mock, p115-2 or PIP-K RNAi constructs, FGF-GFP fusion protein production was induced

by doxycycline after 96 h. FGF-GFP fusion proteins were measured after an additional 24

h as total or cell surface fluorescence. Data are shown relative to the background level of

GFP. Results are expressed as mean±S.D. of three independent experiments.

3.1.7 Role of p115 in MIF Secretion in Macrophages

3.1.7.1 MIF and p115 are Co-Secreted in Activated Macrophages

In order to determine whether p115 is also of importance for stimulated

MIF release in macrophages, THP-1 monocytes were differentiated into

macrophages. Human THP-1 monocytes acquire many of the

characteristics of mature, primary macrophages upon differentiation with

phorbol 12-myristate 13- acetate (PMA), such as an increase in the

expression of the LPS co-receptor CD14 and the induction of cytokine

expression (137). Differentiated THP-1 macrophages thus respond to low

doses of LPS and show a plateau in the MIF secretion response at an LPS

concentration of 0.1 µg/ml. In addition to a lower threshold dose for

stimulating secretion, differentiated THP-1 also secrete ~5 fold more MIF

protein than undifferentiated THP-1 cells (Fig. 19).

Results – 59

Fig. 19: Dose-dependent secretion of MIF from LPS-stimulated THP-1

macrophages. Cells (1x106/ml) were stimulated with the indicated dose of LPS for 4 h

and the supernatants assayed for MIF content by ELISA. Results are expressed as

mean±S.D. of two experiments, each measured in duplicates (n=4).

Of note, ELISA and Western blot analyses of LPS-stimulated THP-1

macrophages show a coordinated increase in secreted MIF and p115, and a

corresponding decrease in the cellular protein levels over the observed

time period of 24 h (Fig. 20). Moreover, the supernatants of unstimulated

macrophages contain only a modest amount of MIF and p115, and no

apparent reduction in the cellular MIF and p115 content is measurable.

These results taken together suggest that MIF and p115 are co-secreted.

Results – 60

Fig. 20: Co-secretion of MIF and p115 from macrophages. A) Differentiated THP-1

macrophages were stimulated with 0.1 µg/ml LPS or PBS (control) and the supernatants

were collected at the indicated time points. MIF concentration was measured by ELISA;

p115 release was determined by immunoblotting and subsequent densitometric analysis.

B) Cell lysates of THP-1 macrophages stimulated with LPS or control (PBS) were

prepared at the indicated time points. Intracellular MIF concentration was measured by

ELISA, and intracellular p115 level was determined by Western blot and densitometric

analysis. The ELISA results are expressed as mean values ±S.D. of two independent

experiments measured in duplicate. Densitometric results are expressed as mean values

±S.D. of at least two independent experiments. Statistical significance was determined

by Student’s t-test comparing LPS-stimulated vs. control time point, *p<0.05, **p<0.01.

Results – 61

Of note, the LPS stimulation of bone-marrow derived macrophages also

led to the export of both MIF and p115 protein (Fig. 21), affirming the

results obtained from THP-1 macrophages.

Fig. 21: Co-Secretion of MIF and p115 in primary macrophages. Bone-marrow

derived primary macrophages (0.5×106/ml) were stimulated with 1 µg/ml LPS or control

(PBS) and the supernatants collected at the indicated time points. MIF concentration was

measured by ELISA and p115 content was determined by immunoblotting.

Further evidence that MIF and p115 are co-secreted came from the

observation that glyburide, an pharmacological inhibitor known to inhibit

the secretion of MIF (105, 133), also shows an inhibitory effect on the

secretion of p115 (Fig. 22).

Fig. 22: The ABC-transporter inhibitor glyburide inhibits the secretion of MIF

and p115. THP-1 macrophages were pre-treated with glyburide (2.5 µM) or solvent

control (DMSO) for 1 h. Subsequently, MIF secretion was stimulated with LPS, and

supernatant were collected 4 h later. MIF concentration in supernatants was measured

by ELISA, and p115 concentration by Western blot analysis. MIF-ELISA: n=3

measurements, p<0.05.

Results – 62

3.1.7.2 Adenovirus-Mediated Depletion of p115

In order to deliver p115 siRNA to differentiated THP-1 macrophages, an

adenovirus construct was utilized. Adenoviruses do not infect monocytes

because of their low expression levels of integrins 3 and 5, which

mediate adenovirus uptake. Upon differentiation to macrophages, the

expression level of the integrins is up-regulated and adenoviruses

interact with the cells (145). The transfection of adenovirus encoding p115

siRNA, but not a control adenovirus led to a reduced intracellular p115

protein level in THP-1 macrophages (Fig. 23, A).

It has been reported that a depletion of p115 below detectable levels

compromises the structure of the Golgi and causes its fragmentation (102,

103). By immunofluorescent microscopy it was confirmed that the

morphology of the Golgi was not affected by the RNAi approach used

herein (Fig. 23, B).

Adenovirus-mediated delivery of p115 siRNA was also a successful

approach to reduce the p115 protein level in human peripheral blood

monocyte-derived macrophages (Fig. 23, C).

Results – 63

Fig. 23: Depletion of p115 by adenovirus-encoded siRNA in macrophages. A)

Differentiated THP-1 macrophages (1x106/ml) were transfected with an MOI of 50 of

adenovirus encoding siRNA against p115 or mock control siRNA. 96 h after transfection,

the cells were lysed and the protein content analyzed by Western blot and densitometry.

The results are expressed as mean values ±S.D. of three independent experiments. B)

THP-1 macrophages were differentiated on culture slides and p115 depleted by

adenovirus transfection. 96 h after transfection, cells were fixed and permeabilized and

stained for p115 (green) and the Golgi marker GM130 (red). Scale bar=10µm. C)

Adenovirus-mediated depletion of p115 reduces p115 protein level in primary human

macrophages. Human peripheral blood monocyte-derived macrophages (1x106/ml) were

transfected with 100 MOI of adenovirus carrying siRNA against p115 or mock control.

The cells were analyzed 48 h later for p115 content by Western blot. Knockdown of p115

was evaluated by densitometry. The results are expressed as mean values ±S.D. of three

independent experiments.

Results – 64

3.1.7.3 p115 is Required for the Secretion of MIF in Macrophages

After p115 was depleted in macrophages via adenoviruses carrying siRNA

targeting p115, the release of cytokines was stimulated with LPS. MIF

secretion in p115 depleted macrophages was reduced in response to LPS

stimulation in comparison to control cells over the 24 h experimental

period (Fig. 24, A). By contrast, the release of TNF-, IL-6 and IL-1 was

not affected by adenovirus encoded p115 siRNA, confirming that partial

p115 reduction does not interfere with the integrity of the Golgi

apparatus, while having a specific role in the release of MIF. Adenovirus-

mediated delivery of p115 siRNA also reduced p115 protein production in

human peripheral blood monocyte-derived macrophages (Fig. 23, C), and

reduced MIF release after stimulation with LPS by almost 50% (Fig. 24,

C).

Results – 65

Fig. 24: LPS-stimulated MIF secretion in macrophages. A) Time course of LPS-

stimulated MIF secretion in control or p115 siRNA treated THP-1 macrophages.

Supernatants were collected at the indicated time points and MIF content was measured

by ELISA. Supernatants also were assayed for TNF-, IL-6 and IL-1 by ELISA. The

results are expressed as mean values ±S.E. of triplicate measurements of two

independent experiments (n=6). Statistical significance was determined by comparing the

curves obtained in p115 knockdown vs. mock control by two-way ANOVA analysis. C)

LPS-stimulated MIF secretion is reduced in p115 siRNA, but not in mock siRNA treated

PBMCs. MIF secretion was stimulated with 10 µg/ml LPS for 4 h and the supernatants

analyzed for MIF content by ELISA. The results are expressed as mean values ±S.D. of

two independent experiments each measured in duplicates (n=4). Statistical significance

was determined by an unpaired Student’s t-test, *p<0.001.

Results – 66

The inhibition of MIF secretion also appeared to be dose-dependent with

respect to the level of p115 depletion (Fig. 25). When compared to controls,

a ~50% depletion of p115 was associated with a ~20% inhibition of MIF

release, while a ~75% depletion of p115 was associated with a ~40%

inhibition of MIF release.

Fig. 25: Dose-dependent effect of p115 depletion on MIF secretion. Differentiated

THP-1 macrophages were transfected with 50 MOI of adenovirus encoding siRNA against

p115 or mock control siRNA. 48 h after transfection (50% depletion of p115 compared to

mock treated cells) and 96 h after transfection (75% depletion of p115 compared to mock

treated cells), the cells were stimulated for 4 h with 0.1 µg/ml LPS. The supernatants

were analyzed for MIF release by ELISA, and data are shown relative to mock treated

cells. Results are representative for two independent experiments measured in

duplicates. Statistical significance was determined by Student’s t-test, *p<0.05, **p<0.01.

In p115-depletion studies, it was shown that the efficient release of MIF

from monocytes/macrophages in response to LPS depends on the presence

of p115. To determine whether an excess of p115 protein leads to an

enhanced secretion of MIF, p115 was overexpressed in THP-1

macrophages utilizing an adenovirus construct carrying a p115 expression

construct. The overexpression of p115 was confirmed by Western blot, and

the effect of p115 overexpression on LPS stimulated MIF secretion was

evaluated by ELISA (Fig. 26). The overexpression of p115 had no effect on

the LPS-stimulated release of MIF or the conventionally secreted cytokine

TNF-.

Results – 67

Fig. 26: Effect of p115 overexpression on MIF secretion. A) Differentiated THP-1

macrophages (1x106/ml) were transfected with an MOI of 100 of adenovirus encoding a

construct for overexpression of p115 or a mock control. 48 h after transfection, the cells

were lysed and the protein content analyzed by Western blot. B) LPS-stimulated MIF

secretion was not affected by the overexpression of p115. MIF secretion was stimulated

with 0.1 µg/ml LPS for 4 h in macrophages overexpressing p115 or control cells, and the

supernatants were collected and assayed for MIF and TNF- content by specific ELISA.

The results are expressed as mean values ±S.D. of two independent experiments each

measured in duplicates (n=4).

3.1.8 p115 Mediates the Release of MIF from Infected Cells

Chlamydiae are gram-negative, obligate intracellular bacteria that infect

and replicate within monocytes/macrophages (146, 147). Infection of

cultured THP-1 cells with Chlamydia trachomatis induces the release of

pro-inflammatory cytokines (148). As shown in Fig. 27 this inflammatory

stimulus also leads to the release of MIF with a peak 48 h post infection.

After establishing that p115 is a required protein for the LPS-stimulated

release of MIF, it was of interest to study the effect of C. trachomatis

infection on MIF production in differentiated THP-1 macrophages that

had been pre-treated with adenovirus encoding either mock control or

Results – 68

p115 siRNA. The depletion of p115 protein had no effect on the infection or

replication rate of C. trachomatis in macrophages, as measured by flow

cytometry for C. trachomatis lipopolysaccharide (Fig. 27, B). In accord

with the previous results using LPS stimulation, macrophages treated

with p115 siRNA showed a significant decrease in MIF secretion after C.

trachomatis infection (Fig. 27). By contrast, the secretion of TNF-, which

peaks 8 h after infection, was unaffected by p115 depletion. These data

indicate that cellular p115 plays an important role in mediating MIF

secretion by macrophages in response to both a defined microbial ligand

such as LPS, as well as life infection by an intracellular pathogen such as

C. trachomatis.

Results – 69

Fig. 27: p115 knockdown inhibits MIF secretion in Chlamydia trachomatis

infected human macrophages. A) THP-1 cells (1x106/ml) were infected with 10 MOI

of C. trachomatis. Supernatants were collected at the indicated time points and analyzed

for MIF release by ELISA. B) p115-depleted or mock control THP-1 macrophages were

analyzed for C. trachomatis infection and replication by flow cytometry. 24 h post

infection, cells were washed, permeabilized, and stained for C. trachomatis using an anti-

LPS antibody. The results are representative for at least three independent experiments.

Statistical significance of infection of p115 depleted vs. mock treated cells was analyzed

by unpaired Student’s t-test. Mock siRNA: 38±5%, p115 siRNA: 39±8%. C) THP-1

macrophages were transfected with 50 MOI of adenovirus encoding a p115 or control

(mock) siRNA construct 96 h prior to infection with C. trachomatis. Supernatants were

collected at the indicated time points and the levels of MIF and TNF- were analyzed by

ELISA. Results are expressed as mean±S.E. of one experiment measured in triplicates.

Data are representative of three independent experiments. Statistical significance was

analyzed by unpaired Student’s t-test, *p<0.01.

Results – 70

3.1.9 Small Molecule Inhibitor Blocks MIF Secretion

Due to the unique structure of MIF and its tautomerase activity, several

prototypic small molecule inhibitors targeting MIF function have been

designed (61, 64). These inhibitors (ISO-1 and NAPQI) bind to MIF’s

amino-terminal proline thus inhibiting the tautomerase activity. Mitchell

and colleagues recently described the suicide substrate, 4-iodo-6-

phenylpyrimidine (4-IPP), and showed by crystallography that 4-IPP

covalently binds to the amino-terminus of MIF (65).

3.1.9.1 Immunorecognition of 4-IPP-Modified MIF

In a prior report by Senter et al. it was observed that the covalent

modification of the amino-terminus of MIF alters its immunorecognition in

a two-antibody sandwich ELISA. Indeed, MIF that had been incubated

with 4-IPP showed increased immunorecognition by a MIF-specific

monoclonal antibody (clone MAB289) when compared to native MIF. By

contrast, when analyzing the 4-IPP/MIF complex with a different anti-

MIF mAb (clone 3H2F), a diminished recognition compared to native MIF

was observed (Fig. 28).

Fig. 28: 4-IPP modifies the immunorecognition of MIF in a two antibody

sandwich ELISA. Quantification of 4-IPP modified MIF (MIF-4-IPP) or unmodified MIF

(MIF) by two distinct ELISAs. A) MIF-4-IPP shows decreased immunorecognition by the

monoclonal anti-MIF antibody 3H2F. B) MIF-4-IPP displays enhanced detection by the

monoclonal anti-MIF antibody MAB289.

Results – 71

By contrast, the immunodetection of MIF by Western blot, which detects

denatured, linearized proteins, was not affected by 4-IPP modification

(Fig. 29).

Fig. 29: Immunorecognition of 4-IPP modified MIF by Western blot. MIF was

incubated with either vehicle control (DMSO), 0.1x molar excess of 4-IPP, or 10x molar

excess of 4-IPP over night. Samples were run on a SDS-PAGE and transferred to a PVDF

membrane. Unmodified and 4-IPP modified MIF were quantified and detected by

Coomassie staining, Ponceau S staining, and Western blot (polyclonal anti-MIF

antibody).

3.1.9.2 4-IPP Targets the MIF/p115 Interaction

Small molecule MIF inhibitors are able to inhibit MIF functions. It was

reported that ISO-1 effectively protects mice from LPS shock and West

Nile virus infection (20, 149). In these models, ISO-1 exhibited similar

protective properties as neutralizing anti-MIF antibody or genetic mif

deletion (21, 149).

To analyze whether the prototypic inhibitor 4-IPP inhibits the release of

MIF after LPS stimulation in THP-1 macrophages, Western blot analysis

was employed to quantify released MIF. Fig. 30, A shows that 4-IPP

effectively blocked the release of both MIF and p115 after LPS stimulation

in macrophages. Analysis of the cytosolic concentration of MIF and p115

in response to LPS treatment showed that the level of both proteins in the

cytoplasm was decreased by ~50% after stimulation with LPS, while in

macrophages pre-treated with 4-IPP the intracellular concentration of

MIF and p115 remained unchanged.

4-IPP also reduced the secretion of MIF from monocytes infected with

Chlamydia trachomatis (Fig. 30, B).

Results – 72

Fig. 30: 4-IPP inhibits MIF release from macrophages. A) THP-1 macrophages

(1×106/ml) were pre-treated with 1 µM of 4-IPP for 1 h prior to stimulation with 0.1 µg/ml

LPS, and MIF and p115 was measured in supernatants and cell lysates by Western

blotting. Western blots were quantified by densitometry relative to β-actin. Mean±S.E.

of at least two independent experiments are stated below the blots. Statistical

significance for the comparison of LPS + 4-IPP vs. LPS alone was analyzed by an

unpaired Student’s t-test, *p<0.05. 4-IPP did not compromise cell viability, supernatants

showed LDH activity of >5 mU/ml LDH. B) THP-1 monocytes were infected with an MOI

of 10 of C. trachomatis and treated after infection with 1 µM of 4-IPP or control over the

time of infection. Supernatants were collected at the indicated time points and analyzed

for MIF content by Western blot. Results are representative for three experiments.

In contrast, 4-IPP did not affect the LPS-stimulated release of TNF-α or

IL-1β (Fig. 31, A); this affirms that 4-IPP has no overall effect on the

secretion mechanism of cells. Furthermore, the influence of the reversible

MIF inhibitor ISO-1 on MIF release from stimulated THP-1 macrophages

was investigated, but no effect was observed (Fig. 31, B).

Results – 73

Fig. 31: 4-IPP does not target release of other cytokines, ISO-1 does not

influence MIF release. A) Supernatants of LPS-stimulated THP-1 macrophages that

had been treated with 4-IPP or control assayed for the release of TNF-α and IL-1β. The

results are expressed as mean±S.D. of one experiment measured in duplicates. Data are

representative for two independent experiments. B) THP-1 macrophages pre-treated

with ISO-1 were stimulated with 0.1 µg/ml LPS. MIF release at the indicated time points

was analyzed by ELISA. The results are expressed as mean±S.D. of one experiment

measured in duplicates. Data are representative for two independent experiments.

The observation that 4-IPP alters the immunorecognition of MIF by anti-

MIF monoclonal antibodies (Fig. 28) suggests that 4-IPP may impart a

significant conformational affect on MIF that may influence its binding to

p115. To test this hypothesis, the impact of 4-IPP on the competitive

binding of MIF to p115 was examined in vitro. As shown in Fig. 32, 4-IPP,

but not the small molecule MIF inhibitors ISO-1 or NAPQI (61, 64),

increase MIF binding to p115. The enhanced binding of MIF to p115 in the

presence of 4-IPP thus may play a role in the inhibitory action of 4-IPP on

MIF release from cells, perhaps by interfering with additional protein

interactions necessary for secretion.

Results – 74

Fig. 32: 4-IPP enhances the binding of MIF to p115. Competitive binding to

immobilized p115 of biotinylated MIF vs. increasing concentrations of MIF pre-incubated

with the small molecule MIF inhibitors 4-IPP, NAPQI, ISO-1, or solvent control. Results

are expressed as mean±S.E. of one experiment measured in duplicate. The data are

representative of three independent experiments.

3.1.9.3 Effect of 4-IPP in LPS-Shock Model

The small molecular MIF inhibitor ISO-1 has a protective effect in the

LPS-shock model (20). Therefore, it was examined, whether the prototypic

inhibitor, 4-IPP, also protects mice from LPS shock. 4-IPP had no

significant effect on the survival following LPS challenge (Fig. 33).

Fig. 33: 4-IPP does not protect mice in a model of endotoxemia. Female BALB/c

mice (6-8 weeks) were intraperitoneally injected with 1 mg 4-IPP or vehicle control

(DMSO and corn oil). 2h later, LPS was injected (17.5 mg/kg). Mice were observed for 6

days. Data points are from one experiment (N=10 mice per group, p=NS), representative

for three independent experiments.

Results – 75

In addition to studying the effect of 4-IPP on the survival of mice in the

LPS-shock model, it was of interest to analyze whether the administration

of 4-IPP modulates the release of MIF and other pro-inflammatory

cytokines. In contrast to the in vitro experiments, 4-IPP did not

significantly inhibit the release of MIF into the serum, although a modest

reduction of MIF levels was observed. Cytokines downstream of MIF

(TNF-α, IL-6) were also not targeted in their release by 4-IPP in the

examined model (Fig. 34).

Fig. 34: 4-IPP does not affect the release of MIF after LPS injection. Female

BALB/c mice (6-8 weeks) were intraperitoneally injected with 1 mg 4-IPP or vehicle

control (DMSO and corn oil). 2h later, a sublethal dose of LPS was injected (15 mg/kg). At

the indicated time points, serum was collected and assayed for cytokine content by

ELISA. (N=5 mice per group per time point), the experiment was performed three times

independently.

Results – 76

3.2 D-Dopachrome Tautomerase (DDT)

Like MIF, D-dopachrome tautomerase (DDT), catalyzes enzymatic

reactions of the non-physiological substrate D-dopachrome in its amino-

terminal tautomerase active site.

Since the “rediscovery” of MIF in the early 90s, the knowledge about MIF

grew steadily, but little is known about DDT. Crystallographic analysis of

DDT revealed that although MIF and DDT share only limited sequence

homology, the three-dimensional structures of the two proteins show

remarkable similarity (69). The gene structure of MIF and DDT also show

similarities: The two proteins are closely linked on chromosome 22 (in

humans) or chromosome 10 (in mice), respectively; moreover, the two

proteins have an identical exon structure (150). Nevertheless, studies

addressing the biological functions of the structural homolog of MIF are

limited (72), and the molecular action of DDT remains unknown.

3.2.1 Purification of D-Dopachrome Tautomerase

3.2.1.1 Purification of Histidine-tagged DDT

In order to expedite the process of obtaining pure DDT protein, mouse

DDT cDNA was cloned into the pET22b vector. The resulting fusion

protein possesses a short carboxy-terminal histidine tag (6x His), and was

overexpressed in E. coli BL21 (DE3). Utilizing the carboxy-terminal

histidine tag, DDT was purified over a Ni2+ column. The purity of the

protein was determined by SDS-PAGE and Coomassie staining (Fig. 35).

Results – 77

Fig. 35: Purification of Histidine-tagged mouse DDT. Purified recombinant mouse

DDT revealed by SDS-PAGE followed by Coomassie staining.

The purified protein was sent to Pocono Rabbit Farm & Laboratory

(PRF&L) for the production of a polyclonal anti-DDT antibody. A rabbit

was periodically injected with 100 µg of DDT protein in Complete Freund’s

adjuvant in the popliteal lymph nodes, serum was collected after day 28,

42 and 70.

3.2.1.2 Purification of Native DDT Protein

From the MIF protein it is known that the amino-terminus is of

importance not only for the tautomerase activity, but also for various

biological actions (ERK 1/2 activation, glucocorticoids overriding ability,

CD74 receptor binding). Similarly, the carboxy-terminus was shown to be

crucial for the trimer formation (51), thus it was of importance to purify

recombinant, native DDT protein, to successfully perform characterization

studies.

Two stop codons were cloned after the DDT cDNA in pET22b to prevent

the expression of the carboxy-terminal histidine-tag and obtain native

DDT protein; the insertion was confirmed by DNA sequencing. After the

overexpression in E. coli BL21 (DE3), DDT was sequentially purified

utilizing anion exchange chromatography, followed by gel filtration. Fig.

36 shows the purification steps performed for obtaining pure protein.

Results – 78

Fig. 36: Sequential purification of mouse D-Dopachrome tautomerase. A) DDT

was overexpressed in E. coli BL21 (DE3) and lysed by French press. The lysate was

loaded on a Q-Sepharose anion exchange chromatography column and proteins were

eluted over a salt gradient. The collected fractions were analyzed by SDS-PAGE and

Coomassie staining. B) Fractions 2 – 5 of the anionexchange chromatography were

pooled and concentrated. The combined fractions were subjected to a size exclusion

chromatography. The DDT-containing fractions were collected and protein content and

purity was determined by SDS-PAGE and Coomassie staining. C) SDS-PAGE and

Coomassie staining of fraction 8 of the gel filtration.

The identity of the purified protein was confirmed by mass spectroscopy

(Fig. 37). Mass spectroscopy showed two peaks: one at 12.947 kd, and one

at 13.079 kd. The peaks correspond to the calculated molecular weight of

DDT: 12.946 kd, when the amino-terminal methionine is post-

translationally cleaved off, 13.077 kd, when DDT still possesses the

amino-terminal methionine.

Results – 79

Fig. 37: Mass spectroscopy of D-Dopachrome tautomerase. Electrospray ionization

mass spectrometry of murine DDT showing a molecular mass (m/z) that lies within

0.02% accuracy of the predicted m/z (13.077 kd and 12.946 kd).

3.2.2 Establishment of a DDT-ELISA

MIF and DDT share 28% sequence identity and the overall topology and

trimeric formations of DDT are similar to those of MIF. Therefore, the

newly produced polyclonal anti-DDT antibody was analyzed for cross

reactivity with both human and murine MIF protein in Western Blot

analysis. Fig. 38 demonstrates that the antibody detects DDT protein,

while exhibiting no cross reactivity for murine or human MIF.

Results – 80

Fig. 38: Polyclonal anti-DDT antibody shows no cross reactivity for MIF in

Western blot. 100, 10 and 1 ng protein (DDT, mMIF or hMIF) were separated on a

SDS-PAGE gel and transferred on PVDF membrane. The membrane was probed with the

IgG fraction of the serum obtained from PRF&L.

In order to analyze and quantify, if DDT is released from cells, an indirect

ELISA for DDT was established. The produced and purified anti-DDT

antibody served both as a capture and detection antibody; the antibody

was biotinylated when used as a detection antibody. The developed ELISA

detects DDT protein in a range of 100 – 5000 pg/ml, and shows no

measureable cross reactivity with MIF (Fig. 39).

Fig. 39: Specific detection of DDT protein in a newly established ELISA. A)

ELISA plate was coated with polyclonal anti-DDT antibody (15 µg/ml). After incubation

with blocking solution, either DDT or MIF protein was added in a range of 100 – 5000

pg/ml. For the detection of proteins, first biotinylated anti-DDT, then a streptavidin-HRP

conjugate was added to the plate. Protein concentration was detected by colorimetric

measurement of TMB conversion. B) DDT ELISA does not significantly cross react with

mouse MIF. A microtiter plate was coated with anti-DDT antibody and either DDT alone

(100 – 5000 pg/ml) or DDT (100 – 5000 pg/ml) plus 50 ng/ml MIF was added. Protein was

detected by colorimetric measurement at 450 nm.

Results – 81

3.2.3 DDT is released from macrophages

To date, little is known about the biological function of DDT. Both MIF

and DDT are leaderless proteins, and it was of interest to analyze whether

DDT like MIF is released from cells in response to a pro-inflammatory

stimulus. Fig. 40 illustrates that not only MIF, but also DDT is released

from bone marrow-derived macrophages in response to LPS.

Fig. 40: Release of DDT and MIF in response to LPS. Bone marrow-derived

macrophages were stimulated with LPS (1 µg/ml) ore PBS (control). At the indicated time

points, supernatants were collected and assayed for DDT and MIF by specific ELISA. The

results are representative for three independent experiments.

The pattern of release of DDT and MIF after stimulation with LPS is very

similar. Both proteins peak at 16 h, exhibiting a bell-shaped curve often

observed for released cytokines.

Results – 82

3.2.4 Effect of mif-gene deletion on DDT production

Both MIF and DDT contribute to the secretion of CXCL8 and VEGF (72)

and both proteins are released in response to LPS stimulation of

macrophages. Given these overlapping functions of the two proteins it was

considered that a loss of the mif-gene might be compensated by higher

expression levels of DDT. Therefore, the intracellular DDT concentration

in wildtype and mif-/- macrophages was measured (Fig. 41, A), but no

difference was observed. These results were also reflected when comparing

DDT concentration in serum of wildtype vs. mif-/- mice (Fig. 41, B). Last, it

was analyzed whether the deletion of the mif-gene had an effect on the

stimulated release of DDT, but again no difference between wildtype and

mif-/- macrophages was measurable (Fig. 41, C).

Fig. 41: Comparison of DDT production. A) Bone-marrow derived macrophages of

wildtype (mif+/+) and MIF knockout (mif-/-) were lysed and DDT concentration was

measured by ELISA, total protein concentration was analyzed using Bradford, (N=8 mice

per group). B) Serum samples of mif+/+ and mif-/- were collected, and DDT concentration

was analyzed by Bradford (N=6 mice per group). C) Bone-marrow derived macrophages

of wildtype (mif+/+) and MIF knockout (mif-/-) were stimulated with LPS (1 µg/ml) or

control (PBS). At the indicated time points, supernatants were collected and assayed for

DDT and MIF by specific ELISA. The results are representative for three independent

experiments.

Results – 83

The functional role of DDT is still unclear. In general DDT might either

have similar functions as MIF, thus supporting MIF in its pro-

inflammatory, anti-apoptotic role or it might be a counter-regulator of

MIF, attenuating the effects of MIF.

Discussion – 84

4 Discussion

4.1 Role of p115 in MIF Secretion

Macrophage migration inhibitory factor plays an important upstream role

in the regulation of diverse cellular responses (8, 151, 152) and its role in

human pathology has been emphasized by the finding that high

expression MIF alleles are associated with the incidence or the severity of

inflammatory and oncologic diseases (12-14, 153).

MIF is constitutively expressed in a large number of tissues,

predominantly in those that are in contact with the host’s environment.

(19, 154, 155). The release of MIF, especially from immune cells, occurs in

many inflammatory diseases, but the precise mechanism underlying the

secretion of MIF, which lacks an amino-terminal signal sequence, has

remained enigmatic (4, 5). MIF has been reported to be associated with

vesicles (134-136), and its release from stimulated monocytes is reduced

by glyburide and probenicide, which are pharmacological inhibitors mainly

targeting the ABCA1 transporter (105, 133).

In many inflammatory diseases, the amount of released MIF positively

correlates with disease severity, e.g. in sepsis, high MIF levels in the

serum are in direct relation with a fatal outcome (156-159). Moreover, the

administration MIF-neutralizing agents such as anti-MIF antibodies or

the MIF inhibitor ISO-1, were shown to be protective in diverse disease

models (20, 149). Together, these data clearly demonstrate the importance

of understanding the molecular mechanisms underlying MIF secretion.

To this end, a yeast two-hybrid screen was performed to identify

intracellular proteins that interact with MIF. In this screen, p115 was

found as a new binding partner of MIF. The specificity of the screen was

confirmed by the finding that 93% of the specifically interacting clones

were MIF itself, which associates into a non-covalently associated

homotrimer (50, 51). However, a cDNA clone encoding the carboxyl

terminal 260 amino acids of the Golgi-associated protein p115 also was

Discussion – 85

identified. Co-immunoprecipitation studies confirmed the interaction

between MIF and p115.

4.1.1 MIF is Released from a Pre-Formed Pool

Lipopolysaccharide (LPS) was one of the first stimuli reported to cause the

secretion of MIF from cells (5, 8). In order to determine the pathway by

which MIF is released from immune cells, it was indispensable to

establish a reliable system to monitor the stimulated release of MIF.

Monocytes stimulated with LPS effectively release MIF, while the mRNA

level of both MIF and p115 remain unchanged. These findings are in

accord with previous observations in murine tissue (19, 140) and clearly

show that MIF is secreted from pre-formed stores and is not synthesized

de novo. In contrast, other cytokines, such as TNF-α and IL-1β, show a

dramatic increase in their mRNA expression level 1 – 3 h post LPS

stimulation (160, 161). A possible explanation for the difference in mRNA

expression profiles may be that TNF-α and IL-1β operate exclusively as

cytokines, whereas MIF has cytokine functions, but in addition also has an

important intracellular role. For instance, it was reported that MIF

contributes to cell proliferation via direct interaction with the cell cycle

regulators p53 and JAB1 (43, 45, 47).

4.1.2 MIF and p115 Associate at the Perinuclear Site

Previous studies have demonstrated that p115 is localized at the Golgi

(95). Immunofluorescent microscopy analysis in macrophages showed that

MIF is localized throughout the cytoplasm, and that MIF and p115

associate most notably in the area of the Golgi. The comparison of MIF

and p115 distribution in unstimulated vs. LPS-stimulated macrophages

furthermore showed that upon stimulation MIF is found predominantly in

a plasma membrane proximal area, whereas unstimulated cells stain for

MIF predominantly in the perinuclear ring. p115 is localized at the Golgi,

Discussion – 86

and upon stimulation a portion of p115 is dispersed in the cytoplasm. This

suggests that the interaction between MIF and p115 occurs early in the

secretory pathway and that MIF and p115 are co-secreted in vesicles. p115

may be essential in the transport of MIF from the perinuclear ring to the

plasma membrane and then out of the cell. This hypothesis is supported

by data showing a co-secretion of MIF and p115 in LPS stimulated cells.

It was reported that MIF is detected in vesicles in pituitary cells, cells of

the testis, and β-cells of the pancreas (134-136). It is of interest to further

characterize the location of MIF and p115 in immune cells. Electron

microscopy images labelled for MIF and p115 might help to understand

whether MIF and p115 localize together at vesicles or if p115 just

momentarily associated with MIF, presumably to help transfer MIF into

vesicles.

4.1.3 MIF and p115 Interact Directly

The interaction between MIF and p115 was discovered in a yeast two-

hybrid screen and affirmed in several co-immunoprecipitation studies in

mammalian cell systems. Furthermore, the interaction of the two proteins

was confirmed biochemically in a competition binding assay utilizing

recombinant MIF and p115702-962. The assay demonstrated that the two

proteins bind directly and specifically without necessarily requiring

additional, intermediary proteins for the interaction. The percent of

competition measured was between 50 and 60%. In contrast, the percent of

competition reached between MIF and its receptor CD74 is near 100%

(58).

A likely explanation for the levelling of the competition curves at 50 – 60%

is that there is some non-specific background binding to the plate which

cannot be fully blocked by the Superblock reagent. Alternatively, the

biotinylated species (both biotin-p115 and biotin-MIF) may have higher

non-specific binding than the non-modified, native protein. It also is

Discussion – 87

possible that there exist two conformational species of the immobilized

protein on the plate, and that only one can be competed for.

4.1.4 p115 Is Required for the Stimulated Release of MIF

p115 is a Golgi-associated protein known to be predominantly localized to

the cis-Golgi and the vesicular tubular clusters (VTC). It was

demonstrated that the cellular depletion of p115 causes a decrease in MIF

export from stimulated cells.

Previous reports describe that the depletion of p115 below detectable

levels and the microinjection of anti-p115 antibodies causes a

fragmentation of the Golgi leading to a delay in protein export (101-103,

162). In contrast, the partial depletion of p115 by the RNAi technique used

in this study neither affected the structural morphology of the Golgi nor

reduced the export of ER/Golgi-dependent proteins such as TNF-α, IL-6

and FGF-4. The release of the unconventionally secreted protein IL-1β,

which occurs via vesicles (118), and FGF-2, which translocates across the

plasma membrane (114) also was not affected by a depletion of p115. That

p115 is necessary for the release of MIF but not other cytokines, whether

conventionally or unconventionally secreted, suggests a specific and

unique role in the export of MIF (Fig. 42).

Discussion – 88

Fig. 42: Scheme of p115-dependent MIF secretion. Left: p115 is localized at the cis-

side of the Golgi where it mediates the tethering of vesicles arriving at the Golgi from the

ER, contributing to the conventional secretion of proteins. MIF and p115 also interact in

the area of the Golgi. Right: p115 is partially depleted, but still mediates the tethering of

ER-derived vesicles at the Golgi. MIF is not longer effectively released from cells.

The role of p115 in the transfer of vesicles from the ER to the Golgi has

been established for some time (97). In this process, the carboxy-terminus

of p115 interacts with the golgin protein GM130 to tether COPII-coated

vesicles to the cis-side of the Golgi. While the depletion of p115 inhibits

the stimulated release of MIF, the depletion of GM130 has no measureable

effect on the release of MIF. This observation indicates that the role of

p115 in MIF secretion is different from its role in the conventional

secretion of proteins. To confirm this hypothesis one can analyze the role

of Rab1, a small GTPase, in the release of MIF, since p115 is a Rab1

effector protein. The active form of Rab1 binds to the amino-terminal head

of p115 (96). Rab1 exists in two isoforms, Rab1A and Rab1B; both have

been suggested to regulate anterograde transport of cargo between the ER

and the Golgi apparatus (163). Therefore, an efficient knockdown of Rab1

for a comprehensive study of its role in MIF secretion is difficult to

accomplish. Another approach to study the effect of Rab1 on MIF release

is the microinjection of guanine nucleotide dissociation inhibitor (GDI). It

Discussion – 89

was demonstrated that the administration of GDI effectively blocks the

function of Rab1 by blocking the transfer of stomatitis virus glycoprotein

(VSV-G) from the ER to the cell surface (164). The disadvantage of this

approach is that in contrast to the previous studies the conventional

secretion pathway is compromised; therefore any effect seen after the

microinjection of GDI could be either contributed to a direct effect on the

MIF secretion or due to the compromising of the cell system, which then

secondarily leads to a reduced secretion rate of MIF.

4.1.4.1 p115 Overexpression does not Affect MIF Secretion

For the intracellular binding partner of MIF, JAB1, it was demonstrated

that both its depletion and its overexpression lead to the modulation of the

MIF signaling pathway (40, 44).

While the depletion of p115 consistently decreased MIF release in various

experimental approaches, the overexpression of p115 had no affect on

either the stimulated release of MIF or TNF-α. These observations

indicate that p115 may not be the only limiting factor in the process of

MIF release, but that other proteins are likely required. To address this

hypothesis it is of interest to screen for MIF interaction partners in the

mammalian cell system. Thus far in efforts to identify new MIF binding

partner only yeast two-hybrid screens were performed. Other approaches,

like tandem affinity purification (TAP) in the mammalian cell system

(165) might lead to the discovery of new MIF interaction partners or even

a multi-protein complex, including MIF and p115, thus possibly

identifying other proteins involved in the pathway of MIF export.

4.1.4.2 MIF and p115 are Co-Secreted

Macrophages stimulated with LPS export both MIF and p115 in a pattern

that suggests the coupled release of the two proteins. This observation is

Discussion – 90

supported by reports linking both MIF (134-136) and p115 (126) to

vesicles. The co-secretion of the two proteins affirms the previous

observations that p115 mediates the release of MIF.

So far it is unknown whether MIF and p115 locate to exosomes in

macrophages. To address this question, supernatants of stimulated

macrophages have to be examined. One potential approach is the isolation

of vesicles by gradient centrifugation and analyzing the vesicles for MIF

and p115. Another method might be the further examination of the two

proteins in the supernatant after cross-linking or co-immunoprecipitation.

A positive outcome of these experiments would support the idea that MIF

and p115 are released together, possible in exosomes.

It is also conceivable that p115 mediates a generalized, non-classical

secretory response by activated monocytes/macrophages that results in the

release of a p115 macromolecular complex containing numerous proteins,

of which MIF is one. Further elucidation of both the signals and the

protein-protein interactions underlying this export pathway may be

informative and enhance our understanding of the acute secretory

response of activated monocytes/macrophages.

4.1.5 4-IPP Inhibits the Release of MIF and p115

4-IPP is the first pharmacological inhibitor for MIF found to block the

secretion of MIF in macrophages in vitro. This was demonstrated via LPS

stimulation and C. trachomatis infection. Furthermore 4-IPP treatment

prevents the export of p115 after LPS stimulation, thus affirming the

hypothesis that MIF and p115 are released in a complex. These data

identify 4-IPP as a potentially selective inhibitor of the p115/MIF

secretory pathway. 4-IPP covalently modifies the MIF amino-terminus,

and alters the protein’s conformational integrity so as to increase its

binding interaction with p115. One possibility might be that MIF’s

enhanced binding to p115 in the presence of 4-IPP disrupts its normal

Discussion – 91

trafficking pathway and secondarily reduces MIF secretion. 4-IPP thus

provides proof-of-concept for the possibility of reducing MIF-dependent

responses in the earliest phase of cell activation by interfering with MIF’s

cytoplasmic release.

The administration of 4-IPP did not improve the survival in a murine

model of LPS shock. Furthermore, 4-IPP did not reduce the serum level of

MIF and other cytokines downstream of MIF in response to LPS shock.

Numerous possibilities might explain why 4-IPP blocks MIF secretion in

vitro, while it does not inhibit MIF release in vivo. 4-IPP is a highly

lipophilic reagent; due to its hydrophobic properties, 4-IPP easily crosses

the plasma membrane, but the solubility in hydrophilic solvents in

concentrations necessary for an in vivo administration is unsatisfactory.

Both MIF inhibitors, 4-IPP and ISO-1, are first dissolved in DMSO, but

while ISO-1 is diluted in water for the intraperitoneal injection, 4-IPP has

to be diluted in corn oil to avoid precipitation. Mitchell and colleagues are

in the process of modifying 4-IPP to improve its solubility in water, and it

is of interest whether a decreased hydrophobicity provides protection in

the LPS shock model. Variables, such as the time point and dose of 4-IPP

administration also contribute to the outcome of the experiment. In this

study, 4-IPP was injected 1.5 h prior to LPS, likewise to the protocol for

the administration of neutralizing antibody and ISO-1 (5, 20). In contrast

to neutralizing antibody or ISO-1, it is not known whether 4-IPP

interferes with MIF’s ability to interact with its receptor CD74, thus a

different time point of administration might be favourable. Once the

solubility of the compound is improved, various concentrations and time

points of administration should to be tested. Furthermore, in the LPS-

shock model, MIF is not only released by specific secretion, but also due to

cell death, and this latter source of MIF cannot be blocked by 4-IPP.

Nevertheless, inhibitors such as 4-IPP may be attractive in clinical

application in those settings, such as severe inflammation, in which rapid

intervention is desired to prevent the initiation of a tissue-damaging,

cytokine cascade (166).

Discussion – 92

4.2 The Structural Homologue DDT

Most cytokines, such as IL-1, belong to large superfamilies. MIF has no

cytokine family, and its protein family comprises only a few members: D-

dopachrome tautomerase in eukaryotes and the two bacterial proteins 4-

oxalocrotonate tautomerase (4-OT) and 5-carboxymethyl-2-hydroxy-

muconate isomerase (CHMI) (167).

The members of the MIF/tautomerase superfamily all possess a

tautomerase active site that has its catalytic base in the amino-terminal

proline-1. To date, the physiological function for this enzymatic activity is

unknown, but in the case of MIF, several biological functions are linked to

this region (61).

The three-dimensional structures of human MIF and DDT are highly

conserved; the protomeric polypeptides of the two proteins consist of six β-

strands and two α-helixes. Four of the β-strands form a β-sheet; the

remaining two β-strands are important for the formation of the trimer by

forming hydrogen bonds to the adjacent protomers (50, 69) (Fig. 1 and Fig.

5). However, while the knowledge about MIF’s prominent role as regulator

of the innate and adaptive immunity has grown tremendously over the

last decades, nothing is known about the physiological role of DDT.

In order to establish the basic tools for a better understanding of the

biological functions of DDT, the native, recombinant protein was purified

and the first specific polyclonal antibody targeting murine DDT was

produced. This antibody does not show any cross reactivity to MIF.

Utilizing this new antibody, an indirect ELISA for DDT was successfully

developed and used to analyze the release of DDT after stimulation of

macrophages with LPS. Both DDT and MIF are secreted from cells in

response to LPS. Additionally it was shown that both proteins are released

constitutively at a low level. However, herein the molecular cascade

triggered by DDT after its release in response to a pro-inflammatory

stimulus was not addressed. Detailed studies have to be conducted to

elucidate whether DDT exerts similar biological activities than MIF, or

whether it acts as a cross-regulator of MIF. Both scenarios are well

Discussion – 93

documented in immunology. For instance, the chemokines CXCL9,

CXCL10, and CXCL11 all bind to the same receptor CXCR3 eliciting an

increase in intracellular Ca2++ level and activating phosphoinositide-3 and

MAPK kinase (168).

Some arguments can be made supporting the hypothesis that MIF and

DDT share some biological abilities. First, the mRNA and protein levels of

DDT are unchanged in mif knockout cells compared to wildtype control

cells. Second, MIF and DDT are released together after stimulation with

LPS from macrophages. Third, the depletion of either MIF or DDT by

siRNA results in similar effects (transcriptional regulation of IL-8), both

involving the receptor CD74 (72).

With the help of the purified, native DDT protein and anti-DDT antibody,

future experiments can be performed to further address these questions.

Additionally, on a molecular level, the details of the release of DDT have

not been addressed. Established protocols from this work might aid in the

determination whether the release of DDT also depends on p115, and

considering the structural conservation of the active sites, whether the

secretion can also be inhibited by the small molecule inhibitor 4-IPP.

Anti-MIF therapies have been shown to be beneficial in many

inflammatory diseases. However, these approaches might not be complete

without considering DDT’s possible role in disease development, therefore

studying the molecular mechanisms underlying DDT might improve

established anti-MIF treatments of inflammatory disease.

Summary and Outlook – 94

5 Summary and Outlook

Macrophage migration inhibitory factor is a pro-inflammatory cytokine

that plays a pivotal role in the pathogenesis of inflammatory disease such

as septic shock and rheumatoid arthritis. The release of MIF from

inflammatory cells is a common feature in these diseases, and a

correlation between the severity of disease and MIF production is

observed. MIF is a leaderless protein that is secreted from cells by a

specialized, non-classical export pathway, possibly involving the

transporter ABCA1. The release of MIF nevertheless is tightly regulated

with respect to its production in response to different inflammatory,

proliferative, and activating stimuli. In recent studies it was demonstrated

that the administration of neutralizing anti-MIF antibodies and small

molecule MIF inhibitors is advantageous in inflammatory diseases. These

approaches have in common that they all target MIF only after it is

released from cells. A more effective approach in treating MIF-related

diseases may be to target MIF before it is released from cells. The

unconventional secretion pathway of MIF might permit to specifically

block the release of MIF without compromising the general secretion

mechanism of the cell. To date, little is known about the molecular

mechanisms involved in the release of MIF.

My thesis demonstrates that MIF associates with the novel MIF

interaction partner p115, a Golgi-associated protein that takes part in the

ER/Golgi secretion pathway. By confocal microscopy, I was able to

demonstrate that MIF and p115 are associating in the cytoplasm;

following activation, macrophages redistribute MIF to the plasma

membrane together with a portion of p115. This finding agrees with

results obtained from Western blot and ELISA analysis, demonstrating

that upon LPS stimulation both MIF and p115 are co-secreted,

accompanied by a corresponding decrease in intracellular protein

concentration. Accordingly, I demonstrated that the depletion of p115 from

monocytes/macrophages by RNAi results in an inhibition of MIF release


after LPS stimulation. Intriguingly, the secretion of other pro-

inflammatory cytokines, both conventionally released (TNF-α, IL-6) and

unconventionally released (IL-1β) was not affected by the partial depletion

of p115. Similarly, the depletion of p115 in HeLa cells did not affect the

conventional (FGF-4) or unconventional (FGF-2) secretion. This

observation leads to the assumption that it is possible to specifically target

the secretion of MIF without compromising the normal and necessary

secretory mechanism of cells. It is important to note that the effect of p115

depletion on MIF secretion was not only observed after LPS stimulation,

but also after bacterial infection with Chlamydia trachomatis. Notably,

the prototypic small molecule MIF inhibitor, 4-iodo-6-phenylpyrimidine,

inhibits MIF secretion by targeting the interaction between MIF and p115.

My data reveal p115 to be a critical intermediary component in the

regulated secretion of MIF from monocytes/macrophages.

After the discovery of GRASP65 in the unconventional secretion of acyl-

CoA binding protein in Dictyostelium (169), p115 is the second protein of

the Golgi complex attributed with a dual function in secretion. On one

hand p115 is involved in the classical secretion pathway tethering vesicles

arriving from the ER to the Golgi, and on the other hand it is involved in

the unconventional secretion of MIF. The discovery that 4-IPP specifically

blocks the secretion of MIF by interfering with MIF’s interaction with

p115 gives hope in the search of a potent approach in treating MIF-related

diseases by blocking its release from cells. It seems feasible to perform a

screening of MIF inhibitors that specifically target MIF’s ability to

interact with p115 and block its release from cells.

In addition, in my thesis I report on the initial characterization of the

structural homolog of MIF, D-dopachrome tautomerase (DDT). After the

successful purification of recombinant, native murine DDT, a specific anti-

DDT antibody was produced and utilized for the establishment of a DDT-

specific ELISA. It was shown that DDT is released from macrophages in

response to LPS in a pattern similar to that of MIF. Furthermore, it was


shown that the deletion of the mif gene has no effect on the production of

DDT. The precise biological function of DDT remains unclear and the

detailed characterization of the secretion pathway of DDT was beyond the

scope of this thesis, but the comparable release patterns of MIF and DDT

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111

Curriculum vitae

Persönliche Daten

Geburtsdatum 20. November 1979

Geburtsort Freiburg im Breisgau

Familienstand ledig

Staatsangehörigkeit deutsch

Bildungsweg

08/90 – 06/99 Gymnasium

Goethe-Gymnasium, Freiburg

Abschluss: Abitur

10/99 – 09/01 Grundstudium Biologie

Albert-Ludwig Universität, Freiburg

Abschluss: Vordiplom

04/02 – 08/05 Hauptstudium Biologie

RWTH Aachen, Aachen

Abschluss: Diplom

Hauptfächer: Molekularbiologie, Bioinformatik

Nebenfächer: Biotechnologie, Mikrobiologie,

Informatik

10/04 – 09/06 Diplomarbeit

Institut für Biochemie und Molekulare Zellbiologie

der RWTH Aachen unter Betreuung von Prof.

Bernhagen

Titel: Charakterisierung der Bindung von Cyclinen

an das Zytokin MIF und seine Phosphorylierung

durch CDKs

01/06 – heute Promotion

Institut für Biochemie und Molekulare Zellbiologie

der RWTH Aachen unter Betreuung von Prof.

Bernhagen in Kooperation mit Yale University

School of Medicine (USA) unter der Anleitung von

Prof. Bucala

112

Auszeichnungen

10/01 – 03/02 Förderung des Deutschen Akademischen

Austauschdienst

01/06 – heute Promotionsstipendium der Studienstiftung des

deutschen Volkes

Publikationen

M. Merk, J. Baugh, S. Zierow, L. Leng, U. Pal, S. Lee, A. Ebert, Y. Mizue,

J. Trent, R. Mitchell, W. Nickel, P. Kavathas, J. Bernhagen, and R. Bucala

(2008). The Golgi-associated Protein p115 mediates the secretion on

Macrophage Migration Inhibitory Factor (MIF). (In Revision Mol Biol Cell)

D. Kamir, S. Zierow, L. Leng, Y. Cho, Y. Diaz, J. Griffith, C. McDonald, M.

Merk, R.A. Mitchell, J. Trent, Y. Chen, Y.K. Kwong, H. Xiong, J.

Vermeire, M. Cappello, D. McMahon-Pratt, J. Walker, J. Bernhagen, E.

Lolis, and R. Bucala. 2008. A leishmania ortholog of macrophage

migration inhibitory factor modulates host macrophage responses. J

Immunol 180:8250-8261.

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