Prolactin regulation of lymphocyteproliferation: An early event

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Authors Vander Hamm, Dale Gene, 1954-

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PROLACTIN REGLT-ATION OF LYMPHOCYTE PROLIFER.\TION:

.\N EARLY EVENT

by

Dale Gene Vander Hamm

A Dissenation Submitted to the Faculty of the

COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE LTMIVERSITY OF ARIZONA

1 9 9 8

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2

THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have

read the dissertation prepared by Dale Gene Vander Hatnm

entitled Prolactin Regulation of Lymphocyte Proliferacion:

An Early Event

and recommend that it be accepted as fulfilling the dissertation

requirement for the Degree of Doctor of Philosophy

I' J. Haionen, Ph.D Date

Douglas F. Larson, Ph. 9

Date

Daniel C. Liebler/'

A./M^Queetl, PtT.D^

JxShn W.

Date

G ̂ ^^ Date ,

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Dissertation Director Douglas F. Larson, Ph.D. Date

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at The University of Arizona and is deposited in the University' Library to be made available to borrowers under rules of the Librziry.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In ail other instances, however, permission must be obtained from the author.

SIGNED: • • • •-/

ACKNOWLEDGMENTS

4

Foremost I express my deepest respect and gratitude to Dr. Hugii E. Laird II for his

guidance and support through the planing and e.xperimental phases of the majoritv' of this

work. My sincere appreciation to Dr. Douglas F. Larson for his insight and patience in

guiding me through the final writing and dissenation defense stages of this program. A

special thanks to Dr. John Regan as a graduate comminee member; in particular for

supporting my final prolactin RT-PCR studies. 1 thank my graduate committee members:

Dr. Dan Liebler. Dr. Charlene McQueen, and Dr. Marilyn Halonen for their time, insight,

and valuable advice. I thank Dr. David Nelson and Dr. Tom Burks for their initial

inspiration and guidance as a graduate student.

In panicular. I thank several special students and lab staff for their friendship,

encouragement, training, and support: Dr. .Ajine Killam. Dr. Linda Comlield. Georgina

Lambert. Elizabeth Ayers. Grace Davis-Gorman. Dr. .A.lpaslan Dedeoglu. Jacqi Evans-

Shields. Karen Achanzar, Kristen Pierce, Todd .\nthony. Ihab Botros and Dr. Jeremy

Richman.

Finally. I express my deepest appreciation to my wife Debi and my daughters

Amanda and Jessica for their long suffering love, endurance, support, and encouragement.

DEDICATION

5

In memory of Hugh E. Laird II. Ph.D. in appreciation for his continual patience,

humor, perseverance, encouragement, guidance, and support. Dr. Laird was truly a man

of integrity and dependable inspiration. His knowledge and e.xperience encompassed life

from many view points to include but not limited to his own experience as a father, a

husband, a pharmacist, a patient, a professor, an administrator, and a mentor. Without Dr.

Lmrd's unwavering long term commitment and his demonstrated belief in my potential and

ability to conduct and evaluate research I would not likely have completed this endeavor.

6

TABLE OF CONTENTS

LIST OF FIGURES 8

LIST OF TABLES 10

ABSTRACT II

1. INTRODUCTION 13

1.1 Hypothesis 13 1.2 Physiology ofProlactin 13 1.3 PRL and Immunology 16 1.4 PRL and Interleukin 2 18 1.5 Clinical Applications 27 1.6 Research Models 31

2. CELL PROLIFERATION 34

2.1 Introduction 34 2.2 Methods 35

2.2.1 Materials 35 2.2.2 PRL-Free Medium 36 2.2.3 PRL-Free Medium NB211c Characterization 37 2.2.4 T-cell Preparation 37 2.2.5 Cell Proliferation 38 2.2.6 Statistical Methods 39

2.3 Results 40 2.3.1 NB21 Ic Assays 40 2.3.2 T-cell Preparation 40 2.3.3 Cell Proliferation 44

2.4 Discussion 45

3. INTERLEUKIN 2 EXPRESSION 54

3.1 Introduction 54 3.2 Methods 56

3.2.1 Collection of mRNA 56 3.2.2 Reverse Transcriptase (RT) 57 3.2.3 Amplification of cDNA(PCR) 57 3.2.4 Analysis ofRT-PCR Products 59 3.2.5 IL-2 Bioassays 59

7

TABLE OF CONTENTS - continued

3.3 Results 60 3.3.1 IL-2 and lL-2r mRNA Expression 60 3.3.2 IL-10 mRNA E.xpression 61 3.3.3 IL-2 Expression 65

3.4 Discussion 73

4. LYMPHOCYTE PROLACTIN LIKE PROTEIN EXPRESSION 79

4.1 Introduction 79 4.2 Methods 79

4.2.1 Dot Blot Analysis 79 4.2.2 RT-PCR Analysis 80 4.2.3 PRL Assays 81 4.2.4 Analysis of Ly-PLP in Cell Lysates 83

4.3 Results 84 4.3.1 PRL mRNA Dot Blots 84 4.3.2 PRL mRNA RT-PCR 84 4.3.3 Ly-PLP NB21 Ic Bioassay 89 4.3.4 Ly-PLP Western Blot 93

4.4 Discussion 97

5. SUMMARY 101

REFERENCES 113

8

LIST OF FIGLHES

1.2.1 rPRL gene and amino acid sequence 15

1.4.1 Pathway for IL-2 gene activation 21

1.4.2 Pathway for PRL action on IL-2 gene 23

2.3.LI MB2IIcrPRLandoPRL 41

2.3. L2 NB21IcrPRL and A-PRL 42

2.3.2.1 F.A.CS analysis of enriched T-cells 43

2.3.3.1 Concentration-dependent response to CON A 47

2.3.3.2 oPRL concentration-dependent effect on restingT-cells 48

2.3.3.3 oPRL concentration-dependent effect with CON .A. 49

2.3.3.4 A-PRL concentration-dependent effect with CON A 50

2.3.3.5 oPRL partially overcomes A-PRL inhibition 51

2.3.3.6 Time course addition of A-PRL 52

2.3.3.7 rhuIL-2 overcomes A-PRL inhibition 53

3.3.1.1 RT-PCR CON A + A-PRL treated for IL-2 and rL-2rp 62

3.3.1.2 RT-PCR 24 hr stimulated and resting 63

3.3.1.3 RT-PCR 24 hr time course 64

3.3.3.1 CTLL-2 rhuIL-2 standard curve 67

3.3.3.2 CTLL-2 concentration-dependent CON A induced IL-2 68

3.3.3.3 CTLL-2 IL-2 production time course 69

3.3.3.4 CTLL-2 concentration-dependent inhibition by A-PRL 70

9

LIST OF FIGURES coniimied

3.3.3.5 CTLL-2 r-PRL partially overcomes A-PRL inhibition ofIL-2 71

3.3.3.6 CTLL-2 A-PRL - rhuIL-2 effect 72

4.3.1.1 Dot blots for (3 .Actin. PRL 86

4.3.2.1 RT-PCR RNAsed Data 87

4.3.2.2 RT-PCR GH3 88

4.3.3.1 NB211 c concentration-dependent response CON .A 90

4.3.3.2 NB2I Ic bioassavs time course of T-cell media 91

4.3.3.3 IL-2 drives NB21 Ic in presence of .A-PRL 92

4.3.4.1 Silver stain protein gel 94

4.3.4.2 Western blot 1 95

4.3.4.3 Western blot 2 96

10

LIST OF TABLES

L3.1 Summary of published data for Ly-PLP and IL-2 19

3.2.3.1 Interleukin RT-PCR primer sequences and annealing temperatures 58

4.2.2.1 Ly-PLP RT-PCR primer sequences 82

5.1 Summary of dissertation data 111

ABSTRACT

II

It has been suggested by immune system studies that pituitan' prolactin (PRL) may

have an immunomodulatory role and that lymphocytes themselves may produce prolactin-

like proteins (Ly-PLP). The studies in this dissertation focused on Ly-PLP production and

PRL/Ly-PLP actions in an enriched population of rat splenic T helper (T^) CD4 and

cytotoxic CDS cells. Studies were plarmed on a dual hypothesis: first, that T-cells produce

Ly-PLP which may act in an autocrine manner and second, that Ly-PLP may be necessary

for interleukin 2 (IL-2) production. To study the possible mechanisms by which PRL and

Ly-PLP regulate lymphocyte proliferation, a PRL-free medium was used and a polyclonal

antibody to rat PRL (A-PRL) was used to inactivate the residual PRL and the expressed Ly-

PLP in the cell culture system. This antibody inhibited T-cell proliferation in both a time

and a concentration-dependent manner. Based on four lines of evidence, the in vitro

inhibitory activity of A-PRL on cell proliferation appeared to be modulated in pan by

inhibition of IL-2 production. First, the A-PRL induced inhibition of T-cell proliferation

was overcome by addition of recombinant human IL-2 (rhuIL-2). Second, A-PRL

treatment of mitogen stimulated T-cells was shown by RT-PCR analysis to inhibit IL-2

mRNA expression. IL-2 receptor beta (IL-2r5) mRNA, which is constitutively expressed

in T-cells, was not inhibited. Third, A-PRL inhibited IL-2 protein production in a

concentration dependent manner. Fourth, the A-PRL induced inhibition of IL-2 production

was overcome by preincubation of the A-PRL with purified rat pituitary PRL (rPRL). Dot

Blot and RT-PCR analysis data in this dissertation were suggestive but were inconclusive

12

in support of the hypothesis that rat lymphocytes and specifically T-cells constitutively

express mRNA for PRL and/or Ly-PLP. Western blot studies of T-cell lysates suggested

that rat T-cells express a Ly-PLP with the apparent molecular weight 46 kD. as well other

variants. In summary the data presented in this dissertation indicate that rat T-cells

expressed Ly-PLP and treatment with A-PRL to inactivate Ly-PLP inhibited cell

proliferation, expression of IL-2 mRNA. and IL-2 protein expression by CD4 cells. The

inhibition of IL-2 production was the likely mechanism by which A-PRL binding of Ly-

PLP inhibited mitogen induced T-cell proliferation in rats. The inhibition of IL-2

production suggests that extracellular pituitary PRL or Ly-PLP through a hormone and/or

an autocrine mechanism respectively may be necessar>' but not sufficient for the induction

of IL-2 mRNA and IL-2 protein expression in activated rat T-cells.

13

CHAPTER 1. INTRODUCTION

1.1 Hypothesis

Immune function studies conducted in humans, mice, and rats have suggested an

immunomodulator\' role for pituitary prolactin (PRL) and also the presence of lymphocvle-

produced prolactin-like proteins (Ly-PLP). The purpose of this dissertation is to summarize

the current literature and to describe my studies to investigate effects of pituitarv' PRL and

Ly-PLP on lymphocyte proliferation and function. Studies were planned based on a dual

hypothesis: First, that a subset of lymphocvies, T-cells. produce a Ly-PLP and that Ly-PLP

may act in an autocrine manner as an essential growth factor or co-mitogen for T-cells.

Second, that Ly-PLP and/or PRL presence may be necessarv' for interleukin 2 (IL-2)

production and/or fimction.

1.2 Physiology of Prolactin

PRL is commonly known to be a 22 to 24 kD peptide produced and secreted by the

anterior pituitary gland. PRL production and release by the pituitary gland are commonly

accepted to be under tonic inhibition by hypothalamic factors such as dopamine and

positive transcription and releasing control by estrogen, thyrotropin releasing hormone

(TRH) (Leong et al., 1983), and exercise induced opioid release (Carr et al.. 1981;

Mandenoff et al.. 1982). PRL is synthesized, stored, and then secreted with diumal

variation in both males and females, with two spikes during the sleep cycle. The highest

14

plasma concentration of approximately 10 ng,''ml in humans occurs upon awakening, with

a half-life of 15 to 20 minutes (Genuth. 1988; Kuret and Murad. 1990). Females exhibit

higher blood concentrations during times of higher circulating estrogen such as certain

phases of menses and post partum with plasma levels rising as much as 20 fold at term

(Genuth. 1988). PRL is generally associated with induction of post partum lactation and

was studied extensively by Eliddle and coworkers for its actions with crop sack milk

production in male and female pigeons in the 1930's (Riddle and Braucher, 1931: Riddle

et al.. 1933: Riddle et al.. 1933). This substance was tlrst named "pro"-lactin in 1932 by

this group of scientists for what was known to be its action in induction of lactation (Riddle

etal.. 1932).

The first publication of the structure of the rat prolactin (rPRL) gene was by

Gubbins et al. in 1980. It was also shortly thereafter analyzed and compared to the rat

growth hormone (rGH) gene by Chien and Thompson (1980) and Cooke and Baxter (1982)

(Figure 1.2.1) with only a few variations between the PRL gene nucleotides as compared

to that of Gubbins. rPRL and rGH are part of a family of polypeptide hormones, which

share many features, are thought to have evolved from gene duplication, and have diverged

greatly since duplication (Gubbins et al.. 1980).

It is now known that both rat and human PRL are e.xpressed as a 227 amino acid

preprolactin which is later cleaved to a 197 amino acid hormone in the rat (Gubbins et al..

1980) and 198 amino acid protein in the human (Truong etal.. 1984). Both rPRL and rGH

have five exons with four intervening introns (Cooke and Ba.xter. 1982). rPRL and rGH

bind to PRL receptors in primate lymphocytes (Fu et al.. 1992).

15

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16

only retain 25% amino acid homolog\' and rPRL is five times larger than rGH (Cooke and

Baxter. 1982). rGH does not cross react with the rPRL receptors whereas primate GH does

The PRL receptor (PRLr) was cloned from the rat liver in 1988 (Boutin et al..

1988). Prior to the cloning of the PRLr in the last decade, there was little understanding

of the biochemical mechanisms by which PRL worked within the cell mostly due to an

inability to identify the target molecules of PRL action in the cell (Doppler. 1994). In

humans one PRLr transcript is expressed of 598 amino acids: in rats two forms, the long

(active) form 591 amino acids and the short form (no assigned function) 291 amino acids;

and in mice four proteins are expressed. 292. 303. 310. and 597 amino acids (review by

Doppler. 1994). The PRLr and the GH receptor (GHr) are grouped into an expanded

cytokine receptor superfamily due to the low but significant sequence homology in their

extracellular domains with a number of cytokine receptors including the interferons (review-

by Doppler. 1994).

1.3 PRL and Immunologv

Prolactin (PRL) has more distinct and diverse effects described than that of any

other pituitary hormone to include but not limited to effects on growth and differentiation

of organs such as the ovaries, testis, prostate, breast, pancreas, involvement in reproduction,

and osmoregulation in lower species (review by Doppler, 1994). For a peptide with such

a diverse repertoire of actions, it is not entirely unexpected to discover a major growth

factor and regulatory role in one of the more highly responsive and complicated regulatorv'

17

systems of mammalian physiology, the immune system.

These immune system actions are diverse and have been well reviewed with PRL

playing roles in immunocompetence. T-cell differentiation, cancer, and autoimmune

diseases (Gala, 1991: Reber 1993; Wilder 1995: Murphy et al.. 1995: Yu-Lee. 1997").

Historically it was shown that PRL could reverse the immunodeficiency that followed

hypophysectomy in rats (Nagy and Berczi. 1978: Nag\' et al., 1983). .A.lso. with

hypophysectomized rats. PRL and GH have been shown to increase in vitro splenocyte

proliferation (Berczi et al.. 1991). In mice, in vivo administration of bromocriptine, a

dopamine receptor agonist that selectively inhibits pimitary PRL release, inhibits

lymphocyte response to antigenic stimulation in a manner correlated with serum PRL levels

(Hiestand et al.. 1986). The inhibition of murine lymphocyte proliferation caused by

bromocriptine administration can be overcome by administration of ovine PRL (oPRL)

(Bemton et al.. 1988). ftmction for PEIL in lymphocyte proliferation and/or activation

is fiirther supported by the presence of high affinity PRL receptors on both T and B human

lymphocytes (Russell et al., 1984: Russell et al., 1985) and expression on primary rat

splenocytes including CD4. CD8, and B lymphocytes (Viselli and Mastro. 1993; Koh and

Phillips, 1993). In rat peripheral lymphocytes in vivo elevated serum PRL levels have been

shown to be negatively linked to expression of the long form, but not the short form of

PRLr mJRNA. Decreased serum PRL levels have also been shown to increase the

expression of PRLr mRNA (DiCarlo et al.. 1995). In women. PRLr levels on lymphocytes

can vary up to 10 fold on different days of the menstrual cycle (Athreya et al.. 1994).

It has been suggested by Hartmarm et al.. (1989) that lymphocytes may produce a

18

prolactin like protein (Ly-PLP) which may act in a paracrine or autocrine manner. Ly-PLP

presence has now been shown by several authors and is summarized in Table 1.3.1. The

exact role or roles that Ly-PLP may play in die myriad of immune system signals both

extracellular and intracellular remains to be determined. It is possible that autocrine action

by Ly-PLP may augment or replace pituitary PRL during fluctuations in plasma

concentrations or it may play a role in pathological conditions such as autoimmune

diseases.

1.4 PRL and Interleukin 2

Lymphocyte proliferation is a generally accepted measure of immune fiinction and

interleukin 2 (IL-2) is a well-documented cytokine that is necessary for T-cell growih and

proliferation. IL-2 is a 15-18 kD lymphokine synthesized and secreted primarily by

activated CD4 lymphocytes of the TH1 T-cell subset (Mosmarm and Coffman. 1987). IL-2

has also been called T-cell growth factor. It has both autocrine and paracrine actions as it

stimulates clonal expansion of antigen-specific T-cells and influences activity of B-cells.

[L-2 receptors are made up of three distinct membrane proteins known as the a chain

(IL2ra), p55 or Tac antigen; P chain (IL2rP), p70/75; and the y chain (IL2rY), p64. The

genes for the IL2ra and the IL-2 protein are not expressed in resting cells but are detectable

in activated cells (Taniguchi and Minami, 1993). IL2rP is expressed constimtively in CDS

cells but not in CD4 cells where it is induced upon activation. In contrast. IL2r7 is

expressed constitutively in all lymphoid cells (Taniguchi and Minami. 1993. Takeshita et

19

Table 1.3.1

Published data for human, mouse, and rat lymphocytes for the presence of PRL mRNA. the presence of Ly-PLP with t respective published molecular weights, and studies demonstrating PRL effects on IL-2 production showing increases 1 or decreases 1 in IL-2.

Species mRN.A Ly-PLP/MW IL-2

HUMAN northern (1) RT-PCR (3.4.6)

RIA (1) 23 & 36 kD (4) 23.5 & 25.5 kD (9) 27 kD (7) 29 kD (2) 11 & 24-27 kD (5) 60 kD (3.8)

MOUSE dot blot (13) 24&48kD (11) 22.33.6.35.6 kD (12) 46 kD (10) 46 kD (13)

RAT dot blot (14.20) RT-PCR (17)

44kD (18) 43 kD (19) 46 kD (20)

ovx-rPRL 1 (16) age^GH3 ' (15) .APRL i (20)

1. DiMattia et al.. 1988 2. Baglia et al., 1991 3. Sabharwal et al.. 1992 4. Pellegrini et al.. 1992 5. Gutierrez et al.. 1995 6. Wu and Malarkey. 1996 7. Matera et al.. 1996 8. Larreaetal.. 1997 9. Matera et al., 1997 10. Montgomery. 1987

11. Kenner et al.. 1990 12. Montgomery et al., 1990 13. Shahetai., 1991 14. Hiestand et al.. 1986 15. Kelly etal.. 1986 16. Viselli et al.. 1991 17. Jurcovica et al.. 1992 18. Mastenetai.. 1996 19. Masten & Shiverick. 1997 20. Vander Hamm. unpublished

20

al., 1992). In humans the combination of IL2rY and IL2rP gives rise to a functional

intermediate affinity (10''°M) receptor but addition of IL2ra is required to reach the highest

affmit>' (10'"M) heterotrimer (Taniguchi and Minami. 1993. Voss et al.. 1993).

Induction of the IL-2 gene is under the control of multiple factors as shown in figure

1.4.1. The following summary- description for IL-2 gene activation can be read in greater

detail (see Male et al., 1996.) Resting T-cells are at of the cell cycle. Following

interaction vvith antigen or other ligands such as plant lectins or antibodies for certain cell

surface markers, they enter the G, phase of the cell cycle and as a result of IL2r expression

and IL-2 production then enter into the G, phase and proceed on through G^ and mitotic

phases.

Initial activation of CD4 lymphocytes begins when an antigen presenting cell (APC)

associated with a membrane bound major histocompatibility complex 2 (MHCII) molecule

presents an antigen to the cell surface T-cell receptor (TcR). The TcR does not have

intrinsic protein tyrosine kinase (PTK) activity but it is associated with activity' of at least

two PTKs of the Src- family of kinases: p56''^'' (Ick) and p59'^"(fyn). Also, ZAP-70 kinase

(zap-70), which has a specialized SH2 binding domain for phosphotyrosine residues of the

TcR complex, is involved in downstream activation events. The TcR coupled with a

membrane bound CD3 molecule activates phospholipase Cy (PLCy) which in turn cleaves

phosphatidylinositol 4,5-biphosphate (PIP,) into a pair of second messengers: membrane

bound diacylglycerol (DAG) and water-soluble inositol 1,4,5-triphosphate (IPj). PIP2 was

previously made from phosphoinositol (PI). DAG activates protein kinase C (PKC), a

calcium (Ca") dependent enzyme which is one of a family of PKC enzymes. PKC can

21

APC

MHC II

Ca' TcR

CD3 PLCy

Ca' DAG

Cm c-fos+c-Jun PKC

CnArrCYP

"Tfcnffl (jKEn-NF^

I NFAT I INFAT

AP-1 Transcnption NFAT

/L- Zgene

Figure 1.4.1 lL-2 gene activation pathways (provided by Douglas F. Larson. PhD. 1998)

22

activate MAP kinase whicti can induce the expression of c-fos and c-jun. Together, c-fos

/c-jun form the AP-1 transcription factor complex. PKC also phosphoryiates and cleaves

IKB from NF-KB. NF-KB then acts as a transcription factor itself IP3 stimulates Ca" ion

mobilization from internal stores and is auemented by influx of external Ca". Ca" binds

with calmodulin (Cm) and with a subunit of calcineurin (CnA). CnA can then act as a

phosphatase to dephosphorylate nuclear factor of activated T-cells (NF.A.T).

Dephosphorylated NF.A.T is then translocated to the nucleus where it interacts in

conjunction with c-fos/c-jun at the .\P-l and NF.\T promoter sequences to induce lL-2

gene expression. Cyclophilin (CYP) is a protein that functions as target for

immunosuppressive therapy in that it binds with the widely used immunosuppressive drug

cyclosporin A (CsA). The CsA/CYP complex binds to and inactivates CnA and therefore

blocks [L-2 gene induction and IL-2 expression.

Of particular interest are the theoretical possibilities where PRL and/or Ly-PLP may

play a modulating role in these [L-2 gene activation pathways. .A.s shown in figure 1.4.1,

multiple steps and pathways are generally accepted as necessary for activation of IL-2 gene

transcription. PRL/Ly-PLP may have at least two sites of action within lymphocytes;

ligand binding to the cell surface PRLr to initiate tyrosine kinase events and also

translocation to the nucleus where it is associated with late gene expression of a yet

unknown mechanism (Prystowsky and Clevenger. 1994). These two possible PRL

mechanisms for modulation of IL-2 gene activation and cell proliferation are shown in

figure 1.4.2 with Ly-PLP as the ligand. Ly-PLP is expressed by the endoplasmic reticulum

and secreted into the extracellular space where it can act in an autocrine manner on the

23

'' \

' \

CA^

PLCy

Ca' •AG

Cm IX Ly-PLP MAP I PKC

I CYP

NFAT ! NFATL

NF-icB Transcnption AP-1 NFAT

IL- 2gene Ly-PLP

Figure 1.4.2 Poteniiai Ly-PLP meciianisms modulating IL-2 gene activation and cell proliferation.

24

PRLr. It has been shown that PRL acts as a ligand with high afflnit\- binding at the level

of the surface membrane PRLr to induce dimerization and activation of which is

associated with both resting and stimulated PRLr (Clevenger and Medaglia. 1994). It has

recently been shown that the PRLr may be directly linked to T-cell activation with the T-

cell receptor (TCRVCD3 complex and that tyrosyl phosphorylation of ZAP-70. a key

signaling peptide intermediate in T-cell activation was strongly induced by 100 ngyml of

PRL in human thymocvies and lymphocytes (Montgomery et al.. 1998). ZAP-70 is known

to be involved in steps leading to phospholipase C action which leads to formation of PKC.

It has also been shown that p59'^" is the kinase that phosphorylates and activates ZAP-7G

(Fusaki et al.. 1996). This ability of PRL and/or Ly-PLP to stimulate PKC is of particular

importance since intracellular PfCC activation is one of the primary* signals required for IL-2

gene activation via the c-fos + c-jun/AP-l and NFAT sites and also the NF-KB pathway to

act on the NF-KB promoter region of the IL-2 gene as described earlier (Isakov et al.. 1987;

Modiano et al., 1991; Male et al.. 1996).

In normal resting lymphocytes PRL or Ly-PLP alone is not sufficient to act as ftill

mitogen and therefore it appears that Ly-PLP may act as co-mitogen necessary for

additional PKC activation. In cells that are in altered hormonal environments or have a

large number of expressed PRLrs in the cell surface membrane it may by possible for PRL

or Ly-PLP to act alone as full mitogen by achieving enough PKC activation to push the

cells over the threshold into an activated state. It has also been shown that PRL activates

nuclear protein kinase C (PKC) in splenocytes and that this activation can be reversed by

treatment with antibodies against rPRL (Russell et al., 1990).

25

PRL and IL-2 have several intracellar actions or pathways in common. They both

have been shown to share a common signal transducer and activator of transcription.

STAT5 (Gilmour et al.. 1995). STAT 5 was originally identified as a mammary gland

factor induced by prolactin in lactating breast cells. PRL also reverses the

immunosuppression induced by CsA that is known to inhibit IL-2 production at the level

of CnA and to compete with PRL for binding to the PRL receptor on T-Iymphocytes

(Hiestand et al.. 1986). Glucoconicoids are known to inhibit IL-2 production at the .AP-l

promoter (Boumpas et al.. 1991). and PRL reverses the suppressed lymphocyte

proliferation induced by hydrocortisone administration (Bemton et al.. 1989).)

Separate from the above stimulatory events. PRL is often negatively linked to cyclic

AMP (c.AMP) production (Buckley et al.. 1988). Inhibition of cAMP may be supportive

of IL-2 production since it has been shown that increases in cAMP levels inhibit IL-2

production but not IL-4 (Novak and Rothenberg, 1990). This suggests that cAMP levels

in cells may be involved in the switch from THl to TH2 subset production of cytokines.

Separate from the above membrane receptor events, Clevenger et al. (1991) have

shown a requirement for PRL to be transported to the nucleus in murine cytotoxic T-cells

driven by IL-2. Also, nuclear PRL appears to be essential for IL-2 to stimulate murine T-

cell proliferation (Clevenger et al., 1990. Clevenger et al., 1991; Clevenger et al.. 1992;

Prystowsky and Clevenger. 1994). In contrast to the work in this dissertation, Clevenger"s

studies were not conducted in CD4. IL-2 producing cells, but were in clonally expanded

murine cytoto.xic T-cells driven by IL-2 to proliferate. Clevenger also has utilized the NB2

immature rat pre-T lymphocyte cell line, which is dependent on PRL for proliferation. In

26

his NB2 studies with transfected genes producing various types of PRL. he has shown that

wild-t\-pe PRL produced by these cells acted alone as a mitogen at the surface membrane

receptors. Its action could be inhibited by extracellular antibodies to PRL which suggested

that intemally produced PRL was secreted and then inactivated. His work also showed that

at the level of the NB2 nucleus, before PRL was able to activate the cells to proliferate, a

preliminary cell activation step at the level of the cell membrane was required, such as

stimulation by extracellular IL-2. a weak mitogen in NB2 cells. This is in agreement with

Davis and Linzer. (1988) who have shown that NB2 cells proliferate in response to PRL

action with cell membrane receptors, but not with intracellular PRL alone. In contrast to

Clevenger's work, it has been reported by Perrot-Applant et al. (1991) that PRL and PRLr

were not translocated to the nucleus of PRLr transfected COS cells.

These potential intracellular mechanisms for PRL modulation of IL-2 e.xpression

are yet undefined and only a few published studies suggest a direct role for PRL in the

regulation of IL-2 production. This limited number of studies has all been conducted with

rats. No published human and murine studies have shown a direct correlation between PRL

and IL-2 production. PRL has been shown to increase IL-2 production and IL-2 receptor

alpha (IL2ra) numbers in splenic lymphocytes of ovariectomized female rats, but not in

normal male rats (Mukeijee et al., 1990; Viselli et al.. 1991). Pituitary adenoma cell

transplants in rats producing PRL and GH increase the number of T^ cells, and enhance

both cell proliferation and IL-2 production (Kelley et al.. 1986). Later studies by this group

using GH alone failed to change IL-2 production, suggesting the possibility that either PRL

or hormones from the thymus enhanced IL-2 production (Davila et al.. 1987). One reason

27

for this inconsistent or lack of GH response may be that there are only a low number of

specific GH receptors on human T-cells which decreases the amount of stimulation

initiated by ligand^receptor events (Badolato et el.. 1994). Many previously reported GHr

studies did not account for the cross reactivity of GH with PRLr on immunocompetent cells

(Gala. 1995).

In chapter 2 and 3 of this dissertation it is shown that addition of an anti-prolactin

polyclonal antibody to stimulated rat splenocytes inhibited both cell proliferation and IL-2

production in vitro (Vander Hamm et al., 1992).

1.5 Clinical Applications

Hyperprolactinemia associated with pituitary tumors has historically been the main

pathological condition generally accepted to be directly associated with prolactin.

However, recent studies in autoimmune disorders, cancer, and organ transplant rejection

have raised several potential areas for research and development of new therapeutic

approaches involving modulation of pituitary PRL and/or Ly-PLP.

Hyperprolactinemia is a pathological condition in which PRL serum levels may be

elevated by pituitary- adenomas, therapeutic use of dopaminergic antagonists (antipsychotic

medications), infiltration disorders of the hypothalmus or pituitary that interfere with

regulation of PRL secretion by dopamine, hypothyroidism with accompanying increases

in TRH. and the use of oral contraceptives (Kuret and Murad. 1990). In women

hyperprolactinemia can induce galactorrhea, amenorrhea and infertility", and in men.

28

impotence. The most commonly accepted pharmacological treatment for

hyperprolactinemia is with the semisynthetic ergot alkaloid derivative bromocriptine

mesylate (Parlodel e; by Sandoz). Bromocriptine is a selective dopamine receptor agonist

that activates postsynaptic dopamine receptors. It acts as prolactin inhibitory factor at the

level of the tuberoinfiindibular process, with action on the dopamine receptors on the

anterior pituitary lactotrophs. Bromocriptine significantly lowers circulating PRL levels

in both normal individuals and in treatment of most pimitary adenomas.

Several autoimmune diseases have been known to become exacerbated during times

when PRL levels are elevated such as post partum and include but are not limited to;

rheumatoid arthritis, multiple sclerosis, and systemic lupus erythromaiosis (SLE). Wilder.

1995 has provided a thorough review of neuroendocrine-immune system interactions and

autoimmunity. It is well known that women have a much higher prevalence (ratio 9:1) of

SLE than do men. Estrogen and testosterone play opposing roles in these autoimmune

processes and work in balance with corticosteroids and pimitary PRL (Homo-Delarche et

al., 1991). Estrogen, acting through an estrogen response element on the PRL gene,

stimulates pituitary PRL production, which may work in concert to enhance humorally

mediated autoimmune processes (Berczi et al. 1993).

PRL may play a role in the development of T-cell subsets. Elevated serum PRL

levels in humans increases the CD4/CD8 ratio (Koller et al.. 1997; Neidhart, 1997).

Corticosteroids suppress the production of CD4 subset THl cytokines. IL-2 and INFy

(Vacca et al.. 1992) and have negligible inhibitory effects on the production of TH2

cytokines such as IL-4 (Daynes and .^raneo, 1989). During pregnancy, the production of

29

THl cytokines IL-2 and INFy decrease while the TH2 cytokine IL-4 increases.

Subsequently, during the post partum period, the balance of hormone shifts as

corticosteroid, estrogen, and progesterone decrease to subnormal levels and PRL levels rise

along with increases in THl activit>' (Berczi et al., 1993). The balance of these potent

regulators of nuclear transcription is vital to control of hyper and hypo immune fiinction.

The decrease in activitv' of one or more of these hormones may allow the remaining

hormone to act unopposed.

[t has been recently shown that lymphocytes from SLE patients have elevated levels

of Ly-PLP (Gutierrez et al.. 1995; Larrea et al.. 1997). This suggests the possibilit>' of a

non pimitary PRL not either being suppressed or being over expressed, which may be

activating cells in an autocrine/paracrine maimer. Paracrine secretion of Ly-PLP from lL-2

stimulated peripheral blood mononuclear cells has been shown to promote in vitro

lymphokine-activated killer (LAK) cells (Matera et al.. 1996).

The stimulatory action of PRL on LAK cells may be detrimental in organ transplant

rejection and beneficial in antitumor therapy. In heart transplant research use of

bromocriptine in conjunction with cyclosporine. azathioprine and prednisone significantly

decreased rejection episodes during the first two months after transplantation (Carrier et al..

1990). In metastatic renal carcinoma, therapeutic intravenous doses of IL-2. generic name

aldeslukin. (Proleukin© by Chiron) are associated with vascular leak syndrome. Following

the work of Clevenger et al.. (1990 & 1992) that PRL is essential at the nuclear level for

IL-2 driven T-cells to properly fimction. it may be possible that manipulations of the PRL

and/or Ly-PLP environment could enhance IL-2 tumorocidal activity of LAK cells to allow

30

use of lower. less toxic IL-2 doses.

After age tiiirty human T-cell production of IL-2 decreases and IL-4, IL-5. and IL-6

increase, which suggests a progressive increase in the TH2 subset of CD4 cells in

comparison to THl (Wilder. 1995). These age related shifts toward TH2 versus THl may

increase other problems, such as allergy and asthma complications with increased IgE

release (Umetsu and DeKruyff. 1997"). Is it possible that site selective phzirmacologic

manipulation ofPRL and Ly-PLP may modulate THl TH2 subset shifting and cytokine

production? Will such modulation be applicable for management of allergies or acquired

immune deficiency syndrome (AIDS) to enhance T-cell proliferation and fiinction? Can

the effectiveness of vaccines be increased by enhancement of humoral immunit\' through

T cell/ B cell activity?

In summary, clinical medicine has historically focused therapeutic interventions on

decreasing pituitary production of PRL for hyperprolactinemic conditions. The increasing

body of evidence mentioned above would suggest that immune system regulation is finely

tuned and modulated in a positive manner by PRL and Ly-PLP, in balance with other

hormones and steroids. Further study is warranted into the specificity of PRL and Ly-PLP

affinities for the lymphocyte PRLr. the intracellular cascade of events, and also into the

regulatory events controlling Ly-PLP production by both normal and abnormally

ftmctioning lymphocytes.

1.6 Research Models

31

The purpose of the studies in this dissertation was to investigate the possible

mechanisms by which both pituitary PRL and Ly-PLP may exert a tandem

hormone/cytokine regulation of the early events responsible for T-cell activation and

proliferation. We chose the male Sprague Dawley rat as our model over the Fischer 344

or Lewis rats, which both have robust and blunted hypothalamic pituitarv- adrenal axis

responses (Murphy et al.. 1995; Wilder. 1995). Thespontaneously hypertensive rat (SHR)

exhibits moderate immune dysfunction as compared to the Wistar-kyoto (WTCY) and F344

rats (Purcell et al.. 1993). We used our Sprague Dawley rats to compare their normal

splenic T-cell response to that of the NB21 Ic T-cell lymphoma line, which is stimulated

to proliferate by PRL (Tanaka et al., 1980) and also to avoid the confounding cross

reactivity of GH with PRLr(s) noted in primate cells (Fu et al.. 1992).

In the process of studying PRL effects on lymphocytes, it has been difficult, for at

least two possible reasons, to show direct in vitro or in vivo PRL responses by exogenous

administration of PRL without first experimentally inducing hormonal deficiencies. First,

PRL is present in vivo in serum and in vitro in culture medium from sources such as fetal

calf serum where it may react directly with cell surface receptors and/or is taken up. stored

and released (Gala and Shevach, 1994; Giss and Walker. 1985; Clevenger et al.. 1990).

Second, lymphocytes produce their own functional prolactin-like peptides. It has been

shown in several recent studies that rat, murine and human lymphocytes may produce

various prolactin-like proteins (Ly-PLP) with important yet undefined roles in regulating

immune function (Table 1.3.1). One approach to the experimentally confounding factor

of residual PRL is to use antibodies to remove or inactivate extracellular PRL. These

antibodies bind to exogenous PRL that is present in culture medium and Ly-PLP released

from cells (Hartmann et al.. 1989). Binding of these residual and secreted peptides may

inhibit PRL/Ty-PLP interactions with cell surface receptors and also prevent their

endosomal internalization into the cell. .Antibodies to PRL have been shown to inhibit

murine, human, and rat lymphoc\te proliferation in vitro (Montgomery et al., 1987;

Hartmarm et al.. 1989; Vander Hamm et al.. 1992) and [L-2 driven proliferation in murine

cytotoxic T-cells and T^ clones (Clevenger et al.. 1990; Hartmarm et al., 1989; Clevenger

et al., 1990; Clevenger et al.. 1991; Clevenger et al.. 1992).

Previously, authors from this laboratory reported that stimulated murine splenocytes

produced a ftinctional Ly-PLP with an apparent molecular weight of 46 kD (Shah et al..

1991). In chapter 4 of this dissertation it is shown that stimulated rat splenocytes produce

Ly-PLP with apparent molecular weights of 24 and 46 kD with other variant forms.

Further evidence for the production of Ly-PLP has been shown by the presence of mRNA

for Ly-PLP in rat. murine, and human lymphocytes (Table 1.3.1). The detection of the low

quantities of mRNA for PRL in lymphocvtes was difficult with traditional northern blot

methods and also with RT-PCR analysis. Pellegrini et al.(1992) were able to amplify

human lymphocyte PRL mRNA but not that of the rat lymphocvies. Chapter 4 discusses

my attempts to amplify and detect T-cell Ly-PLP mRNA.

The purpose of this dissertation is to test the hypotheses diat T-cells produce a Ly-

PLP and that Ly-PLP may act in an autocrine manner as an essential growth factor or co-

mitogen and that Ly-PLP and/or PRL presence may be necessary for IL-2 production

and/or function. Although not directly tested in the studies in this dissertation I propose

that Ly-PLP and PRL may act at the cell surface PRLr to itself directly initiate or to support

an antigen initiated cascade of events through p59'^" 'ZAP-70 pathways stimulating PKC

activation that is supponive of AP-1. NF-KB. andNFAT promotion of IL-2 gene activation.

Also, that nuclear activity of PRL may be necessary but not sufficient for lymphocytes to

proceed through the proliferative phases of the cell cycle. The work presented in this

dissertation involved in viiro T-cell proliferation smdies (Chapter 2"). interleukin 2 (IL-2)

production smdies (Chapter 3). rat T-cell PRL gene activation and expression of Ly-PLP

protein (Chapter 4).

34

CHAPTER 2. CELL PROLIFERATION

2.1 Introduction

It has been difficult to elucidate the effects of PRL in normal healthy immune cells.

Many earlier studies used systems with altered physiological states, such as

hypophysectomy. ovariectomy, dwarfism, pharmacologic treatment, or antibodies to

remove or inhibit PRL. We propose that the difficult\' found in showing an in vitro

response with administration of exogenous PRL is. at least in part, due to the fact that

lymphocytes produce their own lymphokine PRL-like proteins (Ly-PLP). The lymphocyte

production of Ly-PLP substances and their necessity' for cell proliferation has been well

documented in several reviews (Gala. 1991; Reber. 1993. Wilder. 1995, Murphy et al.,

1995. and Yu-Lee, 1997. see also Table 1.3.1).

In an effort to eliminate several complicating factors firom using mixed lymphocyte

populations, these studies were conducted in an enriched population of rat splenic T-cells.

In this enriched population, I performed similar smdies to the numne mixed splenocyte

PRL antibody studies of Montgomery et al. (1987), Hartmarm et al. (1989), and others who

have suggested the presence of an immunoreactive Ly-PLP. We have also shown that A-

PRL inhibits T-cell proliferation in a PRL-free medium, suggesting that a PRL-like

immunoreactive substance is produced by the T-cells in this PRL-free system that promotes

proliferation.

35

2.2 Methods

2.2.1 Materials

Horse Serum (gelding) was obtained from Crowther Serum Supplier. La Jara. CO.

Nutridoma SP and Tris Base were obtained from Boehringer Mannheim Corp..

Indianapolis. IN. Penicillin, streptomycin. RPMI 1640 and rhuIL-2 were obtained from

Gibco BRL. Gaithersburg. MD. Na pyruvate. 2-mercaptoethanol. concanavalin A (CON

A), polyvinylpyrrolidone, glycine and SigmaFast DAB tablets were obtained from Sigma.

St Louis. MO. Hepes was obtained from Calbiochem. La Jolla. CA. NaHCOjwas obtained

from Fisher. Fair lawn. NJ. Ficoll-Paque, Pharmacia LKB Biotechnology Inc.. Piscataway,

NJ. Nylon wool fiber was obtained from Polysciences. INC.. Warrington. PA. Mouse-anti-

rat CDS antibody were obtained from Serotec distributed by Harlan Bioproducts for

Science. Indianapolis. In. Fluorescein conjugated CD4 and CDS Mouse-anti-rat antibodies

were obtained from Caltag Laboratories, San Francisco. CA. Rats were ordered

throughThe University of Arizona Health Sciences Center Animal Care Facility from

Harlan Sprague Dawley, Inc., Indianapolis, IN. CTLL-2 cells, a murine cytoto.xic cell line

that is dependent on IL-2, were obtained from the American Type Culture Collection,

Rockville, MD. NB211 c cells, a rat lymphoma cell line from estrogenized noble rats, were

kindly provided by Drs. Peter W. Gout and Charles T. Beer, Dept. of Cancer

Endocrinology, Cancer Control Agency of British Columbia, Vancouver. BC, Canada were

used as a bioassay for PRL production (Tanaka et al., 1980). Rabbit anti-rat PRL (IC-4).

monkey anti-rat growth hormone (IC-1), ovine growth hormone (S-12), ovine PRL (17),

36

rat PRL (B-6) were generously provided by National Hormone and Pituitary Program,

Bethesda, MD. X-OMAT AR film and N,N'-Methylene-bisacrylamide were obtained from

Eastman K-odak Co.. Rochester, NY. Electroblot apparatus, Trans-Blot nitrocellulose

membranes. Ammonium persulfate, TEMED, acrylamide and molecular weight standards

were obtained from Bio-Rad, Richmond, CA. SE 600 vertical electrophoresis apparatus

was manufactured by Hoefer Scientific Instruments, San Francisco, CA. Peroxidase-

conjugated goat-anti-rabbit IGG was obtained from Cappel Research Products, Durham,

NC. Blast-HRP Blotting Amplification System, [^H] thymidine and [^^S]-L-methionine

were obtained from New England Nuclear, Boston, MA. Glass fiber filters were obtained

from Schleicher & Schuell, Keene. NH. Brandel Cell Harvester was from Gaithersburg,

MD. Silver Stain-DPC Protocol was from Integrated Separation Systems, Hyde Park, MA.

2.2.2 PRL-Free Medium

Medium is prepared using 2% Horse Serum (gelding) shown to be PRL-free in our

NB211c bioassay, 1% Nutridoma SP (a nutrient supplement for serum free media), I mM

Na pyruvate, 5 nM 2-mercaptoethanol (2ME) (pyruvate and 2ME added as recommended

supplements to Nutridoma), RPMI 1640 , 25 mM Hepes, 23.8 mM NaHC03, 50 U/ml

penicillin and 50 |ig/ml streptomycin.

37

2.2.3 PRL-Free Medium NB21 Ic Characterization

NB21 Ic cells grown in 75 cm" Falcon flasks in PRL-free medium, supplemented

with 0.5 ng/ml oPRL. were used as a bioassay for PRL production (Tanaka et al.. 1980).

The NB21 Ic cells were washed three times with PRL-free media and "stepped-down."

brought to a resting G,y G, phase, by 48 hour culture without the oPRL. The stepped-down

cells are then cultured in 96 well micro titer plates at 1 x 10^ cells/well in 100 ul of PRL-

free medium and 100 |il of conditioned test medium. After 24 hours of culture, at hl^C.

5% C0J^5% air, cells are pulsed with 0.5 jiCi [^H] thymidine per well for 16 additional

hours and harvested by rapid filtration. PRL content was measured as the product of the

test medium dilution multiplied by the comparable DPM value of the oPRL standard curve

for that assay.

2.2.4 T-cell Preparation

Male Sprague-Dawley rats weighing approximately 200 g, housed on 12 hour

light/dark schedules were killed by CO, inhalation in a dry ice chamber at the beginning

of the 12-hour light phase. Their spleens were rapidly removed and splenocytes were

collected by dislodging them from the tissue on a fine wire mesh. Splenocytes were rinsed

from the wire mesh with dilute bovine serum albumin and layered over Ficoll-Paque and

centrifuged for 35 minutes at 400 xg. The cloudy lymphocyte layer above the Ficoll-Paque

was removed and red blood cells were lysed with 20 ml of hypotonic ammonium

38

chloride/phosphate buffer centrifuged for 10 minutes at 400xg. The lymphocyte pellet was

then subjected to two wash cycles of dilution with 20 ml PBS and centrifuged for 10

minutes at 400xg. Adherent monocytes were removed by incubation of the mixed

lymphocytes in plastic flasks at 37°C in 95% air/ 5% CO. tor one hour. The non adherent

T and B cells were then incubated on a nylon wool column at 37°C to remove B cells

(Julius et al.. 1973; Kappler and Marrack. 1977). Negative parming for CDS cells was

conducted using the procedure of Mason and coworkers (Mason et al.. 1987). Fluorescence

Activated Cell Sorting (FACS) (F.A.CStar''-^'^. Becton Dickinson. Braintree. .VIA) is used to

characterize the enriched rat T-cells using mouse anti-rat antibodies to CDS and CD4

conjugated to phycoerythrin (PE) and biotin tagged with streptavidin-fluorescein-

isothiocyanate (FITC) respectively.

2.2.5 Cell Proliferation

T-Cells were cultured (1 x 10^ cells/well) in 96 well Falcon micro titer plates for a

total of 72 hours in 5% CO; and 95% air at 37°C. After 48 hours, cultured cells were

pulsed with 0.5 |iCi [^H] thymidine and then harvested 24 hours later by rapid filtration,

a process of collection onto glass fiber filters. Proliferation was measured by the

incorporation of [^H] thymidine into the DNA of replicating cells. Radioactivit>' was

measured by scintillation spectrophotometry (Beckman Instruments. Fullerton. CA). In

each experiment, cells were routinely stimulated with submaximal 10 ^ig/ml or maximal

15 fJ.g/ml doses of the plant lectin. CON A which binds to and cross links cell surface

39

proteins to initiate an activation cascade in lymphocytes. Changes in proliferation were

measured as an increase or decrease in [^H] thymidine incorporation, a measure of new

DNA synthesis, compared to a standard CON A response for that e.xperiment.

2.2.6 Statistical Methods

Cell proliferation data values were determined by measurement of cell sample

disintegrations per minute (DPM) of [^H] thymidine by scintillation spectrophotometry.

Cell proliferation experiments were conducted using 96 well plates with each treatment

administered to three consecutive wells. The mean DPMs of each three wells were

calculated as the data value for that specific treatment for one e.xperiment. The null

hypothesis that several population means are equal was statistically tested using analysis

of variance (ANOVA). .A.11 experimental groups were independent therefore One-Way

ANOVA was used to determine if any two means were unequal. TTie Bonferroni post hoc

multiple comparisons test was used to test and identify which means were statistically

different between treatment groups. SPSS for windows, release 6.0, (SPSS Inc., Chicago,

IL) sofbvare was used for the above statistical comparisons. Graphical representations of

data are displayed as the mean of a select number of experiments (n=?) with error bars

representing the standard error of the mean (SEM). Probability less than <0.05 was

considered significant.

40

2.3 Results

2.3.1 NB21 Ic Assays

The medium used for T cell proliferation and IL-2 production studies (Chapter 3)

was PRL-free/non-mitogenic in tests with the NB211c cell line that is dependent on

lactogenic hormones for growth. Figure 2.3.1.1 shows the concentration-dependent

proliferation response induced by rPRL and ovine PRL (oPRL) compared to the lack of

stimulation from the culture media alone. Figure 2.3.1.2 shows that A-PRL inhibits the

proliferation induced by rPRL and that the A-PRL induced inhibition can be overcome by

increasing concentrations of rPRL with near full recovery. Together these results showdiat

the culture medium alone does not induce proliferation in a lactogen dependent cell line and

that rPRL and oPRL have similar biological activity in this cell culture system. These

results also show that A-PRL inhibits rPRL stimulation ofNB21 Ic cells, an inhibition that

can be overcome by increasing concentrations of rPRL.

2.3.2 T-cell Preparation

The test cell population consisted of 85% enriched rat mixed T-cell population

(n=7) consisting of 47.5% ± 1.5% T^ (CD4-f-) and 39.3% ± 1.6% (CD8+) cells (Figure

2.3.2.1), and a population of up to 78% CD4+ cells with a single anti-CD8 panning

procedure as determined by flow cytometric analyses.

41

200000

180000

160000

140000

120000

100000

80000

60000

40000

20000 -

0 0 . 0 0 1 0 . 0 1 0 . 1 0.0010.01 0.1

oPRL ng/ml rPRL ng/ml

Figure 2J.1.1 NB21 Ic cell concentration-dependent proliferation response induced by rPRL and ovine PRL (oPRL) and lack of response with to lactogen free media. Representative of several separate experiments of standard oPRJL and rPRL responses (shown n=l experiment of triplicate samples ). Vertical bars show NB21 Ic proliferation as mean DPM and SEM, measured by [^H] thymidine incorporation. Increasing concentrations of rPRL and oPRL are shown left to right in ng/ml.

42

160000

140000

120000

100000 -

S: 80000 -

60000 - A-PRL 1/12000 + rPRL

rPRL

40000 -

20000 -

1000 100 1 0 1 .001 .01 1

rPRL ng/ml

Figure 2.3.1.2 NB211c ceil concentration-dependent proliferation response induced by rPRL and inhibition by A-PRL 1/12.000 (lU/ml in 1000s). Cumulative data from n=3 separate experiments of triplicate samples. Vertical bars show NB21 Ic proliferation as mean DPM and SEM, measured by [^H] thymidine incorporation. Increasing concentrations are shown left to right for rPRL in ng/ml. One way ANOVA P<0.00001 for all samples. Multiple range Bonferroni, P<0.05 rPRL cur%'e concentrations 3, 10, 15. 20 and 100 ng.'ml (*) are all significantly different from corresponding A-PRL - rPRL concentrations. 30 ng/ml concentrations are not significantiy different.

43

Figure 2.3.2.1 Flow cytometric analysis data of harvested rat splenocytes from one experiment, representative of the test cell population of several experiments. Quadrant I shows 31.7% phycoerythrin (PE) tagged CDS cells. Quadrant IV shows 51.8% streptavidin-fluorescein-isothiocyanate (FITC) tagged CD4 cells, and quadrant III shows 14.8% non CD4/CD8 cells. Quadrant II shows 1.7% double tagged CD4/CD8 cells.

2.3.3 T-cell Proliferation

44

The test population of enriched T-cells proliferated in a concentration-dependent

manner in response to increasing concentrations of CON .A., a T-cell specific mitogen. The

most consistent submaximal stimulations were obtained with 10 jig/ml and maximal

stimulation with 15 |j.g'mlof CON A/culture media (Figure 2.3.3.1). Resting cells showed

low levels of increased ['"H] thymidine incorporation at supra physiological concentrations

of oPRL (Figure 2.3.3.2). .addition of increasing concentrations of oPRL to submaximal

concentrations of CON A gave a pattern of [""H] thymidine incorporation similar to that

seen at supra physiological concentrations of oPRL (Figure 2.3.3.3).

A-PRL induced a concentration-dependent inhibition of CON .A. stimulated T-cell

proliferation (Figure 2.3.3.4). The inliibitory effect consistently began at dilutions of

1/16000 (1 part .VPRL per 16,000 parts media) and completely inhibited proliferation at

the higher concentration, lowest dilution, of 1/2000. When added to non stimulated cells

this antibody had no effect on its own except at the highest concentration of 1/2000 where

it slightly inhibited the basal level of thymidine incorporation. The inhibition caused by

A-PRL was not consistently overcome by oPRL in some experiments and required high

non-physiological concentrations (1 |ig to I mg/ml) ofoPElL (Figure 2.3.3.5). These same

high oPRL concentrations only moderately enhanced non-stimulated and CON .A.

stimulated proliferation. Also, it was shown that A-PRL had its greatest inhibitory effect

when available to block the early events of activation and cell proliferation, but a delayed

addition of A-PRL still inhibited some process necessary for cell proliferation (Figure

45

2.3.3.6). In these T-cell population studies, recombinant human IL-2 (rhuIL-2) had only

a minimal effect on proliferation in non-stimulated cells, but produced a concentration-

dependent enhancement of proliferation in cells previously stimulated by CON .A. (data not

shown). The inhibition of proliferation caused by A-PRL can be completely overcome by

the addition of rhuIL-2 (Figure 2.3.3.7).

2.4 Discussion

This chapter presents data from rat splenocyte studies conducted to extend the

murine mi.xed splenocyte PRL antibody smdies of Montgomery et al. (1987). Hartmann et

al. (1989). and others who suggested the presence of an immunoreactive Ly-PLP. In an

effort to examine whether rat T-cells themselves produced the Ly-PLP and to eliminate

several complicating factors from using mixed ceil populations, these studies were

conducted in an enriched population of rat splenic T-cells using PRL free media. These

results are in agreement with the results of Hartmann et al. (1989) in that the studies

presented here have shown that A-PRL inhibits T-cell proliferation in a PRL-free medium,

suggesting that an immunoreactive substance is present in and released by the cells that is

necessary for promotion of cell proliferation.

In these smdies it was shown that exogenous addition of oPRL in normal healthy

male rat splenocytes is only weekly mitogenic and that oPRL only partially overcomes the

inhibition of proliferation induced by A-PRL. It is also shown that over a time course

addition of A-PRL inhibited cell proliferation with the greatest effect in the early hours.

46

specifically time zero and six hours, and had a decreasing effect over the first 48 hours.

This suggests that PRL and/or Ly-PLP are necessary' for early, first few minutes to six

hours. ligand/PRLr tyrosine kinase activation events as described in Chapter 1. section 1.4

and also an undefined late cell cycle progression event. These time course experiments also

demonstrated that A-PRL did not interfere with other test parameters since when added to

the culture media just prior to harvesting, it had no effect on the subsequent harvesting,

spectrophotometry, or IL-2 production bioassays (shown in chapter 3).

The most significant result from these studies was the finding that rhuIL-2

overcomes the A-PRL induced inhibition of cell proliferation. This suggests that intact IL-

2 receptors were present, were coupled to intracellular activation cascades, and that the

inhibition of IL-2 production may be responsible for the reduced cell proliferation induced

by antibodies to PRL/'Ly-PLP.

47

CL Q

160000

140000

120000-

100000-

80000 -

60000 -

40000 -

20000 -

0 10 15

CON A UG/ML 2 0

FIGURE 2.3.3.1 T-Cell proliferation concentration-dependent response to CON A. Accumulated data of n=4 separate experiments of triplicate samples shown. Vertical bars show T-cell proliferation as mean DPMs and SEM. measured by [^H] thymidine incorporation, at 72 hrs after addition of concentrations of CON A. One way ANOVA P<0.00001 for all samples. Multiple range Bonferroni. P<0.05 for all concentrations of CON A (*) compared to non stimulated cells (0 CON A) and for CON .A. 10 (**), P<0.05, compared to 15 and 20 ug/ml.

48

3000

2000 -

Q. Q

1000-

1 0 1 0 0

oPRL ug/ml 1 0 0 0

FIGURE 2.3.3.2 T-Cell proliferation concentration-dependent response to oPElL. Accumulated data n=2 separate experiments of triplicate samples. Vertical bars show T-cell proliferation as mean DPMs and SEM. measured by [^H] diymidine incorporation, at 72 hrs after addition of increasing concentrations of oPRL from left to right in p.g/ml.

49

200000

180000

Q_ 100000-Q

160000

140000

120000-

80000 -

60000 -

40000

20000 -

1 0 0 0 CON A

oPRL ug/ml

FIGURE 2.3.3.3 T-Cell proliferation concentration-dependent response to oPRL in conjunction with CON A 10 ^ig/ml. Accumulated data n=2 separate experiments of triplicate samples. Vertical bars show T-cell proliferation as mean DPMs and SEM. measured by [^H] thymidine incorporation, at 72 hrs after addition of CON A 10 |j.g/ml and increasing concentrations of oPRL left to right in [ig/ml. C+o = CON .A -i- oPRL.

50

160000 -

140000 -

120000-

100000-

Q 80000 -

60000

40000 -

20000 -

1 / 4 0 0 0 1 / 8 0 0 0 1 / 1 6 0 0 0 CON A

A-PRL DILUTIONS

FIGURE 2.3.3.4 A-PRL concentration-dependent inhibition of CON A induced stimulation of T-cell proliferation. Representative of 4 experiments (shown n=2 separate experiments of triplicate samples). Vertical bar #l shows T-ceil proliferation as mean DPM and SEM, measured by [-"H] diymidine incorporation, at 72 hrs after CON A 15 jag/ml stimulation. Shown, left to right, bars #2 through #4. CON A 15 |ig/'ml plus A-PRL (C+.A) concentration-dependent inhibition with increasing concentrations of A-PRL shown as dilunon ratios of A-PRL/media x 1000.

51

200000

180000

g 100000

160000

140000 -

120000-

80000 -

60000

40000 -

20000 -

CON A 4 5

C+A C+A 1 0 . 0 1 0 0 . 0

oPRL ug/ml 1 0 0 0 . 0

FIGURE 2.3.3.5 oPRL overcomes A-PRL inhibition of CON A induced stimulation of T-cell proliferation. Representative of 3 experiments (shown n=l separate experiment of triplicate samples ). Vertical bar #I shows T-cell proliferation as mean DPM and SEM, measured by [^H] thymidine incorporation, at 72 hrs after CON A 15 ug/'ml stimulation. Bar #2 CON A 10 ug/ml plus A-PRL (C+A) 1/8000. Bars ^3-6 show left to right increasing concentrations of oPRL in fig/mi.

52

140000 -

120000 -

100000-

80000 -

60000 -

40000 -

20000 -

0 6 1 2 1 8 2 4 3 6 4 8 6 0 7 2

TIME POINT OF A-PRL ADDITION (HRS)

FIGURE 2.3.3.6 T-cell proliferation time-dependent inhibition by A-PRL of CON A induced stimulation. Accumulated data from n=2 experiments of triplicate samples. Vertical bars show T-cell proliferation as mean DPM and SEM. measured by [^H] thymidine incorporation, at 72 hrs after CON A 15 ug/ml stimulation. Left to right time points show time of addition of A-PRL 1/8000 dilution to CON A 15 ug/'ml stimulated T-cells.

53

Q 80000 i

160000

140000

120000

100000-

60000 -

40000 -

20000 -

CON A 4 5

C+A C+A 1 . 0 1 0 . 0 rhulL-2 U/ML

1 0 0 . 0

FIGURE 2.3.3.7 IL-2 overcomes A-PRL inhibition of CON A induced T-cell proliferation. Representative of 3 separate experiments, data shown (n=l). Vertical bar #1 shows T-cell proliferation, as DPM and SEM measured by [^H] thymidine incorporation, at 72 hrs after CON A 10 |j.g/ml stimulation. Bar #2 (C+A) shows A-PRL 1/8000 concentration induced inhibition of CON A stimulation. Bars #3 through #6 show, left to right, CON A plus A-PRL (C+A) with recovery of proliferation by addition of increasing concentrations of rhuIL-2 shown as dilutions of international units/ml of media.

54

CHAPTER 3. INTERLEUKIN 2 EXPRESSION

3.1 Introduction

The results from the cell proliferation studies described in Chapter 2 suggest that

A-PRL may be inhibiting IL-I production by rat T-cells. The following studies were

designed to address this same IL-2 inhibition hypothesis both at the level of IL-2 mRNA

induction and protein expression.

As noted in Chapter 1, and summarized Table 1.3.1, the only published data

suggesting a role for PRL in IL-2 production has been in rats. Although much study in the

field of immunolog>' is conducted with murine and human systems, rats may prove to be

good tools for the smdy of PRL interactions with IL2 responses for both in vivo and in vitro

primary cell cultiure. Many of the earliest PRL studies of immune regulation were

conducted in rats (Nagy and Berczi, 1978; Nagy et al., 1983). The first published evidence

of PRL RNA presence in lymphocytes was produced in rats (Hiestand et al., 1986). The

presence of PRL receptors on rat lymphocytes further completes the necessary system for

paracrine and autocrine Ly-PRL actions (Russell et al., 1985, Viselli and Mastro, 1993;

Koh and Phillips, 1993). Pimitary adenoma cell transplants in rats, producing PRL and

GH, increase the number of T-cells, and enhance both cell proliferation and IL-2 production

(Kelley et al., 1986). Later studies by this group using GH alone failed to change IL-2

production, suggesting the possibility that either PRL or hormones from die thymus

enhanced IL-2 production (Davila et ai., 1987). Rat PRL and GH do not cross react at the

PRL receptor as they do in humans therefore eliminating one confounding factor (Badolato

55

et al., 1994). PRL has also been shown to increase IL-2 production and IL-2 receptor alpha

(IL2ra) numbers on splenic lymphocytes of ovariectomized rats (Mukherjee et al.. 1990;

Viselli et al.. 1991). We have previously shown in rat T-cells that treatment with a rabbit

anti-rat PRL antibody to inactivate extracellular PRL and Ly-PRL inhibits IL-2 production

(Vander Hamm et al.. 1992). Rat IL-2 has a sequence more homologous to human IL-2

than does murine IL-2 (McKjiight et al.. 1989). Sprague Dawley rats may respond to PRL

in a typical THl pattern of cytokine expression and therefore may be appropriate for cell

mediated immunity and/or autoimmune studies (Renzi et al.. 1996; Hancock et al.. 1995;

Purcell et al., 1993).

Although there has been little supportive evidence in the published literature for a

direct role of PRL in the modulation of IL-2 expression, the data presented in this chapter

suggest that normal mature rat T-cells may by a valuable study tool. These data suggest

that extracellular, Ly-PLP peptide activity was necessary for the induction of IL-2 mRNA

£ind expression of IL-2 protein. It was also shown that IL-2rP mRNA induction was

constitutively expressed and unaffected by A-PRL treatment. These results are supportive

of the hypothesis that PRL or Ly-PLP play a necessary role in modulation of IL-2

production in the rat.

56

3.2 Methods

3.2.1 Collection of mRNA

Enriched T-cell and panned rat splenocytes were obtained as described in Chapter

2 of this dissertation and were stimulated with CON A 15 (ig/ml in PRL-free medium at

a concentration of 5x 10® cells/ml. Rabbit anti-rat PRL antibody IC-4 (A-PRL) was added

for a final concentration of 1:6,000 (U/ml). Cells were collected at 0. 6. 12. 18. and/or 24

hrs, washed twice with PBS, pelleted and stored at -70°C. Frozen cells were warmed in

a 37°C water bath, transferred to microflige tubes and 1 ml TRIzol was added. Cells were

incubated 5 minutes in TRIzol, 200 ul of chloroform added, tubes were shaken and

incubated for 3 minutes at room temperature. TRIzol mixture was centrifiiged at 12.000xg

for 15 minutes at 4°C. The aqueous layer was removed by pipetting and 500 ul of 70%

isopropyl alcohol was added to remaining mixture. The contents were gently mixed by

inversion and incubated at room temperature for 10 minutes. Samples were centrifiiged at

4°C, 12,000xg for 10 minutes. Solvent was decanted with pellet remaining in tube. Pellet

was washed once with 1 ml 70% ethyl alcohol, centrifiiged at room temperature, 7,500xg,

for 5 minutes and air dried. Pellet was dissolved in 100 ul DEPC treated water by heating

in 37°C water bath. Samples were quantitated as a 200x dilution at 260 and 280 nm by

spectrophotometry.

3.2.2 Reverse Transcriptase (RT)

57

Total RNA (1 ugj was mixed with 1.0 ul Oligo dT primer (500 ugymO with distilled

water to make 12 ul. Samples were heated to 70 °C for 10 minutes, quick chilled and 4 ul

of 5x first strand buffer. 2 ul 0.1 M DTT and 1 ul mixed dNTP (1 OmiVI stock) were added.

Samples were vortexed. centrifuged and incubated at 37°C for 2 minutes. Superscript 11

RT. 1 ul (200 Units/ul) was added, samples were centrifuged. incubated consecutively at

37°C for 10 minutes. 42^0 for 50 minutes. 70''C for 15 minutes, with cDN.A. then placed

on ice.

3.2.3 Amplification of cDNA (PCR)

The following were mixed with appropriate amounts of distilled water to make 15.5

ul of reaction components: 0.5 ul dNTP (lOmM). 0.5 ul MgCl (50mM"). 1.0 ul primer

(20iiiVt) (Table 3.2.3.1). and 2.0 ul PCR buffer (lOx). 4 ul (1 ug) cDNA from RT reaction

was added. Mineral oil, 20 ul was gently layered over top of cDNA and reaction mixture.

Samples were placed in thermocycler for 5 minutes at 95 °C to denature. Samples cooled

to 80°C then 0.5 ul (5 Units/ul) Taq DNA Polymerase was added to reaction mixture below

the mineral oil. The reaction was run for 30 - 40 cycles, at 95°C, 1 minute, (optimal

armealing temperature specific to primers 55 or 60°C. see (Table 3.2.3.1) 30 seconds, and

72°C, 30 seconds. Final RT-PCR products were held at 4''C.

58

Table 3.2.3.1

Primer Sequences and PCR Annealing Temperatures

PRIMER SEQUENCE BASE P.-MRS PCR X

IL-2 5TCA ACA GCG CAC CCA CTT CAA G3' 403 60 5'GTT GAG ATG ATG CTT TGA CAG A3'

IL2rP 5'GCC AGC TGC TTC ACC AAC CA3' 224 60 5'GAG AAG AGC AGC AGG TCA TC3'

ILIO 5'GCT CTT ACT GGC TGG AGT GA3" 570 55 5'GAT CTC TGG ATC AGA TTT AG3'

/?actin 5'CAC TGG CAT TGT GAT GGA3' 427 55 5'ACG GAT GTC AAC GTC ACA3'

IL-2 and IL2rP primers by McDiarmid et al. (1994), ILIO primers by Sunet al. (1995), and /?actin primers by Gunes and Mastro (1996). For each set of primers the upper sequence shown is sense and the lower sequence is anti-sense.

3.2.4 Analysis of RT-PCR Products

59

RT-PCR products mixed with loading buffer were separated on a 2% agarose gel

containing ethidium bromide, at 60 volts, with dye front moved approximately 5 cm. Gels

were viewed under UV light, the image was digitalized. and quantified with Molecular

Analyst"" software with subtraction for surrounding background. Quantitati\ e PCR was

not conducted and statements made in this chapter pertaining to potential up-regulation or

down-regulation of mRNA induction are to be taken as visual observation and not

quantitative due to the uncertaintv' of RT-PCR procedures with low levels of mRNA and

the fact that these procedures were carried out beyond the linear PCR amplification range.

Semiquantization of sample bands was calculated by first obtaining the Molecular Analyst""

percentage of intensity of each P actin band as compared to the other P actin bands as a

group. The percentages and total counts were calculated for each set of cDN A bands within

a sample group. To calculate the sample loading quantities in reference to p actin the total

counts of the sample band were divided by the percentage calculated for the corresponding

treatment group P actin band.

3.2.5 lL-2 Bioassavs

CTLL-2 cells are a cytotoxic murine T-cell line that proliferates in response to and

is dependent on lL-2 for growth. CTLL-2 cells grown in PRL-free medium in 75 cm*

F a l c o n f l a s k s w e r e u s e d a s a b i o a s s a y f o r I L - 2 p r o d u c t i o n ( G i l l i s e t a l . . 1 9 7 8 ) . . A f t e r 3 - 4

60

days in culture (initial concentration 1 X lO"* ceils/ml with 10 U/mi rhuIL-2). the cells were

subcultured or washed 2 times and used in a bioassay. For the bioassay. CTLL-2 cells were

subcultured in 96 well micro titer plates at 1x10"* cells/well in 100 fil of PRL-free medium

and 100 |j.1 of conditioned test mediimi. After 20 hours of culture, at 37°C. 5% C02/95%

air. cells were pulsed with 0.5 ^iCi [^H] thymidine per well for 4 additional hours and

harvested by rapid filtration. IL-2 content is measured as the product of the test medium

dilution multiplied by the comparable value of the lL-2 standard curve for that assay.

3.3 Results

3.3.1 IL-2 and lL2r6 mRNA Expression

RT-PCR amplification of mRNA harvested ft-om T-cells at 12 hours after

stimulation with CON A 15 ug/ml showed significant induction of lL-2 mRNA

demonstrated by a 403 base pair band and a 224 base pair IL2rP band separated on a 2%

agarose gel. Resting non stimulated T-cells harvested at 12 hrs did not induce any

detectable IL-2 mRNA but did show the most intense [L2rP band. Cells treated at the onset

of CON A stimulation with A-PRL (1 ;6000 dilution) and harvested at 12 hrs. demonstrated

a total inhibition of IL-2 mRNA induction by a lack of detectable IL-2 mRNA (Figure

3.3.1.1). This same treatment group of cells did however show a possibly up regulated

induction of a band for IL2rP mRNA. RT-PCR reactions and analysis of same three

treatment groups of one experimental set (n=l) were conducted in two separate RT-PCR

analyses for the cell population analyzed to be 57.7% CD4 / 27.7% CDS cells (Figure

61

3.3.1.1). Additional studies on separate cells (67.3% CD4 ' 17.9% CDS) treated for 24

hours demonstrated no IL-2 mRNA for non stimulated cells with significant induction at

24 hrs (Figure 3.3.1.2). These studies together both showed the opposite results for IL2rP

as compared to IL-2, as lL-2 message increased in stimulated cells at both 12 and 24 hours

the lL2rP mRNA decreased. n=2 (Figure 3.3.1.2). A separate set of cells analyzed at for

6. 12. 18. and 24 hours after stimulation showed that IL-2 message was not detected until

the 18 and 24 hour time points even though this same set of cells produced detectable

quantities on IL-2 peptide in the culture mediimi. detected by CTLL-2 bioassay as early as

12 hours (Figure 3.3.1.3).

3.3.2 IL-10 mRNA Expression

RT-PCR for IL-10 showed mRNA present as a 570 base pair band in 24 hr

stimulated cells. n=2 (Figure 3.3.1.2 and Figure 3.3.1.3). No IL-10 mRNA was detected

in resting cells or in 12 hour stimulated cells, n=3. Studies for rat interleukin 4 (IL-4), and

IFNy mRNA using sense and anti-sense primer sequences published by Sun et al. (Sun et

al.. 1995) did not produce any detectable cDNA product in our studies.

62

N S SA N S SA N S SA

B-ACT[N IL-2 IL2r

FIGURE 3.3.1.1 A-PRL inhibition of IL-2 niRNA expression shown by RT-PCR cDNA products fractionated in 2 % agarose of T-ceils harvested at 12 hrs. Lanes are Non stimulated (N) cells, mitogen stimulated (S) cells with CON A 15 |ig/ml. and CON A stimulated plus A-PRL 1:6000 treatment (SAP). Marker standards (STD) are Llambda DNA cut with PSTI. /?actin bands are 427 bp. the IL-2 band of403 bp is present in S lane only, and IL-2rP bands are 224 bp.

63

N S N S N S N S

IL-10 IL2r DL-2 B-ACTIN

FIGURE 3.3.1.2 T-cell mRNA expression at 24 hrs stimulation shown by RT-PCR cDNA products fractionated in 2 % agarose. Lanes are Non stimulated (N) cells and mitogen stimulated (S) cells with CON A 15 jag/ml. Marker standards (STD) in outside lanes are Llambda DNA cut with PSTl. Shown from left to right; the IL-10 band is 570 bp in S lane only. IL-2rP bands are 224 bp. the IL-2 band of 403 bp is present in S lane only, and /? actin bands are 427 bp.

64

6 12 18 24 6 12 18 24 6 12 18 24

STD B ACTIN IL-2 IL-10 STD

TIME COURSE (hours)

FIGURE 3.3.1.3 T-ceil mRNA expression time course in CON A 15 ^ig/mi stimulated cells. RT-PCR cDNA products are fractionated on 2% agarose gels. Lanes show time course smdy samples collected at 6,12.18 and 24 hours after mitogen stimulation. Shown from left to right; (3 actin bands are 427 bp, IL-2 bands present in lane 18 and 24 are 403 bp . and the IL-10 band present in lane 24 only is 570bp. Lambda PST markers show base pair size migration patterns in outside lanes.

3.3.3 IL-2 Expression

65

The CTLL-2 cells gave a consistent concentration-dependent increase in

proliferation in response to rhuIL2 {Figure 3.3.3.1). The population of rat T,, cells when

cultiu"ed at 4x 10' cells/ml produces lL-2 in response to CON .A in atime and concentration-

dependent manner with detectable levels in the media begirming at 24 hours peaking at 48

to 60 hrs after CON .A. stimulation (Figure 3.3.3.2 and Figure 3.3.3.3). Cultured at higher

concentrations of 1 .x 10" cells/ml. we measured IL-2 within 12 hours (data not shown). At

5 X lO" cells/ml we measured IL-2 within 3 hours with quantities exceeding 100 U/ml

within 12 hours (data not shown). In our CTLL-2 bioassay, .A.-PRL exerts a concentration-

dependent inhibition of IL-2 presence in media from cultured T,^, 4 x 10' cells/ml (Figm-e

3.3.3.4). Exogenous addition of non-physiologically high doses, up to 1 mg/ml of oPRL

moderately increased IL-2 release from CON A stimulated cells (data not shown),

correlating with our T-cell proliferation results. .AJI antibody to rat GH failed to produce

a consistent IL-2 response from dilutions of 1/16,000 up to 1/2,000. The A-PRL induced

inhibition of IL-2 presence in media can be reversed by preincubation of the antibody with

O.I - 10.0 ng/ml concentrations of r-PRL (Figure 3.3.3.5). A-PRL alone did not affect the

IL-2 driven [^H] thymidine incorporation in CTLL-2 cells, except at concentrations higher

than those used in our bioassay (Figure 3.3.3.6). When characterized in NB21 Ic cells, low

dose rat PRL induced proliferation was completely inhibited by A-PRL at a dilution of

1/12.000 (see chapter 2. Figure 2.3.1.2). This inhibition was consistently overcome at r-

PRL concentrations starting at 20 ng/ml (n=4) (see chapter 2, Figure 2.3.1.2). Also, in

66

rhuIL-2 driven NB211c studies, the A-PRL 1/12,000 had only a slight but consistent

inhibitory effect across all doses of rhuIL-2. similar to that seen with high dose rat PRL

studies. This indicates that this dose of A-PRL did not significantly react with rhuIL-2 (see

Chapter 4. Figure 4.3.4.3). .^.t 1/4.000 A-PRL concentration, the rhuIL-2 response in

NB21 Ic cells was inhibited by approximately 50% (data not shown).

67

80000

60000 -

Q. 40000 -Q

20000 -

3 10 30

rhulL-2 U/ML

Figure 3.3.3.1 CTLL-2 standard proliferation curve in response to rhuIL-2. Combined data from n=4 experiments conducted in triplicate wells is shown. Vertical bars show CTLL-2 response to IL-2 presence in media as mean DPM and SEM of [^H] thymidine incorporation. Increasing rhuIL-2 concentrations are shown left to right as IL-2 units/ml. One way ANOVA P<0.00001 for aJI samples. Multiple range Bonferroni. P<0.05 for concentrations 3. 10. and 30 U/ml (*) as compared to 0 and I U/ml. P<0.05 for concentrations 10 and 30 U/mi as compared to 3 U/ml (**).

68

16000-

14000

12000-

10000-

6000 -

4000

2000 -

Q 8000

2 3 1 0 . 0 1 5 . 0

CON A UG/ML IN T-CELL MEDIA

Figure 3.3.3.2 IL-2 production by Con A stimulated T-ceils measured in CTLL-2 ceils. Combined data from n=3 experiments conducted in triplicate wells is shown. Vertical bars show CTLL-2 response to IL-2 presence in harvested T-cell media as mean DPM and SEM of [^H] thymidine incorporation. Increasing CON A concentrations are shown left to right as |ig/ml presence in T-cell media. One way ANOVA P<0.00001 for all samples. Multiple range Bonferroni. P<0.05 for concentration 10 ng/ml (*) as compared to 15 and 20 ^ig/ml. P<0.05 for concentration 0 fig/ml (**) as compared to all concentrations 10. 15, and 20 |ig/ml.

69

30000

24000 -

18000-

0. Q

12000-

6000-

1 2 1 8 2 4 3 6 4 8 6 0

TIME POINT OF T-CELL MEDIA

COLLECTION (HRS)

Figure 3.3.3.3 IL-2 production over 72 hour time course by Con A stimulated T-cells measured in CTLL-2 cells. Combined data from n=2 experiments conducted in triplicate wells is shown. Vertical bars show CTLL-2 response to IL-2 presence in harvested T-cell media as mean DPM and SEM of [^H] thymidine incorporation. Times of T-cell media harvest are shown left to right as hours after CON A 15 ng/ml stimulation.

70

10000

8000-

CON A C+A C+A C+A 1 / 2 4 0 0 0 1 / 1 2 0 0 0 1 / 6 0 0 0

A-PRL DILUTIONS

Figure 3.3.3.4 IL-2 production inhibition induced by A-PRL in 46 hr stimulated T-cells measured in CTLL-2 cells. Combined data from n=4 experiments conducted in triplicate wells is shown. Vertical bars show CTLL-2 response to IL-2 presence in harvested T-cell media as mean DPM and SEM of [^H] thymidine incorporation. Increasing A-PRL concentrations are shown left to right as dilutions (1 part/mis in 1000s) presence in T-cell media. One way ANOVA P<0.00001 for ail samples. Multiple range Bonferroni. P<0.05 for concentration 1/24000 Bar #2 (**) as compared to Bar #1 (CON A 15 |ig/ml with out A-PRL) and ?r4. P<0.05 for dilution Bar #3 (*) as compared to concentration Bar I; Bar #4 (*) as compared to all concentrations Bars #1 and 2 .

71

10000

8000-

6000-

4000

2000

CON A C+A C+A C+A C+A 0 . 1 1 . 0 1 0 . 0

rPRL UG/ML

Figure 3.3.3.5 rPRL overcomes A-PRL inhibition of IL-2 production in 48 hr. CON A stimulated T-cells. Combined data from n=4 experiments conducted in triplicate wells is shown. Vertical bars show CTLL-2 response to IL-2 presence in harvested T-cell media as mean DPM and SEM of [^H] thymidine incorporation. Bar #1 shows IL-2 production in CON A 15 [ig/ml stimulated T-cells. Bar #2 A-PRL concentration 1/12000 inhibition of CON A (C+A). Bars #3. 4, and 5 show, left to right, recovery of IL-2 production with increasing concentrations of rPRL preincubated with A-PRL prior to addition to T-ceil media, shown as rPRL (ig/ml in T-cell media. One way ANOVA P<G.00001 for all samples. Multiple range Bonferroni. P<0.05 for Bars i¥2.3,and 4(*) as compared to Bar # I. P<0.05 for Bar #5 (**) as compared to Bar ir2.

y->

80000

60000 -

Q_ 40000 Q

20000 -

rh ul L2

1 / 3 2 0 0 0 1 / 2 4 0 0 0 1 / 1 6 0 0 0 A-PRL DILUTIONS

1 / 1 2 0 0 0

Figure 3.3.3.6 .Absence of.VPRL induced inhibition of IL-2 standard CTLL-2 response. Combined data from n=2 e.\periments conducted in triplicate wells is shown. Venical bars show CTLL-2 response to rhuIL-2 1.5 L' ml presence as mean DPM and SEM of ["H] thymidine incorporation. Bars ̂ 2 through =5 (I-.-\) show increasing .A-PRL concentrations left to right as dilutions (1 part/mis in 1000s) presence in CTTL-2 media.

3.4 Discussion

73

I have previously shovm in rat T-cells that treatment with A-PRL inhibits cell

proliferation and [L-2 peptide production in a concentration dependent manner (Vander

Hamm et al., 1992). In this study. A-PRL treatment of the mitogen stimulated IL-2

mRNA producing T-cells totally inhibited detectable induction of lL-2 mRNA. This

inhibition suggests that extracellular, free Ly-PLP acting as an autocrine factor is necessary

for the induction of IL-2 mRNA by rat CD4 cells. This further strengthens my previous IL-

2 peptide inhibition findings. In these present studies resting non stimulated T-cells did not

show any detectable IL-2 mRNA and the mitogen stimulated T-cells did show induction

of IL-2 message as would be expected for a population of T-cells containing a high

percentage of CD4 cells (Taniguchi and Minami. 1993). These cells where also partially

depleted of CDS cells which has been shown to enhance IL-2 production possibly by

lowering the concentration of inhibitory factors produced by CDS cells (Kaempfer. 1994).

In these studies IL-2rP mRNA induction was detectable in all cell samples;

resting/non stimulated, stimulated, and stimulated cells treated with A-PRL. IL-2rP mRNA

was not inhibited by treatment with A-PRL. When IL-2 mRNA was induced, the IL-2rP

mRNA appeared to be decreased when compared to non stimulated cells or mitogen

stimulated/A-PRL treated cells. One confounding factor in our IL2rP results may relate to

the fact that both CD4 and CDS cells express IL2r. Since our cell samples contained both

CD4 and CDS cells, our IL2rP mRNA response is a summation of the responses of this

mixed population. One suggestion for these diverging responses is that the IL-2rP mRNA

74

is constitutively induced and therefore receptor peptides are most likely also expressed and

stored. Upon stimulation of these T-cells it is necessary during the earlier hours for the

cells to express active lL-2r in the surface membrane. These cells may have sufficient IL-2r

proteins available to shift the bulk of the remaining cell resources to induction of IL-2

mRNA. IL-2 peptide production, and IL-2 release.

These studies also provided evidence that under these cell culture conditions. CON

.A. stimulated splenic rat T-cells demonstrate characteristics of the THl subset by induction

of IL-2 mRNA. the prominent TH1 cytokine, with limited and late induction of interleukin

10 (ILIO) mRNA. a TH2 cytokine. These smdies did not detect IL-10 until the 24 hour

time point after mitogen stimulation. IL-10 is known to be inhibitory to the THl subset of

cells and may act as a latent counterbalance to control overstimulation of the THl subset

of T-cells which can lead to autoimmune disease (Umetsu and DeKruff. 1997). I was

unable to detect the presence of IL-4 or IF>JY message under the conditions in these tests.

This lack of e^'idence may be attributed to the lack of detectable mRNA or the lack of

appropriate primer design and/or RT-PCR conditions for these products.

These studies suggest that Ly-PLP may exert a tandem modulatory role with

pituitary PRL in the proliferation of activated TH cells and possibly the regulation of IL-2

actions, including IL-2 production. Both in vivo and in vitro studies with immune system

function indicate the need for pimitary PRL for effective inflammatorv' responses and

lymphocyte proliferation (Nagy et al.. 1983; Berczi et al.. 1991; Hartmann et al., 1989).

There is suggestive evidence firom the literature to implicate a role for pituitary PRL in the

action and/or production of IL-2, a principal lymphokine and growth factor produced by

75

stimulated ceils. First, lympliocv"tes from ovariectomized rats, but not tiiose of estradiol

treated or intact rats, produce IL-2 and iL-2 receptors in response to PRL (Mukherjee et ai..

1990; Viselli et ai., 1991). Second, pituitary adenoma cell transplants in rats, producing

GH and PRL. increase cell numbers, and enhance both cell proliferation and IL-2

production (Kelley et al., 1986). Third. PRL activates nuclear protein kinase C (PKC) in

splenocytes (Russell et al.. 1990) and PKC acti\ation is one of the signals required for IL-2

gene activation (Isakov et al.. 1987: Modiano et al.. 1991). (see also Chapter I. in section

1.4 PRL and Interleukin 2. also Figures 1.4.1 and 1.4.2). Fourth. PRL is often negatively

linked to cyclic AMP (c.AMP) production (Buckley et al.. 1988) and increases in cAMP

levels inhibit IL-2 production (Novak and Rothenberg. 1990). Fifth. PRL reverses the

immunosuppression induced by cyclosporine that is known to inhibit IL-2 production and

to compete with PRL for binding to the PRL receptor on T-lymphocytes (Hiestand et al..

1986). Finally, glucocorticoids are known to inhibit IL-2 production (Boumpas et al..

1991). but PRL reverses the suppressed lymphoc>ie proliferation induced by hydrocortisone

administration (Bemton et al.. 1989). In summary. PRL may play a modulatory role

acting through PKC as a second messenger to support IL-2 production and act also as a

counter regulatory factor against steriod inhibition of IL-2 production/lymphocyte

proliferation.

Preincubation of the A-PRL with r-PRL reverses the inhibition of IL-2 production

and indicates that the antibody causes its inhibitory effects by interacting with an

immunoreactive PRL-like protein or proteins. The fact that |ig/ml concentrations of rPRL

are needed to reverse the effect on IL-2 production, when only ng/ml concentrations

76

overcome this antibody in the NB2llc system, suggests at a minimum that there are

differences between these two cell systems. NB211 c cells are immature pre-T cells that do

not produce their own PRL and they never come to a complete resting stage in the cell

cycle, .\nother difference between these two cell culture systems is the number of PRLr

present which is much higher on NB2 cells than on lymphocytes. (Russell et al.. 1985"). In

our studies, rat GH antibody had no consistent effect on IL-2 production. High

concentrations of oPRL moderately but not significantly increases IL-2 production in CON

stimulated cells and T-cell proliferation in both non stimulated and CON A stimulated

TH cells. The observed lack of effect with low levels of exogenous PRL. may indicate that

stored or lymphokine PRL-like peptide may fulfill basic cellular requirements. With high

doses of exogenous PRL. the effect may be a concentration-dependent gradient-driven

penetration of the cell membrane with a site of action at the nucleus.

Because rhulL-2 overcomes A-PRL's inhibition of proliferation in our system, this

suggests that an intact IL-2 receptor system is present and that the inhibition of proliferation

may be due to a deficient IL-2 production or release. Our results show that A-PRL inhibits

IL-2 production by cells in a concentration-dependent manner. That may be one

mechanism of the concentration and time-dependent inhibitory effect on proliferation.

These findings do not rule out a role for PRL in IL-2 receptor expression suggested by

Mukherjee et al. (1990) and Viselli et al.(1991). IL-2 and IL-2R's are regulated

independently by glucocorticoids (Boumpas et al.. 1991) and cyclosporine (Modiano et al..

1991) and the fact that IL-2 up-regulates the expression of IL-2 receptors clouds the issue

of which is the controlling factor, the IL-2 protein or its receptor (Smith and Cantrell. 1985;

77

Depper et al.. 1985). PRL is translocated to the nucleus and is required by IL-2 driven

murine cells (Clevengeretal.. 1990: Clevenger et al.. 1991). This nuclear requirement in

IL-2 driven cells may also by a major contributing factor in our results within IL-2

producing cells. The inhibitory effect shown by A-PRL in our studies may be the result of

several different mechanisms, singly or in combination. First, the removal of PRL and Ly-

PLP may directly inhibit early IL-2 peptide production or release by uncoupling

intracellular or nuclear events. The time course studies for cell proliferation and IL-2

production presented here support the necessity for PRL and Ly-PLP in early events since

they show that A-PRL had its statistically significant effects when administered in the first

36 hours after stimulation and the greatest effect when added at the onset (time 0) of

stimulation. Second, the reduction shown in IL-2 production may result from the failure

of IL-2 initially produced after gene activation to act as an autocrine factor in driving

further IL-2 production and IL-2 receptor responses as shown in IL-2 driven cells. Third,

A-PRL may directly inhibit cell membrane high affinity [L-2r e.xpression or IL-2r response

to IL-2. This IL-2/IL2r interaction could possibly be studied further by developing

experiments using a knock out of the PRLr. In summary, it appears that PRL and/or Ly-

PLP are/is essential for the early activation of events leading to IL-2 gene activation in rat

T-cells and that the involvement of the IL2r in this activation PRL modulated cascade

requires further study.

The purpose of these studies was to investigate rat T-cell gene activation for

production of Ly-PLP and possible mechanisms by which Ly-PLP may exert a tandem

hormone/cytokine regulation of the early events responsible for IL-2 production. The

78

Sprague Dawley rat serves as a good model for study of in vivo and in vitro PRL/IL-2

interactions within the immune system. In these cell culture studies, the isolated T-cells

from ±e Sprague Dawley rats produce Ly-PLP. These cells appear to preferentially express

or activate the THl subset of CD4 cells to induce IL-2 mRNA and produce IL-2 peptide.

These data suggest that under these cell culture and mitogen activation conditions. Ly-PLP

may play a necessary cofactor role in the activation of CD4 T-cells through enhancement

of induction of IL-2 mRNA. These data also suggest that Ly-PLP presence was not

necessary for induction of IL-2rp mRNA.

79

CHAPTER 4. LYMPHOCYTE PROLACTIN LIKE PROTEIN EXPRESSION

4.1 Introduction

Data from the earlier chapters in this dissertation suggested the possible presence

of Ly-PLP. The purpose of the studies in this chapter was to investigate rat T-cell PRL

gene activation for production of PRL mRNA and Ly-PRL. In these studies we provide

suggestive but not detinitive in vitro evidence of PRL mRNA induction in young adult

male Sprague Dawley rat T-lymphocytes. Previous woric in this laboratory showed that a

46 kD Ly-PLP is synthesized and secreted by murine lymphocytes at 72 hrs stimulation

(Shah et al., 1991) and similar sized PRL-like proteins have been shown in murine cell

lymphocytes by several others Chapter 1. Table 1.3.1. In this chapter. I present evidence

for an immunoreactive Ly-PLP of approximately 46 kD produced by rat T-cells after 24

hours stimulation with CON A. Also shown is evidence of the possible presence of

additional 24. 29.5. 43, and 66 kD Ly-PLPs. It is suggested from these data that

immunoreactive PRL-like substances are produced by rat T-cells.

4.2 Methods

4.2.1 Dot Blot .Ajialvses

mRNA was isolated from T-cells collected, treated and stored at -70''C as shown

in chapter 2 with MiniRiboSep (Collaborative Biosciences. Becton Dickenson) Oligo dT

cellulose columns. A rat PRL probe described in (Mauer. 1980) was obtained from Dr.

80

Mauer. University of Iowa, grown in E coli DH5a and plasmid BR322. Dot Blots where

developed with chemiluminescent detection (PolarPlex™. Millipore. Burlington. MA.) on

X-OMAT AR film (Eastman Kodak Co.. Rochester. NY). GH3 cells, a rat pituitary

adenoma cell line (American Type Culture Collection (ATCC). Rockville. MD). were used

as a positive control for pituitary PRL mRNA expression.

4.2.2 RT-PCR Analyses

Total RNA was isolated from T-cells collected, treated and stored at -70°C as shown

in chapter 2 using TRIzol (GibcoBRL) according to manufacturer's instructions. The final

pellets were resuspended in water and the concentration was determined by UV

spectroscopy (O.D. 260/280nm). RNA harvested using MiniRibosep (Collaborative

Biosciences. Becton Dickenson) from GH3 cells, a rat pituitary adenoma cell line (ATCC.

Rockville. MD), was used as a positive control. Reverse transcriptase was done using

superscript™ II (GibcoBRL) according to the manufacture's specifications, using 200 ng

of Random primer mix (hexamers), 500 ng oligo dT (12-18-mer) and 0.9 ug of total T-cell

RNA per reaction. To rule out amplification of genomic DNA, RT reactions were done in

parallel with a preincubation with RMase for I hr at 37°C. PCR reactions were performed

using 2 nl of the RT reaction as template. The reactions consisted of an initial hot start at

95°C and 36 cycles of 95°C for 1 min, 55°C for I min and 72°C for I min followed by a

final incubation at 72°C for 7 min. Products were separated on 1.2% agarose gels and

stained with ethidium bromide. Primers crossing introns were chosen from Jurcovicova

81

et al., (1992) and for their uniqueness (determined by BLAST search at the National Center

for Biotechnology Information). Primers were synthesized by Genosys Biotechnologies.

Inc. (The Woodlands, TX) and GibcoBRL (Life Technologies. Inc.. Gaithersburg, MD), see

Table 4.2.2.1.

4.2.3 PRL Assays

NB2I Ic cells grown in 75 cm- Falcon flasks in PRL-free medium, supplemented

with 0.5 ng/ml oPRL. were used as a bioassay for PRL production (Tanaka et al.. 1980).

The NB211 c cells were washed 3 times with PRL-free media and "stepped-down". brought

to a resting GQ/G, phase, by 48 hour culture without the oPRL. The stepped-down cells

were then cultured in 96 well micro titer plates at 1 .x 10^ cells/well in 100 |il of PRL-free

medium and 100 |il of conditioned test medium. After 24 hours of culture, at 37°C, 5%

COJ95Vo air. ceils were pulsed with 0.5 [iCi ['H] thymidine per well for 16 additional

hours and harvested by rapid filtration. PRL content was measured as the product of the

test medium dilution multiplied by the comparable value of the oPRL standard curve for

that assay.

82

Table 4.2.2.1

Primer Sequence

PRIMER SEQUENCE BASE PAIRS

PRL exl ex4

5'ACC ATG WC AGC CAG GTG TCA GCC3' 5' GC TTC ATG GAT TCC ACC TAG TCC AG3'

405

PRL ex4 ex5

5'GGT GCA CTC CTG G.\A TGA CCC TCT G3' 5' CG GAT GTA CAT GGA ATG .-^LAT GTA GGC

365 t J

PRL ex5 ex5

5'TAT CCT GAA GCC .AAA GGA -A.AT GAGS' 5' TA CAT GGA ATG AAT GTA GGC TTA G3'

213

(3 actin 5'CAC TGG CAT TGT GAT GGA3' 5'ACG GAT GTC ,\AC GTC AC A3'

All

PRL primer sets exon 1 & exon 4. exon 4 & exon 5 were designed by Jurcovicova et al. (1992), PRL exon 5 primers not crossing an intron were designed by Karen Achemzar (unpublished), and /? actin primers by Gunes and Mastro (1996). For each set of primers the upper sequence shown is sense and the lower sequence is anti-sense.

4.2.4 Analysis of Lv-PLP in cell Ivsates

83

Enriched T-cell and panned rat splenocytes were stimulated with CON A 15 |ig/ml

in PRL-free medium at a concentration of 5x10'' cells/ml. Cells were collected at 24 hrs

washed twice with PBS. pelleted and stored at -70°C. Cells were lysed by slow warming

on ice and vigorous vortex in gel-loading buffer, run on 15% SDS-PAGE according to a

modified procedure of (Laemmli. 1970 and Shah et al.. 1989V Rabbit anti-rat PRL

antibody IC-4 (A-PRL) was used at 1;25.000. Primary and secondary antibody exposure

times were 1 hour each. Western blots were amplified and developed with Blast-HRP

biotinyl-tyramide/streptavidin amplification system and SigmaFast DAB tablets and/or total

silver stain of the gel.

Protein synthesis was detected by culttire of enriched rat T-cells stimulated with

CON A 15 M-g/ml in 1 ml PRL-free and methionine-free medium at a concentration of 5 x

lO'' cells/ml in 24 well plates, for 24 hours followed by the addition of ["S] methionine

incorporation (100 fiC/ml) culture for an additional 12 hours. Cells were collected at 36

hrs washed twice with PBS, pelleted and stored at -70°C. Cells were lysed nm on 15%

SDS-P AGE as described above. Gels were dried and exposed to x-ray film.

84

4.3 Results

4.3.1 PRL mRNA Dot Blots

RNA han'ested from resting and mitogen stimulated T-cells; and GH3 pituitary

cells all gave positive evidence of mRNA for PRL (Figure 4.3.1.1).

4.3.2 PRL mRNA RT-PCR

RT-PCR products for the predicted size 213 bp were obtained for the primers

spanning exon 5 only. As shown in Figure 4.3.2.1. these products were absent or greatly

decreased when reactions were done with RNA that had been digested with RNase.

indicating that they arose from RNA and not from contaminating DNA. Primers spanning

exon 1 - exon 4 and exon 4 - exon 5 both produced the appropriate 405 bp and 365 bp

products respectively in the GH3 positive PRL mRNA control cells but did not show any

detectable product from T-cell RNA on a repeatable basis under the conditions used in

these PGR reactions. Evidence of a faint band of 365 bp was possibly detected in one

experiment from stimulated cells that corresponded with the 365 bp in the GH3 control lane

using primers spanning exons 4 and 5 (Figure 4.3.2.2).

RT-PCR amplification of mRNA harvested from T-cells at 12 and 24 hours after

stimulation with CON A 15 ug/ml showed significant induction of a PRL mRNA

demonstrated by a 213 base pair band separated on a 2% agarose gel. Resting non

stimulated T-cells also induce detectable mRNA. Cells treated with both CON A

85

stimulation and A-PEIL (1:6000 dilution) and harvested at 12 hrs also demonstrated a 213

bp band. RT-PCR reactions and analysis were for the same three treatment groups of one

set (57.7% CD4 / 27.7% CDS) cells (Figure 4.3.2.1). Additional studies on a separate set

of cells (67.3% CD4 / 17.9% CDS) stimulated with mitogen for 24 hours demonstrated

PRL mRNA for both non stimulated and stimulated (Figure 4.3.2.1). Studies for rat

prolactin receptor long and short form using sense and anti-sense primer sequences

published by Gunes and Mastro (1996) did not produce any detectable cDNA product in

these studies.

86

A

GH3 (2 ui:

^ non srimulatedf 12 ul)

^ <5» '• stimulated (3 ui. 7 ul) i

mtemai control (1 ul i

: ® ® "• GH3

non stimulated

•

f, stimulated ^5'- •

B

4.3.1.1 Dot blot chemiluminescent detection for PRL mRN.A. n=2. Figures show detection

for both resting, non stimulated and CON .\ 15 ug/ml stimulated rat T-cells with mRN.-\

harvested at 12 hours after stimulation. Figure .A. shows different quantities (ul) loaded on

dot based on calculated mRN.A. concentration in samples. Figure B shows serial dilutions

left to right. GH3 pituitar\' adenoma cells were cultured. mRN.A harvested, and blotted as

positive PRL mRN.A controls.

87

B ACTIN

Figure 4.3.2.1 RT-PCR cDNA products for rat PRL primers spanninu e.xon 5. Upper 2.0 % agarose gel shows left to right cell products for P actin. 599 bp. and PRL. 213 bp. from 12 hr CON .A. !5 jig/ml stimulated (S) and stimulated plus .A-PRL 1.6000 dilution (S.APl. Middle 2 agarose gel shows 213 bp band for 24 hour CON .A non stimulated (N") and s t i m u l a t e d c e l l s ( S V L o w e r 1 . 2 % a g a r o s e g e l s h o w s l e r t t o r i g h t : b p l a d d e r ( 1 " 4 R F DNA-Hae III fragments). RNase lane (R) for same sample in adjacent N lane, non stimulated (N). and stimulated cells (S). RNased upper band over dye front has greatly decreased compared to its corresponding adjacent Ni lane but did not disappear.

88

ONSG5

Figure 4.J.-2.2 RT-PCR cDNA products separated in 1.2 % agarose gel for primers spanning exon 4 and 5 of the PRL gene. Lanes left to right are: bp ladder (4>X174 Rf DNA/Hae III fragments), blank control (0). non stimulated (N). stimulated 24 lir (S). GHJ positive PRL control, and p actin. The P actin shows a dark band at 427 bp. The GH3 lane shows a dark, band at 365 bp. The stimulated (S) lane shows a possible faint band at 365 bp.

4.3.3 Lv-PLP NB21 Ic Bioassav

89

As described in Chapter 2 the NB21 Ic bioassav was used to verify that the cell

culture medium was PRL and lactogen free. NB21 Ic cells did not proliferate in fresh

media and gave consistent and similar concentration-dependent responses to both oPRL and

rPRL fsee Chapter 2. Figure 2.3.1.1). The NB21Ic cells proliferated in response to test

media taken from stimulated T-cells in a mitogen concentration-dependent (Figure 4.3.3.1)

and time dependent (Figure 4.3.3.2) manner which suggests the presence of PRL in the

media produced by T-ceils. However, in these studies the NB21 Ic cells also responded to

rhuIL-2 with and without oPRL (Figure 4.3.3.3). Also, conditioned T-cell media induced

a panem of NB21 Ic proliferation which correlated closely with that of IL-2 production

dose dependent and time course responses shown in CTLL-2 cells.

90

30000

24000 -

18000-

Q. G

120001

6000-

10 15

CON A ug/ml

2 0

Figure 4.3.3.1 NB21 Ic cell concentration-dependent response to T-cell media stimulated with Con A. Cumulative data from n=4 experiments. Vertical bars show NB21Ic proliferation as mean DPM and SEM. measured by [^H] thymidine incorporation. Increasing concentrations of CON A in the T-cell media are shown left to right in ^xg/ml. One way ANOVA P<0.00001 for all samples. Multiple range Bonferroni. P<0.05 non stimulated (0) (**) is significantly different as compared to all concentrations. CON -A. 10 (*) is different than 15 and 20 (ig/ml.

91

30000

24000 -

18000-

Q. G

12000-

6000-

1 2 1 8 2 4 3 6 4 8 6 0

TIME POINT OF T-CELL MEDIA COLLECTION (HRS)

Figure 4.3.3.2 NB211 c cell response to a time course collection of T-cell media stimulated with Con A. Cumulative data from n=2 experiments. Vertical bars show NB211c proliferation as mean DPM and SEM, measured by [^H] thymidine incorporation. Time point of media collection after stimulation is left to right in hours (HRS).

92

rhulL-2 36000 -

30000 -

rhulL-2 + A-PRL 1/12000

24000 -

18000-

12000-

6000

2 5 0 3 0 0 200 5 0 1 5 0 0 1 0 0

rhulL-2 U/ml

Figure 4.3.3.3 NB2I Ic cell response to rhuIL-2 and A-PRL 1/12000 dilution (part/ml 1000s). shown n=l experiment. Vertical bars showNB21 Ic proliferation as mean DPM and SEM, measured by [^H] thymidine incorporation. Increasing concentrations of rhuIL-2 are shown left to right in U/ml. IL-2 concentrations were: 0. 3,10,30,100, and 300 U/ml. Open boxes show rhuIL-2 concentrations alone and solid dots show curve shift to the right by addition of A-PRL 1/12000.

4.3.4 Lv-PLP Western Blot

93

Silver stain of total protein for stimulated and resting T-cells is shown in Figure

4.3.4.1. A predicted band appears at appro.ximately 46 kD. Western blot analyses of T-cell

lysates showed a consistent 46 kD band and possible 24. 29.5, 43 and 66 kD bands in 24

hour CON A stimulated ceils, but less prominent or not at all in non stimulated cell lysates.

under reducing conditions Figure 4.3.4.2. A separate blot revealed a prominent 46 kD band

in mitogen stimulated cells but not resting cells, it also did not show the 24 kD band.

(Figure 4.3.4.3). Electrophoresis of radio labeled proteins in enriched T-cells with ["'S]

methionine shows bands at 46 kD in both stimulated and non stimulated cell lysates (data

not shown).

94

R - P R L N S S T D

'J 15-^.

1^-ISTR-.T-J-'SRI

IE-V F TS*

•RR:.":®

C^-VR I'-'-•• •» '/I R-^ * *«

9 7 , 4 0 0

6 6 , Z O O

4 5 , 0 0 0 31,000

2 1 , 5 0 0

1 4 , 4 0 0

Figure 4.3.4.1 Silver stain of gel with total protein of T-ceil lysate from 48 hr culture. 5 X 10" cells in 2 ml media, left to right lanes: purified rat pituiiarv" PRL control (R-PRL). non-stimulated (N). Con A 15 |j.g/ml stimulated (S"). and prestained standards (STD).

95

JL06.} 000 : SO, 000

49,500 --vj-** V'-^N ^V.'* ^ O]!;'R '

32,500 > ^

27,500 ' " X '̂FI: - • — " • '̂ '-y

X8:.500

»'>«5»>.^S -N •••V»»XV»' •^5 . <V« . ^•J.V.V.\>JV.:. I- ""t

^.•S;:<-;->?;<TV..

S N P G

Figxire 4.3.4.2 Western blot of gel with T-cell lysates (67.3% CD4 and 17.9% CDS) from 24 hr culture, 5x10® cells in 2 ml media, left to right columns: prestained standards (STD), CON A 15 ng/ml stimulated (S), non-stimulated (N), purified rat pituitary PRL control (?) and GH3 cells (G).

96

97,40a

14,400

Figxire 4.3.4.3 Western blot of T-cell lysates (76% CD4) from 24 hr culture, 5x10® cells in 2 ml media, left to right columns: standards (STD), Con A 15 |ig/ml stimulated (S), non-stimulated (N), purified rat pituitary PRL control (r-PRL).

4.4 Discussion

97

It has been difficult to elucidate the effects of PRL in normal healthy immune cells.

Many earlier studies used systems with altered physiological states, such as

hypophysectomy. ovariectomy, dwarfism, pharmacologic treatment, or antibodies to

remove or inhibit PRL. This dissertation proposes that the difficult}' found in showing an

in vitro response with administration of exogenous PRL is. at least in part, due to the fact

that lymphocytes produce their own lymphokine PRL-like proteins (Ly-PLP). The

lymphocyte production of Ly-PLP substances and their necessity for cell proliferation has

been well documented (Gala. 1991; Reber. 1993; Wilder. 1995; Murphy et al.. 1995; Yu-

Lee. 1997).

Previous work in our laboratory using traditional northern and dot blot techniques

and chemiluminescent detection was able to detect dot blot presence of PIU, mRNA but

was unable to detect in northern blots the low level of PRI, mRNA in rat T-cells. In these

studies we were unable to definitively demonstrate PRL mRNA using RT-PCR

amplification. These unpublished dot blot results are in agreement with, Heistand et al

(1986), and Jurcovicova et al (1992), however only the primers for exon 5 of the rat PRL

gene, not crossing an intron. where able to repeatably produce a 213 bp product in T-cells

under the conditions in these studies. . As shown in Figure 4.3.2.1. these products were

absent or greatly decreased when the reaction was done with RN.A that had been digested

with RNase. indicating diat the cDNA products arose fi^om RNA and not firom

contaminating DNA. In one RT-PCR e.xperiment. using primers spanning exons 4 and 5.

98

evidence of a faint band of 365 bp was possibly detected from stimulated cells that

corresponded with the 365 bp in the GH3 control lane (Figure 4.3.2.2'). These dot blot and

RT-PCR data together suggested that PRL mRNA is constitutively expressed in low levels

in resting T-cells and mitogen stimulated T-cells.

All PRL primer sequences where checked with BLAST at the National Center for

Biotechnology Information to verify that their unique sequence corresponds only with the

rat PRL gene. The sense rPRL primer codon sequence shows no homology for the eight

amino acids coded in rat Growth Hormone (rGH) for the corresponding rOH 5'" exon.

Therefore it is unlikely that we were amplifying GH cDNA (Cooke and Baxter. 1982).

As abioassay for Ly-PLP production, I utilized the PRL-dependent NB21 Ic cell in

a manner similar to that reported by Montgomer%' et al.(1987 and 1990) with NB2 cells.

The NB2I Ic cells proliferated in response to test media taken from stimulated T-cells in

a mitogen concentration-dependent manner which suggests the presence of PRL in the

media produced by T-cells. However, in these studies the NB21 Ic cells also responded to

rhuIL-2 with and without oPRL. Also, conditioned T-cell media induced a pattern of

NB2I Ic proliferation which correlated closely with that of IL-2 production concentration-

dependent and time course responses shown in CTLL-2 cells. This indicates that IL-2 may

be masking a PRL response in NB2I Ic cells similar to that reported with rat mixed

lymphocytes by Roza & Kelly (1988). This IL-2 activity decreases the value of the NB2

cells as a bioassay for PRL production when IL-2 production is possible.

Also, in rhulL-2 driven NB2I Ic studies, the A-PRL 1/12.000 had a only slight but

consistent inhibitor\' effect across all doses of rhuIL-2, similar to that seen with high dose

99

rat PRL studies. This indicates that this dilution of A-PRL did not significantly react with

rhuIL-2 at the concentrations used in T-cell studies. At 1/4.000 .A.-PRL concentration, the

rhuIL-2 response in NB21 Ic ceils was inhibited by approximately 50% (data not shown).

This response is also somewhat different than that reported by Pr\'to\vsky & Clevenger

(1994) in wild t>pe NB2 cells, where they demonstrated that lL-2 driven NB2 cell

proliferation was inhibited by the addition of an antibody to PRL.

Previous work in this laboratory showed that a 46 kD Ly-PLP is synthesized and

secreted by murine lymphocytes at 72 hrs stimulation (Shah et al.. 1991) and similar sized

PRL-like proteins have been shown in lymphocytes by several others (see Chapter 1. Table

1.3.1). In this chapter. I present evidence of an immunoreactive Ly-PLP of appro.ximately

46 kD produced by rat CD4 T-cells after 24 hours stimulation. We also show evidence of

the possible presence of additional 24. 29.5. 43. and 66 kD Ly-PLPs. The presentation of

several variants of PRL or Ly-PLP in cell lysates is not at variance with previous evidence

that thirteen structurally related varijints of PLP are produced by female rat pituitar\' cells

ranging from 17 to 97 kD (Shah and Hymer. 1989). Male Sprague-Dawley rats have been

shown to have size and charge heterogeneity of pituitary and plasma prolactin thought to

be due to post-translational modifications, with die predominant size of 24.3 kD and range

from 21.5 to 30.4 kD. (Briski et al.. 1996). The 24 kD band in my studies may be stored

pituitary PRL or a breakdown product of a 46 kD dimer. however increased density of the

bands identified on our stimulated over non stimulated T-cell lysate suggesedt the increased

production of this peptide. These variant sizes of Ly-PLP where identified in our western

blots using a total protein silver stain and separately using as the primary antibody, the

100

same A-PRL that was used to demonstrate inhibition of cell proliferation and inhibition of

IL-2 production.

The evidence presented in this chapter supports the hypothesis of an autocrine

action for Ly-PLP in T-cells via mRNA and protein expression. Pit-1. the transcription

factor that controls expression of PRL has been shown to be expressed in rat splenocvtes.

which adds evidence to the potential scenario for feedback control of Ly-PLP production

with in T-cells (Delhase et al.. 1993). The facts that lymphocytes express Pit-I, Ly- PLP.

and PRLr creates a fairly complete autocrine or paracrine system with the potential to be

feed back regulated. PRL has also been shown to regulate its own receptors in a tissue

specific manner (Kelly et al., 1993). In lymphocytes PRL serum level increases are

negatively linked to PRLr expression (Athreya et al.. 1994; DiCarlo et al.. 1995).

CHAPTER 5. SUMMARY

101

As stated in the introduction, recent total immune function studies in humans, mice,

and rats have suggested an immunomodulatory role for pituitary prolactin (PRL) and the

presence of lymphocyte-produced prolactin-like proteins (Ly-PLP). The purposes of this

dissertation are to summarize the current literature and to describe my studies to investigate

effects of pituitary PRL and Ly-PLP on lymphocyte proliferation and function. Studies

were planned based on a dual hypothesis: First, that a select population of lymphoc\Tes.

T-cells. produce a Ly-PLP that may act in an autocrine maimer as an essential growth factor

or co-mitogen for T-cells. Second, that Ly-PLP and/or PRL presence may be necessar>- for

interleukin 2 (IL-2) production and/or function by CD4 cells.

Over the last two decades there has been an increasing accumulation of research

and clinical information in support of a modulatory role for pituitary PRL in the proper and

improper functioning of the immune system. Clinically, elevated serum PRL levels have

been correlated with both decreased immune function and overstimulation of the immune

system as is found with some autoimmune diseases such as rheumatoid arthritis, multiple

sclerosis, and systemic lupus erythematosus (SLE). In an attempt to understand PRL's role,

the majoritv' of published experimental work has shown that addition of either physiological

or supra physiological concentrations of PRL had no significant effect in healthy animals

or healthy normal lymphocytes. To achieve consistent and repeatable PRL effects under

experimental conditions it has been necessary to alter the normal environment to decrease

PRL levels in vivo with such procedures as hypophysectomy. ovariectomy, bromocriptine

102

treatment, or the use of antibodies against PRL. This use of antibodies led Hartmann et

al.( 1989) to suggest that lymphocytes produce their own PRL like protein. It is now evident

from Chapter 1. Table 1.3.1. that multiple authors have published evidence supporting the

production of Ly-PLP by the lymphocytes themselves. It has been suggested that PRL and

Ly-PLP may be acting by a hormone, paracrine, or autocrine mechanism on lymphocytes.

This theory is fiirther supported by the identification of PRL receptors (PRLrl on

lymphocytes. The PRLrs are found in the greatest numbers on T-cells as compared to

monocytes and B lymphocytes and are up and down regulated by decreasing and increasing

circulating PRL levels and phases of the menstrual cycle. The balance of complicated

multiple hormone and steroid counter-regulatory mechanisms is likely to be at least in part

responsible for homeostasis of properly functioning immune systems. Estrogen stimulates

PRL expression by the pituitary. Estrogen and testosterone have counterbalancing effects

on autoimmune disease incidences. PRL and corticosteroids may have counter-regulatorv'

effects on stimulation and inhibition of T-cell function. .\ny change in the activity of one

of these hormones may allow the others to act unopposed. Further study involving PRL.

Ly-PLP. IL-2. corticosteroids, estrogen and testosterone counter-regulatory mechanisms

may facilitate our understanding of several immune system related malfimctions.

It is important to review at this point how the resiilts reported in this dissertation

may fit into the known physiological activation of T-lymphocytes and IL-2 production by

the CD4 cells in the test population. In normal resting lymphocytes PRL or Ly-PLP alone

is not sufficient to act as full mitogen and therefore it appears that Ly-PLP may act as co-

mitogen. It has been shown that PRL/Ly-PLP may have at least two sites of action within

103

lymphocytes; ligand binding to the cell surface PRLr to initiate t\'rosine kinase events and

also translocation to the nucleus where it is associated with late gene expression of a yet

unknown mechanism {Pr\'stowsky and Clevenger. 1994). These two possible PRL

mechanisms for modulation of IL-2 gene activation and ceil proliferation are shown in

Chapter 1. Figure 1.4.2. with Ly-PLP as the ligand. Ly-PLP is expressed by the

endoplasmic reticulum and secreted into the extracellular space where it can act in an

autocrine manner on the PRLr. It has been shown that PRL acts as a ligand with high

affinity binding at the level of the surface membrane PRLr to induce dimerization and

activation of p59'^" which is associated with both resting and stimulated PRLr (Clevenger

and Medaglia. 1994). It has recently been shown that the PRLr may be (Montgomery' et al..

1998). ZAP-70 is known to be involved in steps leading to phospholipase C action which

leads to formation of PK.C. It has also been shown that p59'^" is the kinase that

phosphorylates and activates ZAP-70 (Fusaki et al.. 1996). This ability of PRL and/or Ly-

PLP to stimulate PKC is of particular importance since intracellular PKC activation is one

of the primary signals required for IL-2 gene activation via the c-fos + c-jun/AP-1 and

NFAT sites and also the NF-icB pathway to act on the NF-KB promoter region of the IL-2

gene as described in Chapter I.

Based on the historical evidence presented above, the effects of exogenous PRL and

the removal of PRL were initially studied in this dissertation using T-cell proliferation as

a measure of immune function. Studies were conducted using normal resting adult

Sprague-Dawley rat T-cells removed from the spleen and enhanced to approximately 87%

T-cells (48% CD4 and 39% CD8). Cell proliferation was measiu^ed by in vitro

104

incorporation of radioactive thymidine into the newly synthesized DNA in cells as a marker

for cellular preparation for cell division and proliferation. A controlled PRL-free

environment die cell culture media was prepared using PRL-free serum albumin from

gelded horses instead of the commonly used bovine serum albumin which contains bovine

PRL. This media was tested to be PRL-free using a rat lymphoma cell line (NB21 Ic) that

proliferates in response to PRL. .Addition characterization studies using the NB21 Ic cells.

PRL-free media, and rPRL showed that the A-PRL. a polyclonal antibody against rPRL.

specifically recognized and inactivated the rPRL in a concentration-dependent manner and

did not inactivate rhuIL-2 in the concentrations used in these assays.

The experimental results of the cell proliferation studies show that the addition of

exogenous oPRL and rhuIL-2 to resting T-cells was not mitogenic. These cells did respond

in a concentration-dependent manner to increasing concentrations of CON A. a T-cell

specific mitogen. The addition of .A.-PRL to cell culture e.xperiments decreased the CON

A induced cell proliferation in a concentration-dependent maimer. This inhibition was in

agreement with the work of Hartman et al., (1989) and others. Due to the fact that this cell

culture system was PRL-free it was hypothesized that the A-PRL was recognizing and

inactivating either; T-cell stored and released pituitary PEU.. or A-PEIL was inactivating Ly-

PLP produced and secreted by the test population of T-cells. The addition of A-PRL over

a 72-hour time course showed the greatest inhibition of cell proliferation occurred when the

A-PRL was available to inactivate PRL and/or Ly-PLP at the earliest time points,

specifically time zero and over the first six hours following mitogen stimulation. The

inhibition was lower, but still significant over the time course from si.x to 48 hours. This

105

suggested that PRL-T-y-PLP presence in the extracellular media is from PRL and/or Ly-

PLP secreted from the cells during the first six hours after stimulation and that it is

necessary to support the early cell activation events. It also suggested that Ly-PLP secreted

over the next 48 hours plays a maintenance role for progression through cell proliferation.

The inhibition of cell proliferation caused by A-PRL was inconsistently reversed by the

addition of oPRL across experiments, but this inhibition was completely overcome by

addition of rhuIL-2. The abilit\' of rhuIL-2 to overcome the inhibition of cell proliferation

suggested that A-PRL treatment may be decreasing IL-2 production by the CD4 (THI )

cells.

Studies were designed to address both IL-2 mRNA induction and IL-2 protein

expression to further investigate whether or not .A.-PRJL treatment did inhibit IL-2

production. Using a mouse cytoto.xic T-cell line (CTLL-2) that is dependent on IL-2 and

proliferates in response to IL-2 as a bioassay. it was shown that A-PRL treatments produced

concentration-dependent decreases in IL-2 release into the T-cell culture media. This A-

PRL induced concentration-dependent inhibition of IL-2 release from the CD4 cells

correlates with the pattern of inhibition ofT-cell proliferation which suggests that inhibition

of IL-2 production may be a primary cause for the corresponding decrease in cell

proliferation. The A-PRL induced inhibition of IL-2 production was overcome by

preincubating the A-PRL with rat pimitary PRL (rPRL) which suggested that rPRL or Ly-

PLP released from the test population of T-cells was a primary target for action of the

antibody. The inhibition of IL-2 production was ftuther confirmed by RT-PCR analysis

which showed that A-PRL ffeatment was able to inhibit IL-2 mRNA induction and

106

expression in CON A stimulated cells without affecting expression of IL2rp mRNA which

was constitutively expressed by the test cells.

The question of Ly-PLP production was suggested by the previous actions

demonstrated by the use of PRL-free media and A-PRL. Several approaches were taken

to directly address the hypothesized production of Ly-PLP. The NB211 c bioassay was used

initially as a test for biological activity of substances released into the conditioned media

from the test population of T-cells. The NB211c cells proliferated in response to

conditioned media from T-cells which were stimulated with increasing concentrations of

CON A and also in a time course manner for harvested conditioned media. This evidence,

on face value, strongly suggested that the T-cell conditioned media contains PRL or a PRL

like substance, both from resting and stimulated cells. The highest levels of NB2Ilc

stimulation were reached at approximately 60 hrs and slightly dropping off by 72 hrs after

CON A stimulation of the T-cells. However, the confounding issue in this NB21 Ic data

is that [L-2 alfo stimulates NB21 Ic cells to proliferate as shown in this work and that

published by other authors. The studies in this dissertation have shown the production of

IL-2. as would be expected by these T-helper (CD4) cells, and also that the time course

production of IL-2 corresponds with the stimulation pattern of the NB21 Ic cells. It does

however appear that the some PRL-like substance was present in the T-cell conditioned

media before 24 hours and after 60 hours diat gave a response which exceeded the IL-2

production pattern detected in the CTLL-2 bioassay. Therefore, it appeared that IL-2

production may be masking and/or combining with any stimulation induced by Ly-PLP

released into die test media. These NB21 Ic studies in Chapters 2 and 4 were valuable in

107

characterizing the PRL-free status of freshly prepared media. These cells were also

valuable in characterizing the specificity of A-PRL with rPRL in a biological cell culture

system. The NB21 Ic data were inconclusive as a specific test for Ly-PLP due to lL-2

production by the test population.

Western blot studies of T-cell lysates produced a consistent band at approximately

46 kD which is in agreement with the published results of other authors as shown in Table

1.3.1. Molecule weights reported in the 40-*- kD range in rats and mice and the approximate

46 kD band in this dissertation are consistent and repeatable. The slight differences in size

may be due to minimal errors in experimental measurements compared to molecular weight

standards or to post translational modifications such as glycosylation. Published studies

for human, mouse, and rat have shown the consistent molecular weight measurements for

the rat. In on Western Blot, a band in the 23-24 kD range shown in this dissertation

corresponded with the rPRL control band and also a band in a GH3 pituitary cell line,

which is known to produce PRL. This band did not appear on all T-cell lysate western

blots and therefore may have been Ly-PLP or circulating pituitary PRL diat was taken up

and stored in these T-cells. It has been reported by Briski et al.. (1996) that in male

Sprague-Dawley rats the predominant size of circulating PRL was 24.3 kD with a range of

21.5 to 30.4 kD.

In this dissertation the Dot Blot analyses for PRL mRNA were suggestive for the

presence of mRNA in both resting and CON A stimulated cells. .Attempts to identify' the

PRL mRNA by Northern analysis were unsuccessful. It is believed that this difficulty was

due to the extremely low quantity of PRL mRNA that may be present in the T-cells. This

108

same difficulty has been reported by other authors, who were later successful in the

identification of mRNA in lymphocytes using RT-PCR analysis, see Table 1.3.1. My

attempts to use RT-PCR to amplify and identify PE?I- mRNA in the test population of rat

T-cells was also suggestive but inconclusive. Under the conditions used in this dissertation

I was unsuccessful in clearly identifying a mRNA product using primers that crossed at

least one or more introns to rule out the amplification of genomic DNA. Initial RT-PCR

work repeatedly identified a 213 bp product that was obtained using primers that did not

cross and intron. These primers spanned a region within E.xon 5 of the PRL gene.

Digestion with RNase decreased the amplitude of this product vvhen compared to the

nondigested band which was suggestive of mRNA verses genomic DNA reverse

transcription and amplification but was not conclusive. Other well published authors

(Pellegrini et al., 1992) have also reported an inability to amplify PRL mRNA from rat

lymphocytes. As noted in Table 1.3.1 only one author, Jurcovicova et al.. (1992). has

reported RT-PCR amplification of rat lymphocyte PRL mRNA. The mRNA results in this

dissertation taken together for Dot Blots and RT-PCR are suggestive of the presence of

mRNA but are inconclusive.

In summary, the initial dual hypotheses set forth in this dissertation have been

confirmed and a summary of the overall data of this dissertation is shown in Table 5.1.

Based on four lines of evidence the in vitro inhibitory activity of A-PRL on cell

proliferation appears to be modulated in part by inhibition of interleukin 2 (IL-2)

production by CD4 cells. First, the A-PRL induced inhibition of cell proliferation was

overcome by addition of rhuIL-2. Second. A-PRL treatment of mitogen stimulated T-ceils

109

was shown by RT-PCR analysis to inhibit IL-2 mRNA expression but not IL-2r5 niRNA

which is constitutively expressed in T-cells. Third. A-PRL inhibited IL-2 protein

production in a concentration dependent manner shown by CTLL-2 bioassays. Fourth, the

A-PRL induced inhibition of IL-2 protein production was overcome by preincubation of

the A-PRL with purified rPRL.

Dot Blot and RT-PCR analysis data in this dissertation were suggestive but were

inconclusive in support of the hypothesis that lymphocytes and specifically T-cells

constitutively express mRNA for PRL and/or Ly-PLP. Western blot studies of cell lysates

suggest that rat T-cells express a Ly-PLP with the apparent molecular weight of 46 kD, as

well as other variants.

In conclusion the data presented in this dissertation and that published by other

authors since the beginning of this work, suggest that rat T-cells constitutively express a

Ly-PLP of approximately 46 kD, with up regulation of expression induced by CON .A

stimulation. This Ly-PLP is necessary but not sufficient for T-cell proliferation and IL-2

production. Inhibition of IL-2 production is at least one likely mechanism by which .VPRL

binding of Ly-PLP inhibits mitogen induced cell proliferation of rat T-cells. The inhibition

of IL-2 production suggests that extracellular stored pituitary PRL or Ly-PLP may be

necessary through a hormone and/or an autocrine mechanism respectively for the induction

of IL-2 mRNA and IL-2 protein expression by activated rat CD4 cells.

Further studies of the possible roles for Ly-PLP and IL-2 interactions may yield

valuable information to our understanding of the pathophysiology of many

neuroendocrine/immune system related diseases and provide information for future

110

modifications to therapeutic approaches for these diseases. The characterization and

sequencing of these Ly-PLP proteins, the mechanisms regulating their expression in

lymphocytes, and identification of their binding characteristics with lymphocyte PRL

receptors will be valuable steps in understanding Ly-PLP's role in immune system

homeostasis.

I l l

Table 5.1 Summary of evidence in this dissertation for Rat T-cell synthesis of Lv-PLP

and its necessity for T-cell proliferation and lL-2 production

TEST RESULTS

FACS Analysis of test cells averages 85% mixed rat CD4 & CDS cells

Medium does not stimulate NB21 Ic cells

A-PRL inhibits CON-A induced cell proliferation which is partially overcome by the addition of o-PRL

Exogenous rhuIL-2 overcomes A-PRL induced inhibition of cell proliferation and rhulL-2 has no effect on resting cells

RT-PCR analysis shows lL-2r5 but no lL-2 mRNA in resting cells, both are present in stimulated T-cells. and analyses show an absence of IL-2 mRNA in A-PRL treated T-cells but no change lL-2r5

Media from CON-A stimulated T-cells treated wdth A-PRL showed concentration-dependent decreased stimulation of CTLL-2 cells

Exogenous rPRL preincubated with A-PRL before treatment of T-cells overcomes A-PRL induced inhibition of test media stimulation of CTLL-2 cells

A-PRL binds to rPRL in Western Blot and inactivates rPRL in NB21 Ic assay

NB21 Ic cells proliferate when exposed to T-cell conditioned media. PRL. and lL-2 individually

SUGGESTED CONCLUSION

Test cells are normal mature rat T-cells

Medium is PRL/lactogen free

A-PRL inactivates cellularly released PRL and/or Ly-PLP which is required for cell proliferation

A-PRL induced inhibition of cell proliferation is caused by a deficiency in lL-2 peptide pro­duction and/or secretion

A-PRL treatment of stimulated T-cells inactivates a PRL like substance that is necessary for IL-2 mRNA induction and expression by rat CD4 lymphocytes

A-PRL treatment of stimulated T-cells inhibited IL-2 production in a concentration-dependent manner

PRL and/or Ly-PLP is required for IL-2 production by CON A stimulated rat CD4 lymphocytes

A-PRL is specific for rPRL and/or Ly-PLP

T-cells may produce a bioactive Ly-PLP but the NB21 Ic stimulation maybe masked by IL-2.

112

Table 5.1 (continued)

Dot Blot with PRL probe for pituitary PRL mRNA shows presence in all populations

RT-PCR analyses for PRL mRNA results were unclear and not repeatable

Western Blots of T-cell lysate show variant forms of peptides identified by A-PRL

Western Blot 46 kD bands from stimulated cells are more intense than non-stimulated bands

A-PEIL inhibition of cell proliferation and IL-2 production is the greatest when present upon initial mitogen activation and within the first six hours, but also has a lower but still significant inhibitorv' effect over the first 48hrs

PRL mRNA present in test cells is constitutively expressed

Suggestive of PRL mRNA presence but not conclusive

T-cells contain variant forms of immuno reactive PRL and/or Ly-PLP

Ly-PRL is constitutively expressed and activated T-cells up regulate production of a 46 kD protein

Ly-PLP autocrine or paracrine action on the T-cells and specifically CD4 cells is necessary but not sufficient for early T-cell activation events. Ly-PLP presence over the first

48 hrs is necessary for on going support of cell proliferation

113

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