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
DMI Number: 9901700
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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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