0013-7227/90/1261-0088102.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Prolactin Induction of Interleukin-2 Receptors on Rat Splenic Lymphocytes* PINKU MUKHERJEE, ANDREA M. MASTRO, AND W. C. HYMER Department of Molecular and Cell Biology, Penn State University, University Park, Pennsylvania 16802
ABSTRACT. A case for the involvement of PRL in the regulation of the immune system is strong. However, no mechanism by which PRL exerts this regulation has yet been identified. We
studied the in vitro effects of PRL on splenocytes from ovariectomized (OVX) rats and discovered that PRL induced the formation of interleukin-2 (IL-2) cell surface receptors. However, PRL did not induce IL-2 secretion. This response, which was dependent on the concentration of PRL, also depended upon
A
LTHOUGH PRL is usually considered to be a reproductive hormone, a case for its involvement in the regulation of the immune system is strong but controversial. For example, 1) PRL receptors are present on normal T- and B-cells, on Nb2 cells (a thymoma cell line), and on monocytes (1, 2); 2) PRL regulates yinterferon production by T-lymphocytes (3); 3) PRL can reverse the lowered antibody response to sheep red blood cells in hypophysectomized or bromocriptine-treated rats (4, 5); 4) cyclosporine, a drug used to produce immunosuppression in organ transplant patients, interferes with PRL binding to its receptors on lymphocytes (1); 5) serum PRL levels increase before rejection episodes, a result which suggests a role in the allograft rejection process (6); 6) a PRL-like molecule is secreted by Concanavalin-A (Con-A)-stimulated lymphocytes (7); 7) neonatal administration of PRL antiserum or bromocriptine alters the development of mouse thymocytes and splenocytes (8); and finally, 8) lymphocytes produce a protein that has homology to PRL and is critical for progression through the cell cycle (9). However, other investigators have shown that 1) cyclosporin-A does not inhibit the binding of PRL to its specific receptors on rabbit mammary gland membranes (10); 2) the inhibitory effect of cyclosporin-A on the growth of Nb2 cells is due to some step other than the Received July 20,1989. Address all correspondence and requests for reprints to: Dr. W. C. Hymer, Department of Molecular and Cell Biology, 401 Althouse Laboratory, Penn State University, University Park, Pennsylvania 16802. * This work was supported by NIH Grants CA-23248 (to W.C.H.) and CA-24385 (to A.M.M.).
the estrogen status of the splenocyte donor; thus, splenocytes from OVX rats or rats in diestrus responded to PRL, whereas those from estrogen-treated OVX rats or rats in estrus did not. We propose that in vivo exposure of PRL, under certain physiological conditions, may prime a pool of splenocytes to express IL-2 cell surface receptors, allowing these cells to be responsive to variations in local concentrations of IL-2. (Endocrinology 126: 88-94,1990)
binding of PRL (11); and finally, 3) serum PRL levels increases before rejection episodes in heart transplant patients, but the data are variable (12). Since the release of PRL from the pituitary is inhibited by dopamine and stimulated by estrogen (E2), and since E2 suppresses immune cell function (13), we reasoned that the study of immune cells in a low E2 environment might shed light on PRL's involvement in the lymphocyte activation process. We have discovered that PRL induces interleukin-2 (IL-2) receptors on the surface of lymphocytes in vitro and that this response is dependent upon the E2 millieu of the animal.
Materials and Methods Animals Fischer 344 female rats (Charles River Laboratories, Wilmington, MA) were ovariectomized (OVX) between 38-42 days of age, and spleens were used 10-15 days later. In some experiments spleens from animals at either random or defined stages of the estrous cycle, from animals injected sc with 17/?-estradiol on day 15 (0.5 fig) and day 16 (50 ^g) after ovariectomy according to the method of Neill (14), or from 50- to 55-dayold intact or 2-week castrated male rats were also used. Animals were housed under 14-h light, 10-h dark lighting conditions, with food ad libitum. Preparation of splenocytes In each experiment the entire spleen from a single animal was removed aseptically and divided into thirds, and each piece was teased apart with two 18-gauge needles. Liberated cells were recovered, centrifuged, and washed three times in RPMI1640 medium supplemented with 5% fetal bovine serum (con-
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PRL INDUCTION OF IL-2 RECEPTORS trol no. 37N9387, Gibco, Grand Island, NY), 0.1% penicillin (10,000 U/ml), streptomycin (10 /ug/ml; Gibco), and 0.01 mM /3-mercaptoethanol (Sigma Chemical Co., St. Louis, MO). Reagents. Rat PRL B-6 (25 IU/mg), rat PRL RP-3 (33 IU/mg), and rat GH B10 (1.8 U/mg) were kindly provided by the NIDDK, NIH. Other reagents were human recombinant IL-2 (Boehringer Mannheim, Indianapolis, IN); Con-A (Sigma); antirat IL-2 receptor (IL-2-R) antibody (CD25, 0X39, clone MCA 273), anti-CD5 receptor antibody (OX19, clone MCA 52F), anti-CD4 (W3125 clone MCA 55F), and anti-CD8 receptor antibody (OX8, clone MCA 48F; Bioproducts for Science, Indianapolis, IN); and fluorescein isothiocyanate-conjugated F(ab)2 antimouse immunoglobulin G (Jackson Immunochem, West Grove, PA). PRL and GH antisera, with cross-reactivities of less than 0.3% to either GH or PRL, respectively (15), were used in validation studies. IL-2-R induction. IL-2-R expression was based on the ability of cells with IL-2-R to proliferate in response to IL-2 (16). Proliferation in the assay indicates the presence of high affinity sites (both a- and /3-chains) of the IL-2-R. Splenocytes (5 X 10s/ml) were incubated in triplicate in Linbro 24-well plates (Flow Laboratories, McLean, VA) containing 1 ml RPMI-1640, 5% fetal bovine serum (FBS), and PRL or Con-A for 18 h at 37 C in a humidified atmosphere of 5% CO2-95% air. Con-Atreated cells were washed twice in RPMI-1640 containing 5% FBS and a-methylmanoside (20 mg/ml) and replated in triplicate (2.5 x 105 cells/ml; 100 Ml/culture) in round-bottom 96well plates (Corning, Corning, NY). These were incubated for an additional 24 h in RPMI-1640 medium containing 5% FBS with or without 100 MI IL-2 (20 U/ml) or medium. [3H]Thymidine (0.5 fiCi/we\\; 6.7 Ci/mM; ICN Radiochemicals, Irvine, CA) was added for the final 4 h of incubation in all experiments. Cells were harvested onto glass fiber filters using a Skaton Multiwell Harvester, washed with H2O, and dried. Radioactivity was measured using an LKB Betaplate scintillation counter (Rockville, MD) (16). Measurement of IL-2-R expression. After exposure to PRL or Con-A for 18 h, splenocytes were washed and stained with a panel of monoclonal antibodies (see above; 1:800 final dilution) for 25 min at 4 C, followed by centrifugation and incubation in fluorescein isothiocyanate-conjugated second antibody for 25 min at 4 C in the dark. The cells were analyzed for the presence of the a-chain of the IL-2-R (TAC) by flow cytometry using green fluorescence intensity (488 mM; 60 mW; Epics V, Coulter Electronics, Hialeah, FL). Anti-TAC antibody binds to both high and low affinity IL-2 receptors. Forward angle light scatter measurements were made using a 1.0 attenuation filter and a linear amplifier setting of 650+50 gated to include all lymphocytes (17). Thirty thousand cells per treatment group were counted. IL-2 secretion assay. To determine if PRL-treated (1 Mg/ml) or Con-A-treated (1 Mg/ml) splenocytes secreted IL-2, 24-h cellfree culture media were serially diluted and added to CTLL2 cells, an IL-2-responsive cell line (American Type Cell Culture, Rockville, MD). The secretion assay was performed exactly as described by Larsson and Couhnho (16). Human recombinant IL-2 served as the positive control.
89
Data analysis. Differences between control and experimental treatment groups were assessed by analysis of variance (Duncan's multiple range test).
Results PRL induction of IL-2-R Splenocytes from OVX rats, incubated in the presence of PRL for 18 h, responded to synthetic IL-2 by dividing (Fig. 1). This response was repeatable (11 experiments), linearly related to PRL concentration, and seen at doses as low as 20 ng/ml (P < 0.05). Similar results were obtained in cells cultured in low serum-containing medium (0.5% FBS) or in rat serum. Cells incubated in the absence of PRL for 18 h and then exposed to medium in 30 n PRL + IL2
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PRL (ugs/ml) FIG. 1. Preincubation of splenocytes from OVX rats with PRL induces proliferation in response to IL-2. Splenocytes incubated for 18 h in PRL-containing medium were washed and cultured for an additional 24 h in medium containing radiolabeled thymidine plus IL-2 (see Materials and Methods). Data represent the mean ± SEM of 11 experiments. Cells preexposed to medium without PRL for 18 h, followed by incubation in [3H] thymidine-containing medium plus IL-2 had incorporation levels of 4594 ± 592 and 3211 ± 245 cpm, respectively. For each of the PRL doses, the response in the presence of IL-2 was statistically different (P < 0.002) from that in its absence.
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PRL INDUCTION OF IL-2 RECEPTORS
90
the presence or absence of IL-2 for 24 h had background [3H]thymidine incorporation levels of 4594 ± 592 cpm and 3211 ± 245 cpm, respectively. When cells were incubated in PRL for 18 h, followed by culture in [3H] thymidine-containing medium devoid of IL-2, incorporation levels were about one third of those achieved in the presence of IL-2. These levels were also related to the PRL dose (Fig. 1). Finally, responses virtually identical to those shown in Fig. 1 were obtained when splenocytes from OVX rats of the Sprague-Dawley strain were tested (not shown). When a highly purified PRL preparation was used (RP-3), the [3H]thymidine incorporation levels were significantly greater than those induced with the B6 PRL (Fig. 2a). In neither case, however, did the incorporation levels reach those achieved with Con-A, a commonly used mitogen which served as a positive control for these experiments. On the average, exposure of splenocytes from OVX rats to Con-A (1 fxg/nl) caused 1.5-fold more [3H]thymidine incorporation than did exposure to PRL (1 ftg/nl). Thymocytes from these same animals did not respond to PRL, but lymph node lymphocytes responded the same as the splenocytes (data not shown). Hormone specificity Preincubation of PRL with PRL antiserum (1:1000 final dilution) for 18 h markedly reduced the subsequent incorporation levels, whereas preincubation with GH antiserum was without effect (Fig. 2b). The small reFlG. 2. a, Comparison of the ability of two rat PRL preparations (RP-3 and B6; 1 fig/m\ for 18 h) to stimulate the incorporation of radiolabeled thymidine in the presence and absence of IL-2. Data represent the average of three experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (us. medium control, b, Neutralization of the PRL response by preincubation of PRL in PRL antiserum (aPRL). PRL (B6; 1 /ug/ml) was incubated with polyclonal antiserum (1:1000 final dilution) to rat PRL or GH (aGH) for 18 h at room temperature. Both antisera have been validated; their crossreactivity is 0.3% (15). The antibodytreated hormones were added to splenocytes, which were assayed for IL-2 responsiveness as described in Fig. 1 and Materials and Methods. Data represent the average of three experiments. Levels of [3H]thymidine incorporation in cells incubated in the absence of IL-2 were: group 1, 9123 ± 1241; group 2, 4232 ± 896; group 3, 8751 ± 1023; group 4, 4612 ± 803 and group 5, 3919 ± 912 cpm.
Endo • 1990 Voll26-Nol
sponse induced by GH (Fig. 2b, column 5) may be attributed to PRL contamination of the GH preparation, since preincubation of the PRL with both PRL and GH antisera completely abolished the response (Fig. 2b, column 4). Time course Although significant levels of incorporation were detected in splenocytes incubated for 10 h with PRL before the addition of IL-2, maximal responses were found in cells incubated for 18-48 h (Fig. 3). The reason for the marked decline in incorporation rates after a 72-h exposure is not known. Cell viability was more than 90% after a 72-h incubation. Flow cytometry The IL-2-induced proliferation response of PRLtreated lymphocytes implied that PRL induced IL-2-R on the cell surface. This was confirmed directly by flow cytometric analysis of PRL-treated lymphocytes stained with an antirat IL-2-R (anti-TAC) antibody. Thus, 34% of the PRL-treated cells (Fig. 4B) and 56% of the ConA-treated cells (Fig. 4C) exhibited specific membrane IL2-R staining. Cultures incubated only with medium contained 9% IL-2 receptor-postive cells. This experiment was repeated once with similar results. When PRL-treated lymphocytes were incubated in antisera to other T-cell surface antigens, CD5 or CD8, the percentages of stained cells above control levels were also increased. In the case of CD5, PRL (1 /ug/ml) in-
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PRL INDUCTION OF IL-2 RECEPTORS
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FIG. 3. Time course of PRL-induced IL-2 responsiveness in splenocytes from OVX rats in vitro. Data represent the average of three experiments. The PRL concentration was 1 Mg/ml. See Materials and Methods for assay details.
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creased the numbers of CD5-positive cells by 8% and 19% in two experiments; CD8 was increased by 3% and 13%. Neither increase was seen at lower doses of PRL. The percentage of CD4-stained cells ranged from 2128% regardless of the PRL concentration, while untreated cells were 22% CD4 positive. These results suggested that the PRL induction of IL-2-R occurs on Tlymphocytes, and possibly on cells of the CD5-positive subclass. IL-2 secretion To determine if PRL induced IL-2 release from splenocytes, CTLL2 cells [an IL-2-responsive cell line (16)] were cultured in media obtained from splenocytes that had been previously incubated in either PRL or Con-A for 18 h. As expected, both the Con-A-conditioned medium and recombinant IL-2 (used as a positive control) stimulated radiolabeled thymidine incorporation; however, the media from the PRL-treated cells did not (Fig. 5). When medium from the PRL-treated cells was mixed with either medium from Con-A-treated cells or recombinant IL-2, the activity was not affected, thus indicating that media from PRL-treated splenocytes does not inhibit the IL-2 assay (data not shown).
LOG INTEGRATED GREEN FLUORESCENCE FIG. 4. Flow cytometric analysis of splenocytes from OVX rats pretreated with PRL in vitro and then stained with antiserum to IL-2 cell surface receptor. A, Cells incubated in medium alone; B, cells incubated in B-6 PRL (1 Mg/ml) for 18 h; C, cells incubated with Con-A (1 ng/ ml) for 18 h. After incubation cells were stained for the presence of IL2 surface receptors using 0X39 antiserum according to the method of Baldwin et al. (17). Thirty thousand cells were counted per treatment group. The area containing IL-2-R-positive cells is outlined by the rectangular box. The percent positive cells is: A, 9%; B, 34%; and C, 56%. This experiment was repeated once with similar results.
Endocrine status of splenocyte donor
Splenocytes from OVX rats injected with sesame oil for 2 days before death showed the usual PRL-induced
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PRL INDUCTION OF IL-2 RECEPTORS
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Serial Dilutions FIG. 5. Effect of prior exposure (18 h) to PRL (1 /ig/ml) or Con-A (1 /ig/ml) on in vitro IL-2 secretion from splenocytes of 0VX rats. Cellfree media were serially diluted and assayed using CTLL2 cells, an IL2-responsive cell line. Human recombinant IL-2 was used as a positive control.
IL-2 response, whereas cells prepared from OVX and estradiol-injected donors did not (column 1 vs. 2, Fig. 6a). Splenocytes prepared from rats in random stages of the estrous cycle yielded variable results that were not significantly different from those of cells incubated in medium alone (column 3, Fig. 6a). Splenocytes prepared from diestrous, but not estrous, animals repeatedly had significant PRL responses (Fig. 6b). Splenocytes from either intact or castrated male rats failed to show the PRL-induced IL-2 response, but these same cell preparations responded to Con-A (Fig. 6c).
Discussion There is a considerable body of data which establishes that PRL participates in the regulation of immune cell function (1-12); our finding, showing that PRL can induce the formation of IL-2-Rs on rat splenocytes, extends this important and interesting area of study. Both IL-2 and its receptor are commonly recognized as being pivotal to proper T-cell-mediated immune response. Although most resting T-cells do not display IL2 surface receptors, exposure to mitogens such as Con-
Endo • 1990 Vol 126-No 1
A, PHA, antibodies, and antigens will induce IL-2-R formation. Agents that bypass portions of signal transduction mechanics, e.g. calcium ionophore, phorbol esters, and diacylglycerol, can also enhance receptor expression. While our data offer little insight into an understanding of the mechanism(s) by which PRL might exert its IL-2 effect on the lymphocyte, there are three observations that are helpful. First, PRL stimulates incorporation of radiolabeled thymidine in the absence of IL-2. While the magnitude of this response is only about one third of that seen when IL-2 is present, direct lymphocyte activation as a result of PRL exposure is clearly possible under our experimental conditions. Since the splenocyte preparation contains B-cells, T-cells, macrophages, and fibroblasts, the possibility that PRL by itself can activate a subpopulation of T-cells is attractive. Indeed, the flow cytometric data tend to support this idea, since the target lymphocytes seem to be primarily T-cells, with the CD8+ T subsets being somewhat increased and CD4+ T-cells being unaffected. Second, PRL induces IL-2-R formation, but clearly does not cause detectable levels of IL-2 secretion. We are unaware of other agents that act in this fashion. We speculate that PRL exposure, under certain physiological conditions, might prime a sizeable pool of splenocytes (~35%) to express IL-2-R to become responsive to variations in the local concentration of IL2. The net result would be that PRL, a native and ubiquitous peptide hormone, could provide a mechanism for the nonantigen-specific expansion of a large population of splenocytes. This expansion might be more efficient than an antigen-directed one, since a specific antigen commonly activates approximately less than 0.01% of the lymphocyte population (18). Third, there is little question that the PRL IL-2-R induction response would have gone undetected had non-OVX animals been used. Our data clearly document that the effect is dependent upon the E2 millieu. In fact, the responsiveness of splenocytes prepared from diestrous, but not estrous, animals offers strong support for the idea that these findings are physiologically relevant. Since it is generally recognized that estrogens are immunosuppressive (13, 19-21), the ratio of estrogen/PRL at the target splenocyte must be critical for IL-2-R induction. At estrus, circulating levels of serum E2 and PRL average 88 pg/ml and 241 ng/ml, whereas at diestrus, these average 17 pg/ml and 41 ng/ ml (22). Con-A is commonly used to induce IL-2-R on lymphocytes in vitro. In fact, several laboratories have used ConA as a means for testing the effects of hormones on the lymphocyte activation process. For example, in vivo administration of glucocorticoids or estrogens will suppress Con-A-activated mitogenesis in vitro (13). Furthermore, PRL is comitogenic with Con-A when tested on spleno-
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PRL INDUCTION OF IL-2 RECEPTORS a)
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FIG. 6. Effect of hormonal status of the splenocyte donor on in vitro responsiveness to PRL. A, In each of three experiments, three OVX rats (columns 1 and 4), three intact random cycle rats (column 3), or three OVX estrogen-treated rats (column 2) served as splenocyte donors. Those animals in column 2 were injected sc with 17/3-estradiol on day 15 (0.5 ng) and day 16 (50 fig) as previously described (14). Splenocytes were assayed for PRL responsiveness; only cells from group 1 were significantly (P < 0.05) different from controls. Cells incubated in PRL, but in the absence of IL-2 had the following levels of incorporation: column 1, 9,612 ± 2,411; column 2, 894 ± 214; column 3, 5,232 ± 3,946; column 4, 2,526 ± 610 cpm. B, In each of four experiments, splenocytes from cycling intact rats prepared at either diestrus or estrus were used in the assay. Diestrous response, P < 0.05 vs. control. Levels of incorporation in the absence of IL-2 were: diestrus, 9,614 ± 2,212; estrus, 3,340 ± 892; and medium control, 1,568 ± 323 cpm. C, In each of three experiments, three OVX, three male, or three castrated male rats served as splenocyte donors. Splenocytes from all three groups of animals gave a significant (P < 0.05) response when Con-A (1 Mg/ml) was used in the assay. However, only the splenocytes from the OVX animals gave a significant (P < 0.05) PRL response. Levels of incorporation in the absence of IL-2 were (from left to right), 12,341 ± 3,112; 14,023 ± 9,216; 12,146 ± 2,142; 8,723 ± 1,422; 3,242 ± 1,021; 3,562 ± 782; 3,814 ± 921; 3,862 ± 867; and 2,916 ± 812 cpm.
cytes and peripheral lymphocytes in vitro (23). In every experiment in our study, we also tested splenocyte that were exposed to Con-A instead of PRL. In all cases a 24h exposure of Con-A was more effective than PRL in promoting [3H]thymidine incorporation. At this time about 50% more lymphocytes expressed IL-2-R after exposure to Con-A than to PRL. However, Con-A also induced significant amounts of IL-2 secretion, while PRL did not. Our data also show that PRL by itself is mitogenic. Others have reported that PRL was comitogenic with Con-A, but was not mitogenic alone in the systems tested to date (24). This discrepancy may be accounted for by the hormonal status of the splenocyte donor. It is known that IL-2-R and IL-2 are induced in stimulated lymphocytes, but are induced independently (25). Our data can be interpreted to mean that PRL primarily activates IL-2-R expression but not IL-2. At this point we do not know the mechanism of action of PRL. It will be interesting to determine if the PRL receptor is in some way associated with the T-cell-receptor complex. In summary, our observations together with those of many others (1-13) confirm the important relationship between the endocrine and immune systems. They also invite further study into relationships among PRL, estrogen, and lymphocytes during development, aging, and tumorigenesis.
Acknowledgments Some of the immunochemical reagents used in this study were kindly
furnished by the NIDDK. We thank Dr. David J. Hurley and Barbara Bour for help with the flow cytometry.
References 1. Russell DH, Matrisian L, Libler R, Larson DF, Poulos B, Magun BE 1984 Prolactin receptors on human lymphocytes and their modulation by cyclosporine. Biochem Biophys Res Commun 121:899 2. Shiu RPC, Elsholtz HP, Tanaka T, Friesen HG, Gout PW, Beer CT, Noble RL 1983 Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology 113:159 3. Bernton EW, Meltzer SM, Holaday JW 1988 Suppression of macrophage activation and T-lymphocyte function in hypoprolactinemic mice. Science 401:000 4. Berczi I, Nagy E, Kovacs K, Horvath E 1981 Regulation of humoral immunity in rats by pituitary hormones. Acta Endocrinol (Copenh) 98:506 5. Gambel PI, Cleland AW, Ferguson FG, 1980 Alterations in thymus and spleen cell populations and immune reactivity during syngenic pregnancy and lactation. J Clin Lab Immunol 3:115 6. Carrier M, Russell DH, Wild JC, Emery RW, Copeland JG 1987 Prolactin as a marker of rejection in human heart transplantation. Transplant Proc 19:3442 7. Montgomery DW, Zukoski CT, Shah GN, Buckley AR, Pacholczyk T Russell DH 1987 Concanavalin A-stimulated murine splenocytes produce a factor with prolactin like bioactivity and immunoactivity. Biochem Biophys Res Commun 145:2692 8. Russell DH, Mills KT, Talamantes FJ, Bern HA 1988 Neonatal administration of prolactin antiserum alters the development pattern of T- and B-lymphocytes in the thymus and spleen of Balb/c female mice. Proc Natl Acad Sci USA 85:7404
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PRL INDUCTION OF IL-2 RECEPTORS
9. Hartmann DP, Holaday JW, Bernton EW 1989 Inhibition of lymphocyte proliferation by antibodies to prolactin. FASEB J 3:2194 10. Mahajan PB, Ebner KE 1985 Cyclosporin A and rabbit mammary prolactin receptors. Biochem Biophys Res Commun 133;753 11. Varma S, Ebner KE 1988 The effect of cyclosporin A on the growth and prolactin binding to Nb-2 rat lymphoma cells. Biochem Biophys Res Commun 156:233 12. Cosson C, Myara I, Guillerman R, Amrein C, Dreyfus G, Moatti N 1989 Serum prolactin as a rejection marker in heart transplantation. Clin Chem 35:492 13. Luster MI, Hayes HT, Korach H, Tucker AN, Dean JH, Greenlee WF, Boorman GA 1984 Estrogen immunosuppresion is regulated through estrogenic responses in the thymus. J Immunol 133:110 14. Neill JD 1972 Sexual differences in the hypothalamic regulation of prolactin secretion. Endocrinology 90:1154 15. Hatfied JM, Hymer WC 1986 Flow cytometric analysis and sorting of live male rat anterior pituitary cell types by forward angle and perpendicular light scatter. Endocrinology 119:2670 16. Larrson EL, Couhnho A 1980 Mechanism of T-cell activation. I. A screening of "step one" ligands. Eur J Immunol 10:93 17. Baldwin CL, Teaole AJ, Naessens JG, Goddeeris BM, MacHugh ND, Morrison WI 1986 Characterization of a subset of bovine Tlymphocytes that express BoT4 by McAb and function: similarity to lymphocytes defined by human T4 and murine L3T4. J Immunol
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136:4385 18. Weiss A 1986 The role of the T3/antigen receptor complex in Tcell activation. Annu Rev Immunol 3:593 19. Smith EM, Blalock JE 1988 A molecular basis for interactions between the immune and neuroendocrine systems. Int J Neurosci 38:455 20. Kalland T, Strand 0, Forsberg JG 1979 Long term effects of neonatal estrogen treatment on mitogen responsiveness of mouse spleen lymphocytes. J Natl Cancer Inst, 63:413 21. Sobhon P, Jirasattham C 1974 Effect of sex hormones on the thymus and lymphoid tissue of ovariectomized rats. Acta Anat 89:211 22. Butcher RL, Collins WE, Fugo NW 1974 Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17/3 throughout the 4-day strus cycle of the rat. Endocrinology 94:1704 23. Montgomery DW, Russell DH, Buckley AR, Zukovski CF 1987 Conconavalin A (Con-A)-stimulated lymphocytes produce a prolactin (PRL)-like mitogen for Nb2 node lymphoma cells. Fed Proc 46:529 (Abstract) 24. Spangelo BL, Hall NR, Goldstein AL, 1985 Evidence that prolactin is an immunomodulatory hormone. In: MacLeod RM, Scapagnini U, Thorner MO (eds) Prolactin, Basic and Clinical Correlates. Springer-Verlag, New York, pp 343-350 25. Crabtree GR 1989 Contingent genetic regulatory events in T lymphocyte activation. Science 243:355
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Vol. 126, No. 1 Printed in U.S.A.
Prolactin Induction of Interleukin-2 Receptors on Rat Splenic Lymphocytes* PINKU MUKHERJEE, ANDREA M. MASTRO, AND W. C. HYMER Department of Molecular and Cell Biology, Penn State University, University Park, Pennsylvania 16802
ABSTRACT. A case for the involvement of PRL in the regulation of the immune system is strong. However, no mechanism by which PRL exerts this regulation has yet been identified. We
studied the in vitro effects of PRL on splenocytes from ovariectomized (OVX) rats and discovered that PRL induced the formation of interleukin-2 (IL-2) cell surface receptors. However, PRL did not induce IL-2 secretion. This response, which was dependent on the concentration of PRL, also depended upon
A
LTHOUGH PRL is usually considered to be a reproductive hormone, a case for its involvement in the regulation of the immune system is strong but controversial. For example, 1) PRL receptors are present on normal T- and B-cells, on Nb2 cells (a thymoma cell line), and on monocytes (1, 2); 2) PRL regulates yinterferon production by T-lymphocytes (3); 3) PRL can reverse the lowered antibody response to sheep red blood cells in hypophysectomized or bromocriptine-treated rats (4, 5); 4) cyclosporine, a drug used to produce immunosuppression in organ transplant patients, interferes with PRL binding to its receptors on lymphocytes (1); 5) serum PRL levels increase before rejection episodes, a result which suggests a role in the allograft rejection process (6); 6) a PRL-like molecule is secreted by Concanavalin-A (Con-A)-stimulated lymphocytes (7); 7) neonatal administration of PRL antiserum or bromocriptine alters the development of mouse thymocytes and splenocytes (8); and finally, 8) lymphocytes produce a protein that has homology to PRL and is critical for progression through the cell cycle (9). However, other investigators have shown that 1) cyclosporin-A does not inhibit the binding of PRL to its specific receptors on rabbit mammary gland membranes (10); 2) the inhibitory effect of cyclosporin-A on the growth of Nb2 cells is due to some step other than the Received July 20,1989. Address all correspondence and requests for reprints to: Dr. W. C. Hymer, Department of Molecular and Cell Biology, 401 Althouse Laboratory, Penn State University, University Park, Pennsylvania 16802. * This work was supported by NIH Grants CA-23248 (to W.C.H.) and CA-24385 (to A.M.M.).
the estrogen status of the splenocyte donor; thus, splenocytes from OVX rats or rats in diestrus responded to PRL, whereas those from estrogen-treated OVX rats or rats in estrus did not. We propose that in vivo exposure of PRL, under certain physiological conditions, may prime a pool of splenocytes to express IL-2 cell surface receptors, allowing these cells to be responsive to variations in local concentrations of IL-2. (Endocrinology 126: 88-94,1990)
binding of PRL (11); and finally, 3) serum PRL levels increases before rejection episodes in heart transplant patients, but the data are variable (12). Since the release of PRL from the pituitary is inhibited by dopamine and stimulated by estrogen (E2), and since E2 suppresses immune cell function (13), we reasoned that the study of immune cells in a low E2 environment might shed light on PRL's involvement in the lymphocyte activation process. We have discovered that PRL induces interleukin-2 (IL-2) receptors on the surface of lymphocytes in vitro and that this response is dependent upon the E2 millieu of the animal.
Materials and Methods Animals Fischer 344 female rats (Charles River Laboratories, Wilmington, MA) were ovariectomized (OVX) between 38-42 days of age, and spleens were used 10-15 days later. In some experiments spleens from animals at either random or defined stages of the estrous cycle, from animals injected sc with 17/?-estradiol on day 15 (0.5 fig) and day 16 (50 ^g) after ovariectomy according to the method of Neill (14), or from 50- to 55-dayold intact or 2-week castrated male rats were also used. Animals were housed under 14-h light, 10-h dark lighting conditions, with food ad libitum. Preparation of splenocytes In each experiment the entire spleen from a single animal was removed aseptically and divided into thirds, and each piece was teased apart with two 18-gauge needles. Liberated cells were recovered, centrifuged, and washed three times in RPMI1640 medium supplemented with 5% fetal bovine serum (con-
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PRL INDUCTION OF IL-2 RECEPTORS trol no. 37N9387, Gibco, Grand Island, NY), 0.1% penicillin (10,000 U/ml), streptomycin (10 /ug/ml; Gibco), and 0.01 mM /3-mercaptoethanol (Sigma Chemical Co., St. Louis, MO). Reagents. Rat PRL B-6 (25 IU/mg), rat PRL RP-3 (33 IU/mg), and rat GH B10 (1.8 U/mg) were kindly provided by the NIDDK, NIH. Other reagents were human recombinant IL-2 (Boehringer Mannheim, Indianapolis, IN); Con-A (Sigma); antirat IL-2 receptor (IL-2-R) antibody (CD25, 0X39, clone MCA 273), anti-CD5 receptor antibody (OX19, clone MCA 52F), anti-CD4 (W3125 clone MCA 55F), and anti-CD8 receptor antibody (OX8, clone MCA 48F; Bioproducts for Science, Indianapolis, IN); and fluorescein isothiocyanate-conjugated F(ab)2 antimouse immunoglobulin G (Jackson Immunochem, West Grove, PA). PRL and GH antisera, with cross-reactivities of less than 0.3% to either GH or PRL, respectively (15), were used in validation studies. IL-2-R induction. IL-2-R expression was based on the ability of cells with IL-2-R to proliferate in response to IL-2 (16). Proliferation in the assay indicates the presence of high affinity sites (both a- and /3-chains) of the IL-2-R. Splenocytes (5 X 10s/ml) were incubated in triplicate in Linbro 24-well plates (Flow Laboratories, McLean, VA) containing 1 ml RPMI-1640, 5% fetal bovine serum (FBS), and PRL or Con-A for 18 h at 37 C in a humidified atmosphere of 5% CO2-95% air. Con-Atreated cells were washed twice in RPMI-1640 containing 5% FBS and a-methylmanoside (20 mg/ml) and replated in triplicate (2.5 x 105 cells/ml; 100 Ml/culture) in round-bottom 96well plates (Corning, Corning, NY). These were incubated for an additional 24 h in RPMI-1640 medium containing 5% FBS with or without 100 MI IL-2 (20 U/ml) or medium. [3H]Thymidine (0.5 fiCi/we\\; 6.7 Ci/mM; ICN Radiochemicals, Irvine, CA) was added for the final 4 h of incubation in all experiments. Cells were harvested onto glass fiber filters using a Skaton Multiwell Harvester, washed with H2O, and dried. Radioactivity was measured using an LKB Betaplate scintillation counter (Rockville, MD) (16). Measurement of IL-2-R expression. After exposure to PRL or Con-A for 18 h, splenocytes were washed and stained with a panel of monoclonal antibodies (see above; 1:800 final dilution) for 25 min at 4 C, followed by centrifugation and incubation in fluorescein isothiocyanate-conjugated second antibody for 25 min at 4 C in the dark. The cells were analyzed for the presence of the a-chain of the IL-2-R (TAC) by flow cytometry using green fluorescence intensity (488 mM; 60 mW; Epics V, Coulter Electronics, Hialeah, FL). Anti-TAC antibody binds to both high and low affinity IL-2 receptors. Forward angle light scatter measurements were made using a 1.0 attenuation filter and a linear amplifier setting of 650+50 gated to include all lymphocytes (17). Thirty thousand cells per treatment group were counted. IL-2 secretion assay. To determine if PRL-treated (1 Mg/ml) or Con-A-treated (1 Mg/ml) splenocytes secreted IL-2, 24-h cellfree culture media were serially diluted and added to CTLL2 cells, an IL-2-responsive cell line (American Type Cell Culture, Rockville, MD). The secretion assay was performed exactly as described by Larsson and Couhnho (16). Human recombinant IL-2 served as the positive control.
89
Data analysis. Differences between control and experimental treatment groups were assessed by analysis of variance (Duncan's multiple range test).
Results PRL induction of IL-2-R Splenocytes from OVX rats, incubated in the presence of PRL for 18 h, responded to synthetic IL-2 by dividing (Fig. 1). This response was repeatable (11 experiments), linearly related to PRL concentration, and seen at doses as low as 20 ng/ml (P < 0.05). Similar results were obtained in cells cultured in low serum-containing medium (0.5% FBS) or in rat serum. Cells incubated in the absence of PRL for 18 h and then exposed to medium in 30 n PRL + IL2
PRL - IL2
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0.005
0.020
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0.312
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PRL (ugs/ml) FIG. 1. Preincubation of splenocytes from OVX rats with PRL induces proliferation in response to IL-2. Splenocytes incubated for 18 h in PRL-containing medium were washed and cultured for an additional 24 h in medium containing radiolabeled thymidine plus IL-2 (see Materials and Methods). Data represent the mean ± SEM of 11 experiments. Cells preexposed to medium without PRL for 18 h, followed by incubation in [3H] thymidine-containing medium plus IL-2 had incorporation levels of 4594 ± 592 and 3211 ± 245 cpm, respectively. For each of the PRL doses, the response in the presence of IL-2 was statistically different (P < 0.002) from that in its absence.
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PRL INDUCTION OF IL-2 RECEPTORS
90
the presence or absence of IL-2 for 24 h had background [3H]thymidine incorporation levels of 4594 ± 592 cpm and 3211 ± 245 cpm, respectively. When cells were incubated in PRL for 18 h, followed by culture in [3H] thymidine-containing medium devoid of IL-2, incorporation levels were about one third of those achieved in the presence of IL-2. These levels were also related to the PRL dose (Fig. 1). Finally, responses virtually identical to those shown in Fig. 1 were obtained when splenocytes from OVX rats of the Sprague-Dawley strain were tested (not shown). When a highly purified PRL preparation was used (RP-3), the [3H]thymidine incorporation levels were significantly greater than those induced with the B6 PRL (Fig. 2a). In neither case, however, did the incorporation levels reach those achieved with Con-A, a commonly used mitogen which served as a positive control for these experiments. On the average, exposure of splenocytes from OVX rats to Con-A (1 fxg/nl) caused 1.5-fold more [3H]thymidine incorporation than did exposure to PRL (1 ftg/nl). Thymocytes from these same animals did not respond to PRL, but lymph node lymphocytes responded the same as the splenocytes (data not shown). Hormone specificity Preincubation of PRL with PRL antiserum (1:1000 final dilution) for 18 h markedly reduced the subsequent incorporation levels, whereas preincubation with GH antiserum was without effect (Fig. 2b). The small reFlG. 2. a, Comparison of the ability of two rat PRL preparations (RP-3 and B6; 1 fig/m\ for 18 h) to stimulate the incorporation of radiolabeled thymidine in the presence and absence of IL-2. Data represent the average of three experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (us. medium control, b, Neutralization of the PRL response by preincubation of PRL in PRL antiserum (aPRL). PRL (B6; 1 /ug/ml) was incubated with polyclonal antiserum (1:1000 final dilution) to rat PRL or GH (aGH) for 18 h at room temperature. Both antisera have been validated; their crossreactivity is 0.3% (15). The antibodytreated hormones were added to splenocytes, which were assayed for IL-2 responsiveness as described in Fig. 1 and Materials and Methods. Data represent the average of three experiments. Levels of [3H]thymidine incorporation in cells incubated in the absence of IL-2 were: group 1, 9123 ± 1241; group 2, 4232 ± 896; group 3, 8751 ± 1023; group 4, 4612 ± 803 and group 5, 3919 ± 912 cpm.
Endo • 1990 Voll26-Nol
sponse induced by GH (Fig. 2b, column 5) may be attributed to PRL contamination of the GH preparation, since preincubation of the PRL with both PRL and GH antisera completely abolished the response (Fig. 2b, column 4). Time course Although significant levels of incorporation were detected in splenocytes incubated for 10 h with PRL before the addition of IL-2, maximal responses were found in cells incubated for 18-48 h (Fig. 3). The reason for the marked decline in incorporation rates after a 72-h exposure is not known. Cell viability was more than 90% after a 72-h incubation. Flow cytometry The IL-2-induced proliferation response of PRLtreated lymphocytes implied that PRL induced IL-2-R on the cell surface. This was confirmed directly by flow cytometric analysis of PRL-treated lymphocytes stained with an antirat IL-2-R (anti-TAC) antibody. Thus, 34% of the PRL-treated cells (Fig. 4B) and 56% of the ConA-treated cells (Fig. 4C) exhibited specific membrane IL2-R staining. Cultures incubated only with medium contained 9% IL-2 receptor-postive cells. This experiment was repeated once with similar results. When PRL-treated lymphocytes were incubated in antisera to other T-cell surface antigens, CD5 or CD8, the percentages of stained cells above control levels were also increased. In the case of CD5, PRL (1 /ug/ml) in-
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PRL INDUCTION OF IL-2 RECEPTORS
91
30 -i
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PRL - IL2 20-
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72
< Exposure to PRL (hours)
FIG. 3. Time course of PRL-induced IL-2 responsiveness in splenocytes from OVX rats in vitro. Data represent the average of three experiments. The PRL concentration was 1 Mg/ml. See Materials and Methods for assay details.
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creased the numbers of CD5-positive cells by 8% and 19% in two experiments; CD8 was increased by 3% and 13%. Neither increase was seen at lower doses of PRL. The percentage of CD4-stained cells ranged from 2128% regardless of the PRL concentration, while untreated cells were 22% CD4 positive. These results suggested that the PRL induction of IL-2-R occurs on Tlymphocytes, and possibly on cells of the CD5-positive subclass. IL-2 secretion To determine if PRL induced IL-2 release from splenocytes, CTLL2 cells [an IL-2-responsive cell line (16)] were cultured in media obtained from splenocytes that had been previously incubated in either PRL or Con-A for 18 h. As expected, both the Con-A-conditioned medium and recombinant IL-2 (used as a positive control) stimulated radiolabeled thymidine incorporation; however, the media from the PRL-treated cells did not (Fig. 5). When medium from the PRL-treated cells was mixed with either medium from Con-A-treated cells or recombinant IL-2, the activity was not affected, thus indicating that media from PRL-treated splenocytes does not inhibit the IL-2 assay (data not shown).
LOG INTEGRATED GREEN FLUORESCENCE FIG. 4. Flow cytometric analysis of splenocytes from OVX rats pretreated with PRL in vitro and then stained with antiserum to IL-2 cell surface receptor. A, Cells incubated in medium alone; B, cells incubated in B-6 PRL (1 Mg/ml) for 18 h; C, cells incubated with Con-A (1 ng/ ml) for 18 h. After incubation cells were stained for the presence of IL2 surface receptors using 0X39 antiserum according to the method of Baldwin et al. (17). Thirty thousand cells were counted per treatment group. The area containing IL-2-R-positive cells is outlined by the rectangular box. The percent positive cells is: A, 9%; B, 34%; and C, 56%. This experiment was repeated once with similar results.
Endocrine status of splenocyte donor
Splenocytes from OVX rats injected with sesame oil for 2 days before death showed the usual PRL-induced
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PRL INDUCTION OF IL-2 RECEPTORS
92 50 -\ O
human reccmbinant L-2(sUring at 25 irits/ml)
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Serial Dilutions FIG. 5. Effect of prior exposure (18 h) to PRL (1 /ig/ml) or Con-A (1 /ig/ml) on in vitro IL-2 secretion from splenocytes of 0VX rats. Cellfree media were serially diluted and assayed using CTLL2 cells, an IL2-responsive cell line. Human recombinant IL-2 was used as a positive control.
IL-2 response, whereas cells prepared from OVX and estradiol-injected donors did not (column 1 vs. 2, Fig. 6a). Splenocytes prepared from rats in random stages of the estrous cycle yielded variable results that were not significantly different from those of cells incubated in medium alone (column 3, Fig. 6a). Splenocytes prepared from diestrous, but not estrous, animals repeatedly had significant PRL responses (Fig. 6b). Splenocytes from either intact or castrated male rats failed to show the PRL-induced IL-2 response, but these same cell preparations responded to Con-A (Fig. 6c).
Discussion There is a considerable body of data which establishes that PRL participates in the regulation of immune cell function (1-12); our finding, showing that PRL can induce the formation of IL-2-Rs on rat splenocytes, extends this important and interesting area of study. Both IL-2 and its receptor are commonly recognized as being pivotal to proper T-cell-mediated immune response. Although most resting T-cells do not display IL2 surface receptors, exposure to mitogens such as Con-
Endo • 1990 Vol 126-No 1
A, PHA, antibodies, and antigens will induce IL-2-R formation. Agents that bypass portions of signal transduction mechanics, e.g. calcium ionophore, phorbol esters, and diacylglycerol, can also enhance receptor expression. While our data offer little insight into an understanding of the mechanism(s) by which PRL might exert its IL-2 effect on the lymphocyte, there are three observations that are helpful. First, PRL stimulates incorporation of radiolabeled thymidine in the absence of IL-2. While the magnitude of this response is only about one third of that seen when IL-2 is present, direct lymphocyte activation as a result of PRL exposure is clearly possible under our experimental conditions. Since the splenocyte preparation contains B-cells, T-cells, macrophages, and fibroblasts, the possibility that PRL by itself can activate a subpopulation of T-cells is attractive. Indeed, the flow cytometric data tend to support this idea, since the target lymphocytes seem to be primarily T-cells, with the CD8+ T subsets being somewhat increased and CD4+ T-cells being unaffected. Second, PRL induces IL-2-R formation, but clearly does not cause detectable levels of IL-2 secretion. We are unaware of other agents that act in this fashion. We speculate that PRL exposure, under certain physiological conditions, might prime a sizeable pool of splenocytes (~35%) to express IL-2-R to become responsive to variations in the local concentration of IL2. The net result would be that PRL, a native and ubiquitous peptide hormone, could provide a mechanism for the nonantigen-specific expansion of a large population of splenocytes. This expansion might be more efficient than an antigen-directed one, since a specific antigen commonly activates approximately less than 0.01% of the lymphocyte population (18). Third, there is little question that the PRL IL-2-R induction response would have gone undetected had non-OVX animals been used. Our data clearly document that the effect is dependent upon the E2 millieu. In fact, the responsiveness of splenocytes prepared from diestrous, but not estrous, animals offers strong support for the idea that these findings are physiologically relevant. Since it is generally recognized that estrogens are immunosuppressive (13, 19-21), the ratio of estrogen/PRL at the target splenocyte must be critical for IL-2-R induction. At estrus, circulating levels of serum E2 and PRL average 88 pg/ml and 241 ng/ml, whereas at diestrus, these average 17 pg/ml and 41 ng/ ml (22). Con-A is commonly used to induce IL-2-R on lymphocytes in vitro. In fact, several laboratories have used ConA as a means for testing the effects of hormones on the lymphocyte activation process. For example, in vivo administration of glucocorticoids or estrogens will suppress Con-A-activated mitogenesis in vitro (13). Furthermore, PRL is comitogenic with Con-A when tested on spleno-
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PRL INDUCTION OF IL-2 RECEPTORS a)
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FIG. 6. Effect of hormonal status of the splenocyte donor on in vitro responsiveness to PRL. A, In each of three experiments, three OVX rats (columns 1 and 4), three intact random cycle rats (column 3), or three OVX estrogen-treated rats (column 2) served as splenocyte donors. Those animals in column 2 were injected sc with 17/3-estradiol on day 15 (0.5 ng) and day 16 (50 fig) as previously described (14). Splenocytes were assayed for PRL responsiveness; only cells from group 1 were significantly (P < 0.05) different from controls. Cells incubated in PRL, but in the absence of IL-2 had the following levels of incorporation: column 1, 9,612 ± 2,411; column 2, 894 ± 214; column 3, 5,232 ± 3,946; column 4, 2,526 ± 610 cpm. B, In each of four experiments, splenocytes from cycling intact rats prepared at either diestrus or estrus were used in the assay. Diestrous response, P < 0.05 vs. control. Levels of incorporation in the absence of IL-2 were: diestrus, 9,614 ± 2,212; estrus, 3,340 ± 892; and medium control, 1,568 ± 323 cpm. C, In each of three experiments, three OVX, three male, or three castrated male rats served as splenocyte donors. Splenocytes from all three groups of animals gave a significant (P < 0.05) response when Con-A (1 Mg/ml) was used in the assay. However, only the splenocytes from the OVX animals gave a significant (P < 0.05) PRL response. Levels of incorporation in the absence of IL-2 were (from left to right), 12,341 ± 3,112; 14,023 ± 9,216; 12,146 ± 2,142; 8,723 ± 1,422; 3,242 ± 1,021; 3,562 ± 782; 3,814 ± 921; 3,862 ± 867; and 2,916 ± 812 cpm.
cytes and peripheral lymphocytes in vitro (23). In every experiment in our study, we also tested splenocyte that were exposed to Con-A instead of PRL. In all cases a 24h exposure of Con-A was more effective than PRL in promoting [3H]thymidine incorporation. At this time about 50% more lymphocytes expressed IL-2-R after exposure to Con-A than to PRL. However, Con-A also induced significant amounts of IL-2 secretion, while PRL did not. Our data also show that PRL by itself is mitogenic. Others have reported that PRL was comitogenic with Con-A, but was not mitogenic alone in the systems tested to date (24). This discrepancy may be accounted for by the hormonal status of the splenocyte donor. It is known that IL-2-R and IL-2 are induced in stimulated lymphocytes, but are induced independently (25). Our data can be interpreted to mean that PRL primarily activates IL-2-R expression but not IL-2. At this point we do not know the mechanism of action of PRL. It will be interesting to determine if the PRL receptor is in some way associated with the T-cell-receptor complex. In summary, our observations together with those of many others (1-13) confirm the important relationship between the endocrine and immune systems. They also invite further study into relationships among PRL, estrogen, and lymphocytes during development, aging, and tumorigenesis.
Acknowledgments Some of the immunochemical reagents used in this study were kindly
furnished by the NIDDK. We thank Dr. David J. Hurley and Barbara Bour for help with the flow cytometry.
References 1. Russell DH, Matrisian L, Libler R, Larson DF, Poulos B, Magun BE 1984 Prolactin receptors on human lymphocytes and their modulation by cyclosporine. Biochem Biophys Res Commun 121:899 2. Shiu RPC, Elsholtz HP, Tanaka T, Friesen HG, Gout PW, Beer CT, Noble RL 1983 Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology 113:159 3. Bernton EW, Meltzer SM, Holaday JW 1988 Suppression of macrophage activation and T-lymphocyte function in hypoprolactinemic mice. Science 401:000 4. Berczi I, Nagy E, Kovacs K, Horvath E 1981 Regulation of humoral immunity in rats by pituitary hormones. Acta Endocrinol (Copenh) 98:506 5. Gambel PI, Cleland AW, Ferguson FG, 1980 Alterations in thymus and spleen cell populations and immune reactivity during syngenic pregnancy and lactation. J Clin Lab Immunol 3:115 6. Carrier M, Russell DH, Wild JC, Emery RW, Copeland JG 1987 Prolactin as a marker of rejection in human heart transplantation. Transplant Proc 19:3442 7. Montgomery DW, Zukoski CT, Shah GN, Buckley AR, Pacholczyk T Russell DH 1987 Concanavalin A-stimulated murine splenocytes produce a factor with prolactin like bioactivity and immunoactivity. Biochem Biophys Res Commun 145:2692 8. Russell DH, Mills KT, Talamantes FJ, Bern HA 1988 Neonatal administration of prolactin antiserum alters the development pattern of T- and B-lymphocytes in the thymus and spleen of Balb/c female mice. Proc Natl Acad Sci USA 85:7404
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9. Hartmann DP, Holaday JW, Bernton EW 1989 Inhibition of lymphocyte proliferation by antibodies to prolactin. FASEB J 3:2194 10. Mahajan PB, Ebner KE 1985 Cyclosporin A and rabbit mammary prolactin receptors. Biochem Biophys Res Commun 133;753 11. Varma S, Ebner KE 1988 The effect of cyclosporin A on the growth and prolactin binding to Nb-2 rat lymphoma cells. Biochem Biophys Res Commun 156:233 12. Cosson C, Myara I, Guillerman R, Amrein C, Dreyfus G, Moatti N 1989 Serum prolactin as a rejection marker in heart transplantation. Clin Chem 35:492 13. Luster MI, Hayes HT, Korach H, Tucker AN, Dean JH, Greenlee WF, Boorman GA 1984 Estrogen immunosuppresion is regulated through estrogenic responses in the thymus. J Immunol 133:110 14. Neill JD 1972 Sexual differences in the hypothalamic regulation of prolactin secretion. Endocrinology 90:1154 15. Hatfied JM, Hymer WC 1986 Flow cytometric analysis and sorting of live male rat anterior pituitary cell types by forward angle and perpendicular light scatter. Endocrinology 119:2670 16. Larrson EL, Couhnho A 1980 Mechanism of T-cell activation. I. A screening of "step one" ligands. Eur J Immunol 10:93 17. Baldwin CL, Teaole AJ, Naessens JG, Goddeeris BM, MacHugh ND, Morrison WI 1986 Characterization of a subset of bovine Tlymphocytes that express BoT4 by McAb and function: similarity to lymphocytes defined by human T4 and murine L3T4. J Immunol
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136:4385 18. Weiss A 1986 The role of the T3/antigen receptor complex in Tcell activation. Annu Rev Immunol 3:593 19. Smith EM, Blalock JE 1988 A molecular basis for interactions between the immune and neuroendocrine systems. Int J Neurosci 38:455 20. Kalland T, Strand 0, Forsberg JG 1979 Long term effects of neonatal estrogen treatment on mitogen responsiveness of mouse spleen lymphocytes. J Natl Cancer Inst, 63:413 21. Sobhon P, Jirasattham C 1974 Effect of sex hormones on the thymus and lymphoid tissue of ovariectomized rats. Acta Anat 89:211 22. Butcher RL, Collins WE, Fugo NW 1974 Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17/3 throughout the 4-day strus cycle of the rat. Endocrinology 94:1704 23. Montgomery DW, Russell DH, Buckley AR, Zukovski CF 1987 Conconavalin A (Con-A)-stimulated lymphocytes produce a prolactin (PRL)-like mitogen for Nb2 node lymphoma cells. Fed Proc 46:529 (Abstract) 24. Spangelo BL, Hall NR, Goldstein AL, 1985 Evidence that prolactin is an immunomodulatory hormone. In: MacLeod RM, Scapagnini U, Thorner MO (eds) Prolactin, Basic and Clinical Correlates. Springer-Verlag, New York, pp 343-350 25. Crabtree GR 1989 Contingent genetic regulatory events in T lymphocyte activation. Science 243:355
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