Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 28 September 2004 ; accepted in final form 13 December 2004
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ABSTRACT |
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Nb2 cells; cytokines; immune functions
Because of its participation in the regulation of immune functions, PRL has emerged as a potential target for intensive research in diseases in which immune dysfunction is apparent (3, 13, 34). Trauma-hemorrhage (T-H) causes severe and prolonged immunosuppression in young males (36, 39) but not in proestrus females, indicating gender dimorphism in the response to injury. Previous studies have also shown that administration of PRL or metoclopramide (a dopamine antagonist that increases PRL release) in male mice after T-H restores immune functions and decreases mortality from subsequent sepsis (17, 4042). Besides PRL, administration of 17-estradiol (18, 19) and the androgen receptor antagonist, such as flutamide (35), in males after T-H also has been shown to restore immune functions. Thus a close association between the endocrine and immune systems is apparent after T-H.
The restoration of immune functions in males by PRL or metoclopramide administration after T-H suggests an important role of this hormone in immune function after injury. However, the mechanism by which PRL restores immune function remains unclear. Thus evaluation of immunosuppression effects of T-H in male PRL/ mice in which pituitary hormone release is expected to be absent may provide useful information concerning the role of this hormone in the response to injury. Studies were therefore performed to assess changes in the splenic T lymphocyte functions after T-H in the transgenic PRL/ and wild-type PRL+/+ male and female mice. Significant changes in the release of IL-2, IL-10, and IFN- by splenocytes after T-H were observed, especially in the males of both wild-type and transgenic mice. The results demonstrate a release of
25-kDa protein that was active in the proliferation of Nb2 cells in vitro by the concanavalin A (ConA)-stimulated splenocytes of both wild-type and transgenic mice. Because of the splenocyte release and the inherent lymphocyte stimulatory activity, the local release of the
25-kDa protein may play a significant role in the changes in splenocyte function after T-H.
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MATERIALS AND METHODS |
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The results obtained from one such analysis of animals are shown in Fig. 1. The data show that the homozygous PRL+/+ mice exhibited only the 420-bp band, the homozygous PRL/ mice exhibited only the 930-bp band, and the heterozygous PRL+/ mice exhibited both the 420- and 930-bp bands. PRL/ and age-matched PRL+/+ mice (C57BL/6 mice; The Jackson Laboratory, Bar Harbor, ME), 1015 wk old and weighing 2025 g, were used in the experiments. PRL+/ and PRL/ mice, male or female, did not manifest any abnormal behavior. The major phenotypic changes resulting from target disruption of the PRL gene were observed only in the transgenic female mice. The estrus cycles of PRL/ females were irregular, with longer or shorter duration, and these mice were infertile. Although the mammary glands of the mutant females developed a normal ductal tree, the ducts failed to develop lobular decorations. To the contrary, PRL-deficient mice were fertile. Primary lymphopoiesis and myelopoiesis, however, were normal in both mutant mice (14). The females that were found to be in the proestrus stage on the basis of vaginal smear analysis were used in these studies. Animal experiments were conducted in accordance with guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH) and approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
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Trauma-hemorrhage. Animals were anesthetized with isoflurane (IsoSol; Vedco, St. Joseph, MO) and restrained in a supine position. A 2.5-cm midline laparotomy (i.e., induction of soft tissue trauma) was performed and then closed aseptically in two layers using 6-0 sutures (Ethilon; Ethicon, Somerville, NJ). Subsequently, both femoral arteries were aseptically catheterized with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ), and the animals were allowed to awaken. Blood pressure was monitored continuously through one of the femoral catheters using a blood pressure analyzer (Digi-Med BPA-190; Micro-Med, Louisville, KY). Upon awakening, the animals were bled through the other catheter to a mean arterial pressure (MAP) of 35 ± 5 mm that was maintained for 90 min. At the end of that period, animals were resuscitated with four times the shed blood volume in the form of lactated Ringer solution over 30 min. Lidocaine was applied to the groin incision sites, the catheters were removed, the vessels were ligated, and the incisions were closed with 6-0 sutures. Sham-treated animals underwent the same anesthetic and surgical procedures, but neither hemorrhage nor fluid resuscitation was performed. The animals were anesthetized by isoflurane administration at 24 h, after which blood, pituitary gland, and spleen were collected for analysis.
Plasma collection and storage. Blood was obtained by cardiac puncture and centrifuged at 10,000 g for 15 min at 4°C. Plasma was immediately frozen and stored at 80°C until analyzed.
Splenocyte preparation. The spleen was placed in ice-cold, 4°C PBS and gently ground between frosted slides to produce a single-cell suspension. The suspension was centrifuged at 300 g for 15 min, the pellet was resuspended in PBS, erythrocytes were hypotonically lysed, and the remaining cells were washed with PBS by centrifugation at 300 g for 15 min. Splenocyte viability was consistent at >95% determined using a Trypan blue exclusion procedure. The splenocytes were resuspended in RPMI 1640 medium (Life Technologies, Grand Island, NY) with or without 10% heat-inactivated FBS (Life Technologies) to yield a final concentration of 1 x 106 cells/ml.
Analysis of pituitary for PRL content. The presence of PRL in the pituitary was assessed by Western blot analysis. Briefly, the pituitary gland was homogenized, and volume was then adjusted to 100 µl with 1x SDS sample buffer (62.5 mM Tris·HCl, pH 6.8, at 25°C, 2% wt/vol SDS, 10% glycerol, 50 mM DTT, and 0.01% wt/vol bromophenol blue) boiled at 95°C for 5 min. An aliquot of the extract (20 µl) and recombinant mouse PRL (NIH) that acted as a positive control were loaded on a Nu-PAGE 412% Bis-Tris gel and electrophoresed at 200 V. Proteins were electroblotted to a nitrocellulose membrane and blocked with 5% BSA in 10 mM Tris·HCl buffer (pH 7.6) containing 150 mM NaCl and 0.1% Tween 20 for 1 h at room temperature. The membrane was first incubated with rabbit polyclonal anti-mouse PRL antibody (NIH) diluted 1:1,000 with the same buffer for 2 h and later with the secondary antibody (anti-rabbit IgG antibody conjugated to horseradish peroxidase; Santa Cruz Biotechnology, Santa Cruz, CA; diluted to 1:3,000 with 10 mM Tris·HCl buffer, pH 7.6, containing 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk). After the membrane was washed three times, the immunoreactive bands were visualized using chemiluminescence with Chemiglow West substrate (Alpha Diagnostics, San Leandro, CA).
Splenocyte proliferation assay. Splenocytes were cultured at 1 x 105 cells/well in flat-bottomed, 96-well microtiter plates using RPMI 1640 medium supplemented with 10% heat-inactivated FBS for 60 h. Cells were pulsed with 5-bromo-2'-deoxyuridine (BrdU; Amersham, Piscataway, NJ) during the final 12 h of culture. The ability of the cells to proliferate in response to 2.5 µg/ml ConA (Pharmacia/LKB Biotech, Piscataway, NJ) was measured by performing a BrdU incorporation procedure according to the manufacturer's instructions.
Expression of IL-2, IL-10, and IFN- genes in splenocytes by RT-PCR.
Total RNA was extracted from splenocytes using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, cells were lysed in a denaturing solution, and RNA was sequentially isolated using phenol/chloroform extraction and isopropanol precipitation. To prepare cDNA, 1 µg of total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Alto, CA) primed with random hexamers at a final volume of 100 µl. PCR was performed using HotStar Taq DNA polymerase and the Q-Solution kit (Qiagen, Valencia, CA) in accordance with the manufacturer's protocol. The primers used were IL-2, 5'-ACAGGTTCCCCGGGCAGCATCTCTA-3' (forward) and 5'-TCAGCATCTCATCCCAGCCCAAGCA-3' (reverse); IL-10, 5'-ATGCAGGACTTTAAGGGTTACTTGGGTT-3' (forward) and 5'-ATTTCGGAGAGAGGTACAAACGAGGTTT-3' (reverse); and IFN-
, 5'-AGCGGCTGACTGAACTCAGATTGTAG-3' (forward) and 5'-GTCACAGTTTTCAGCTGTTATGGG-3' (reverse).
Briefly, a PCR assay was performed using 5 µl of the cDNA mixture. RT products were used in a total volume of 50 µl containing 5 µl of 10x PCR buffer, 10 µl of Q-Solution, 0.2 mM of each dNTP, 1.5 units of HotStar Taq Polymerase (Qiagen), and 1 µM of each of the primers. PCR reactions were performed in a gradient Mastercycler (Eppendorf, Westbury, NY). For each PCR reaction, preheating was performed at 95°C for 15 min, then at 94°C for 40 s, at 55°C for 45 s, and at 75°C for 1 min, with a final extension phase at 72°C for 10 min. Each reaction was analyzed for amplification between 25 and 40 cycles. The amplification of -actin (Clontech) was used as the housekeeping gene. The PCR products were analyzed using electrophoresis on 1.5% agarose gels in 1x TBE buffer. The intensity of the bands was measured in the 500 Fluorescence ChemiImager (Alpha Diagnostics). The relative absorbance of the PCR products was corrected against the absorbance obtained for
-actin.
Splenocytes IL-2, IL-10, and IFN- release.
Splenocytes were cultured at 1 x 106 cells/well in 24-well plates using RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 2.5 µg/ml ConA for 48 h at 37°C with 5% CO2 and 90% humidity. IFN-
and IL-2 were chosen as representative Th1 cytokines, whereas IL-10 was measured as representative Th2 cytokines. The levels of INF-
, IL-2, and IL-10 in culture supernatants of stimulated splenocytes were determined using sandwiched enzyme-linked immunosorbent assays (ELISA; BD Pharmingen, San Diego, CA). After the incubation period, cell-free supernatants were harvested and kept frozen at 80°C until assayed for the cytokines.
Analyses of splenocytes for PRL-like protein. Splenocytes were cultured at 1 x 106 cells/well in 24-well plates using serum-free RPMI 1640 medium constituted with the serum replacement medium (BD Nu-Serum serum replacements, which provide low-protein alternatives to newborn calf, fetal bovine, and other sera routinely used for cell culture; BD Biosciences, San Jose, CA) and 2.5 µg/ml ConA for 48 h at 37°C in an incubator in 5% CO2 and 90% humidity. The protein content of the supernatant was determined by performing a colorimetric assay (Bio-Rad, Hercules, CA). For the immunoprecipitation reaction, the assay mixture consisted of an aliquot of the supernatant equivalent to 200 µg, 1 µg/ml rabbit polyclonal anti-mouse PRL antibody (provided by Dr. Albert F. Parlow, National Hormone & Peptide Program, NIH), and PBS to a final volume of 200 µl. After overnight incubation in a shaker at 4°C, 50 µl of 10% protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden) were added. The reaction mixture was then shaken at room temperature for 2 h, centrifuged at 12,000 g for 1 min, and washed three times with PBS. The resulting pellet was suspended in 40 µl of 3x SDS sample buffer, boiled for 5 min at 95°C, and centrifuged at 12,000 g for 10 min, and then the supernatant was collected for electrophoresis. Splenocyte extracts (20 µg) and recombinant mouse PRL that acted as a positive control were loaded on a Nu-PAGE 12% Bis-Tris gel and electrophoresed at 200 V. Western blot analysis of the electrophoresed proteins was performed essentially using the procedure described above.
PRL bioassay. Plasma PRL as well as the protein from splenocyte extracts for in vitro proliferation activity was assayed in the laboratories of Dr. Arthur R. Buckley (University of Cincinnati, Cincinnati, OH) using the lymphoma Nb2 cell line (30). In brief, Nb2 cells were growth arrested by removing FBS for 24 h. Cells were plated and grown in 96-well tissue culture plates in the presence of either PRL standard (NIH sheep PRL-20, 31 IU/mg; a gift from the National Hormone and Pituitary Program) or the splenocyte extracts for 48 h at 37°C in a humidified atmosphere. Proliferation of Nb2 cells in the plates was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye conversion assay. The minimum detectable level of PRL in this assay was 1 pg/ml. The minimum detectable level of PRL in this assay was 1 pg.
Statistical analyses. Data are presented as means ± SE for each group. One-way ANOVA for multiple comparisons, followed by Tukey's or Dunn's test, was used to determine the significance of the differences between experimental means. P < 0.05 was considered significant for all statistical analyses.
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RESULTS |
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Expression and release of IFN- by splenocytes.
There was no significant difference in IFN-
expression between PRL+/+ and PRL/ in either male or female shams (Fig. 6A). T-H, however, caused increased IFN-
expression only in male wild-type and transgenic strains, but not in corresponding proestrus female strains (Fig. 6A). Similarly, there was no significant difference in the release of IFN-
between the strains in males and proestrus females (Fig. 6B). However, in contrast to what we observed with IFN-
expression, T-H led to decreased IFN-
release in PRL+/+ and PRL/ male strains. The release of IFN-
by splenocytes in wild-type and knockout males followed the opposite pattern regarding gene expression. The release of IFN-
by splenocytes of PRL+/+ and PRL/ females was not altered after T-H.
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DISCUSSION |
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The PRL/ mouse used in this study is a C57BL/6 transgenic mouse whose pituitary PRL gene is disrupted by homologous recombination with the neomycin cassette insert (14). Earlier studies to assess the effects of T-H on immune responses were performed with the C3H/HeN strain. Because PRL/ transgenic mice of the C3H/HeN strain were not available, C57BL/6 transgenic mice were used in this study. Our recent studies have shown that C57BL6 mice were also immunodepressed, as were the C3H/HeN mice, after T-H, with the exception that the former were slightly hyporesponsive to T-H (25). The behavior of PRL/ male and females was normal, except that the estrus cycle of transgenic females was irregular and they were infertile. PRL expression was absent in the pituitaries of transgenic male and female mice. Furthermore, circulatory levels of PRL in transgenic mice of both sexes were so low that they were not quantifiable using the in vitro Nb2 cell proliferation assay.
Previous studies of PRL/ mice have indicated that PRL is not required for somatic growth, male reproduction, and the maintenance of homeostasis but that it is essential for reproduction and mammary gland development in adult females (11). Moreover, the absence of significant changes in the development and distribution of lymphocyte subpopulations in the thymus, spleen, and lymph nodes of PRL/ mice suggested that the hormone is not required for lymphocyte development (11). This does not, however, exclude the involvement of PRL in lymphocyte function of the transgenic mice. In this study, significant differences in ConA-stimulated splenocyte proliferation were observed in the wild-type and transgenic female (sham) mice. Furthermore, opposite effects were observed in the males and females after T-H. In PRL/ males, a significant decrease in proliferation was observed after T-H, while in PRL/ females, a significant increase in proliferation was observed after T-H (Fig. 3). The negative effect of T-H on splenocyte proliferation in male mice and the acute effect in the transgenic PRL/ mice support our earlier observation that the lack of PRL may be a factor in T-H-induced immunodepression. Furthermore, our study showed increased release of IL-10 in male PRL/ of sham control mice and after T-H (Fig. 5B). The differences observed in the ConA-stimulated splenocyte proliferation and in the cytokine release by splenocytes of transgenic mice suggest that stress or inflammation resulting from T-H may be the reason for alterations in splenocyte function observed in PRL/ mice of both sexes.
Inflammatory response is assessed by Th cell cytokine release. The response is measured by the release of Th1 cytokines, TNF- and IFN-
and Th2 by the release cytokine such as IL-10. The immune response after T-H is thought to produce a shift from Th1 to Th2 cytokines with the release of IL-10 and TGF-
. Studies have also shown that PRL deficiency enhances inflammatory responses in vitro and exhibits Th1-type cytokine-like effects (24, 37). The results of this study do not appear to indicate a Th1-like effect, because splenocyte IL-2 and IFN-
release was lowered in both PRL+/+ and PRL/ male mice after T-H, with no significant changes in release observed in corresponding females (Figs. 4B and 6B). Furthermore, there was no effect of T-H on the release of IL-10 to indicate a Th2 cytokine response.
The critical role of cytokines and growth factors in the regulation of immune functions after T-H is well known. However, IL-2 and IFN- deserve special attention because the former acts in vivo to constrain lymphoid growth by promoting apoptosis and maintain peripheral tolerance by promoting cell-mediated immunity (32). A decrease in these cytokines released by splenocytes is evident after T-H, implying a proinflammatory Th1 response. This observation is corroborated in the present study and shows that both IL-2 and IFN-
release are significantly lowered in both the wild-type and transgenic strains. This is evident in males, but not in proestrus females, after T-H. Studies demonstrate PRL synergism with IL-2 in eliciting IFN-
production as well as the Th1 response (24, 26, 44). Because PRL release is lowered after T-H, it is not surprising that release of IL-2 and IFN-
decreases after T-H. The absence of such effects in proestrus females, in the wild-type as well as transgenic animals, may be due to 1) maintenance of estrogen release in the proestrus females after T-H (33), and 2) the effect of estrogen on the lymphocyte functions, either alone or in synergy with PRL.
Although the majority of the circulating PRL is of pituitary origin, attention is now focused on the local production of the hormone in peripheral tissues or cells. Studies have shown the release of proteins exhibiting activity similar to that of PRL in normal lymphoid cells (2830). We therefore examined whether PRL-like proteins were released by splenocytes from PRL+/+ and PRL/ mice. Male mice were used in these experiments because they are immunosuppressed after T-H and respond to PRL administration to restore immune functions. Our results show the release of a protein (25 kDa) in the ConA-stimulated splenocytes in both wild-type and transgenic male mice. This protein exhibited lymphoproliferative activity in the in vitro Nb2 assay similar to that of PRL. This
25-kDa protein is unlikely to be related in any way to pituitary PRL, because it is found in the splenocyte extracts of PRL/ transgenic mice. Moreover, it is uncertain whether this
25-kDa protein possesses other PRL functions, namely, the lactogenic property and osmoregulation. Purification and chemical characterization of the
25-kDa protein and analysis of it for PRL-associated functions are thus required. Because lymphocyte proliferation is generally associated with immune function, its release may be of significance in immune function after T-H. It should be pointed out that, similarly to PRL, IL-2 and IL-7 are also capable of stimulating Nb2 cells in vitro, and changes in the release of these cytokines are evident after T-H. The
25-kDa protein appears not to be a cytokine, because it is prepared using an immunoprecipitation procedure with PRL antiserum. A detailed analysis of the
25-kDa protein in relation to immune function is essential.
PRL is a paradoxical multifunctional hormone that is present in many discrete forms. It is produced in the pituitary as well as in other tissues and cells, including lymphocytes. PRL reacts with its own receptor, and its mechanism of action is similar to that of a cytokine or growth factor. Because the development and function of the immune system are normal in PRLR/ and PRL/ mice, PRL may not be an obligate lymphopoietic hormone. Previous studies conducted at our laboratory have demonstrated favorable effects of PRL in the restoration of immune function after T-H and decreased lethality as a result of subsequent sepsis (42). In contrast to T-H, Oberbeck et al. (31) showed that exogenous PRL increased mortality in a sepsis model. The reason why PRL decreases lethality as a result of sepsis induced after trauma but increases lethality resulting from sepsis per se, without prior T-H, is unclear. Because PRL receptor-knockout mice have normal immune function (5), it could be suggested that PRL may not play as important a role as previously suggested.
PRL secretion in the pituitary is under hypothalamic control. IL-1, IL-2, IL-6, and estrogen stimulate PRL production in the pituitary, while IFN- is inhibitory. Neurotransmitters released after T-H injury, such as catecholamines (epinephrine and norepinephrine) and dopamine, also are capable of regulating PRL release. Among these, dopamine is known to suppress the release of PRL. In fact, our earlier studies demonstrated salutary effects on the restoration of immune function in males after T-H by a dopamine antagonist, metoclopramide, suggesting the need for PRL in the maintenance of immune function after injury (14, 40). It is unclear whether local production of PRL in tissues, especially lymphocytes, is also influenced by cytokines, neurotransmitters, and sex steroids. The influence of mediators, especially the sex steroids, in the local synthesis and release of PRL has relevance to T-H because the administration of 17
-estradiol, similarly to PRL, restores the immune functions after T-H in male as well as ovariectomized female mice (1541). Unlike males, proestrus females are not immunosuppressed after T-H, owing to continued synthesis of 17
-estradiol in T lymphocytes even after injury (33). Because splenocytes, especially the T lymphocytes, have the intrinsic capability of producing cytokines, sex steroids (32, 33, 43), and PRL (2, 28, 29, 30) and, as observed in this study, also express their receptors, it is essential to know how they interact with each other in the maintenance of homeostasis under normal conditions and how the disruption of homeostasis occurs after stress or disease conditions. The interaction between estrogen and PRL in the regulation of immune function is critical because these hormones reciprocally stimulate each other (27). Both PRL and 17
-estradiol regulate immune function by interaction with their specific receptors present on the T lymphocyte surface. PRL influences the immune response, probably as a cytokine, because its receptor, PRLR, is a part of the cytokine hematopoietic receptor family. Thus PRL as an immunopotentiating agent may have a powerful therapeutic role in the treatment of patients after traumatic injury. Although much attention has been paid to the pituitary and circulatory PRL, none has been paid to the locally produced PRL or to proteins with PRL-like activity. Additional studies of the relationship between PRL-PRLR complexes, locally synthesized PRL, and PRL-like proteins and sex steroids in the regulation of the immune system after T-H may provide significant information concerning the potential of this hormone in the treatment of trauma patients.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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