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Regulation of Lymphocyte Proliferation by Uterine Serpin: Interleukin-2 Mrna Production, CD25 Expression and Responsiveness to Interleukin-2 (44465) 2 1 MORGAN R

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Regulation of Lymphocyte Proliferation by Uterine Serpin: Interleukin-2 Mrna Production, CD25 Expression and Responsiveness to Interleukin-2 (44465) 2 1 MORGAN R Regulation of Lymphocyte Proliferation by Uterine Serpin: Interleukin-2 mRNA Production, CD25 Expression and Responsiveness to Interleukin-2 (44465) 2 1 MORGAN R. PELTIER,WEN-JUN LIU, AND PETER J. HANSEN Department of Dairy and Poultry Sciences, University of Florida, Gainesville, Florida 32611–0920 Abstract. During pregnancy, the endometrium of the ewe secretes large amounts of a progesterone-induced protein of the serpin superfamily of serine proteinase inhibitors called ovine uterine serpin (OvUS). This protein inhibits lymphocyte proliferation in response to concanavalin A (ConA), phytohemagglutinin (PHA), or mixed lymphocyte reaction. The purpose of these experiments was to characterize the mechanism by which OvUS inhibits lymphocyte proliferation. Ovine US caused dose-dependent in- hibition of lymphocyte proliferation induced by phorbol myristol acetate (PMA), an activator of protein kinase C. The PHA-induced increase in CD25 expression was inhibited in peripheral blood mononuclear leukocytes (PBML) by OvUS. However, no effect of OvUS on Con A-induced expression of CD25 was observed. Further analysis using two-color flow cytometry revealed that OvUS inhibited ConA-induced expres- sion of CD25 in ␥␦-TCR− cells but not ␥␦-TCR+ cells. Stimulation of PBML for 14 hr with ConA resulted in an increase in steady state amounts of interleukin-2 (IL-2) mRNA that was not inhibited by OvUS. Ovine US was also inhibitory to lymphocyte proliferation induced by human IL-2. Results suggest that OvUS acts to inhibit lymphocyte prolif- eration by blocking the upregulation of the IL-2 receptor and inhibiting IL-2–mediated events. Lack of an effect of OvUS on ConA-stimulated CD25 expression in ␥␦-TCR+ cells may reflect a different mechanism of activation of these cells or insensitivity to inhibition by OvUS. [P.S.E.B.M. 2000, Vol 223] vine uterine serpin is a member of the serpin su- tive center loop (8, 9). However, OvUS is distinct from the perfamily of serine proteinase inhibitors that is ex- prototypical serpin because it has no antiproteolytic activity Opressed in high amounts in the sheep uterus during to any of the serine proteinases tested (1) and only weak pregnancy in response to progesterone (1–4). Similar pro- inhibitory activity against pepsin A and pepsin C (6), which teins are also produced in pigs (5, 6) and cattle (4, 6, 7). The does not seem to be dependent on an intact reactive center majority of serpins, such as ␣1-antitrypsin, inhibit protein- loop (10). ases by forming a tight tetrahedral complex with their target Functionally, OvUS acts as an inhibitor of lymphocyte proteinase through interaction of the protein with the reac- proliferation. It inhibits a wide variety of events including mixed lymphocyte reactions, mitogen-stimulated lympho- cyte proliferation (11–14) and Poly I и Poly C–induced NK- cell activity and abortion in mice (15). In fact it is believed Funding was received from USDA NRIGP grant no. 96-35203 and NIH grant that OvUS mediates the inhibitory effects of progesterone HD26421. 1 To whom requests for reprints should be addressed at P.O. Box 110920, University on uterine immune function that leads to prolonged skin of Florida, Gainesville, FL 32611–0920. E-mail: [email protected] graft survival (16) and decreased lymphocyte numbers in 2 Present address: Howard Hughes Medical Institute, Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104. the endometrium (17). The mechanism by which OvUS acts to inhibit lymphocyte function is unknown. Concentrations Received April 21, 1999. [P.S.E.B.M. 2000, Vol 223] of OvUS from 125 to 500 ␮g/ml are required for inhibition Accepted July 19, 1999. of lymphocyte proliferation (11, 12). Although these con- centrations are physiological (concentrations in uterine fluid 0037-9727/00/2231-0075$14.00/0 Copyright © 2000 by the Society for Experimental Biology and Medicine are 5 mg/ml or greater), it is likely that OvUS may not function through a classical high-affinity receptor. Nonethe- MECHANISM FOR IMMUNOSUPPRESSION BY OVINE UTERINE SERPIN 75 less, OvUS binds specifically to the lymphocyte surface lection (Salisbury, UK). Ascites for clone 86D was pro- (18). It is possible that OvUS binds proteins on the surface duced by the Hybridoma Core Laboratory of the University of the lymphocyte and that such binding either activates an of Florida Interdisciplinary Center for Biotechnology Re- inhibitory response or blocks events required for proliferation. search and stored at −20°C until use. For indirect labeling of Stimulation of the T-cell receptor with antigen or a lymphocytes, an FITC- or phycoerythrin (PE)-labeled sheep mitogenic lectin induces a signal transduction pathway in- anti-mouse IgG F(ab)2 fragment was purchased from volving protein kinase C, which stimulates IL-2 secretion Sigma. and upregulation of the ␣ subunit of the IL-2 receptor, For some experiments, antibody to the ␥␦-TCR was CD25 (the ␤ and ␥ subunits are constitutively expressed). purified by Protein A affinity chromatography using the Secreted IL-2 functions in an autocrine and paracrine man- FPLC system (Pharmacia, Piscataway, NJ). Briefly, ascites ner by binding to the full IL-2 receptor to initiate mitogen- produced from clone 86D was diluted 1:10 in 3 M glycine, esis of the antigen-stimulated lymphocytes. To understand 1.5 M NaCl, pH 8.9, and 2–6 ml were loaded onto a Protein how immunity may be suppressed in the uterus during preg- A-Sepharose column (1 × 5 cm; BioRad, Hercules, CA). nancy, several experiments were performed to determine at The column was washed with 25 ml of 3 M glycine, 1.5 M what steps OvUS acts to inhibit lymphocyte proliferation. NaCl, pH 8.9, and then antibody was eluted with 0.5 M Specifically, effects on PMA-stimulated lymphocyte prolif- citrate-phosphate buffer, pH 5.5. Column fractions (0.5 ml eration, mitogen-stimulated CD25 expression, mitogen- each) were collected into 12 × 75-mm tubes that contained stimulated IL-2 gene expression, and responsiveness to hu- 0.1 ml of 1 M Tris-HCl, pH 8.8. Fractions corresponding to man IL-2 (Hu-IL-2) were tested. the protein peak were pooled and concentrated using cen- trifugal ultrafiltration devices (Amicon, Beverly, MA). An- tibodies were then buffer exchanged into DPBS using Seph- Materials and Methods adex G-25 desalting columns (Pierce, Rockford, IL). Pro- Materials. Tissue Culture Medium 199, bovine serum tein concentration was determined using the BCA albumin (BSA), concanavalin A (ConA), phytohemagluti- modification of the Lowry procedure (20). nin (PHA), Taq polymerase, glutamine, penicillin-strepto- For FITC labeling, antibodies purified as described mycin, bicinchoninic acid (BCA), and ovalbumin (OVA) above were buffer-exchanged into 0.1 M carbonate pH 9.6, were purchased from Sigma (St. Louis, MO). Trizol reagent and 1 mg of 3.7 mg/ml antibody was incubated with 120 ␮l for RNA purification was purchased from Life Technolo- of 10 mg/ml FITC in DMSO. After 2 hr in the dark at room gies (Gaithersburg, MD). Kits for RT-PCR (Retroscript) temperature, labeled protein was separated from excess were purchased from Ambion (Houston, TX) and included FITC by desalting on a G-25 Sephadex chromatography a pair of PCR primers for the housekeeping gene coding for column. The flourescence:protein ratio was then estimated the ribonucleoprotein S-15 (forward:5Ј-TTCCGCAAGTT- by UV absorbance, and the protein concentration was de- CACTACC-3Ј; reverse: 5Ј-CGGGCCGGCCATGCTTTA- termined by the BCA assay. CG-3Ј; 361-bp product). The primers for PCR of OvIL-2 Purification of OvUS. The ovine uterine serpin was (forward: 5Ј-CTT-GTC-TTG-CAT-TGC-ACT-AAC-TCT- purified from uterine fluid collected from unilaterally preg- TG-3Ј; reverse: 5Ј-TCC-ATT-TGT-TCA-GAA-ATT-CTA- nant ewes. Prior to breeding, one uterine horn was surgi- CAG-CG-3Ј; 413-bp product) were designed to anneal to cally ligated, and the ipsilateral ovary was removed to re- regions of the bovine sequence (19) that are 100% homolo- strict the subsequent pregnancy to one uterine horn (21). At gous to the ovine sequence; these primers were manufac- approximately Day 140 of pregnancy, ewes were sacrificed, tured by Research Genetics (Huntsville, AL). Recombinant and fluid was collected from the nongravid uterine horn. human IL-2 was purchased from Genzyme (Boston, MA). Uterine fluid was clarified by centrifugation (10,000g for 30 Molecular weight markers (100 bp) were purchased from min at 4°C) and stored at −20°C until purification of OvUS Promega (Madison, WI). Flourescein isothiocynate (FITC) by a combination of cation-exchange and gel-filtration chro- monomer for conjugation of antibodies was purchased from matography as previously described (2). After purification, Molecular Probes (Eugene, OR). Horse and mouse sera OvUS was buffer-exchanged into DPBS and concentrated were purchased from Atlanta Biologicals (Norcross, GA). using Centricon ultrafiltration devices (Amicon, Beverly, Falcon tissue culture–treated 96-well plates, Falcon 12 mm MA). Protein purity was confirmed (>95%) by SDS-PAGE × 75 mm polystyrene tubes, and all other chemicals were under reducing conditions on 10% (w/v) polyacrylamide purchased from Fisher Scientific (Pittsburgh, PA). gels and staining with Coomassie Blue R-125. Concentra- Antibodies. Mouse ascites containing antibody to tion of the protein was determined by the BCA modification ovine CD25 (Clone 9–14) was generously provided by Dr. of the Lowry protein assay using BSA as the standard (20). Andrew Nash (Center for Animal Biotechnology, Univer- Lymphocyte Proliferation Assays. Procedures sity of Melbourne, Australia) and was used at a 1:200 di- for purification of peripheral blood mononuclear leukocytes lution in PBS for all experiments. Hybridoma cells that (PBML) using Ficoll-Paque and mitogen-stimulated lym- secrete an antibody to the ovine ␥␦-T cell receptor (Clone phocyte proliferation were as previously described (13, 22).
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