Proinflammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses

This information is current as Dennis W. Thomas, Paulo N. Rocha, Chandra Nataraj, Lisa of October 1, 2021. A. Robinson, Robert F. Spurney, Beverly H. Koller and Thomas M. Coffman J Immunol 2003; 171:6389-6395; ; doi: 10.4049/jimmunol.171.12.6389 http://www.jimmunol.org/content/171/12/6389 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Proinﬂammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses1

Dennis W. Thomas,2* Paulo N. Rocha,2* Chandra Nataraj,* Lisa A. Robinson,* Robert F. Spurney,* Beverly H. Koller,† and Thomas M. Coffman3*

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE2, in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A2 synthesis. To study the immunoregulatory actions of thromboxane A2, we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to Downloaded from alloantigen were substantially reduced in TP؊/؊ spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs. The Journal of Immunology, 2003, 171: http://www.jimmunol.org/ 6389Ð6395.

rostaglandins and thromboxanes (TX)4 that are produced lated to broad-based COX inhibition in tissues such as the through the cyclooxygenase (COX) pathway of arachi- gastrointestinal tract and the kidney, precise identification of proin- P donic acid metabolism influence many biological pro- flammatory prostanoids provides a template for the development cesses (1, 2), including inflammation and immune responses (3). of more specific anti-inflammatory therapies.

During inflammation, prostanoids may affect disease pathogenesis TXA2 is one prostanoid with actions that appear to be primarily by modulating tissue responses and/or by directly regulating im- proinflammatory. TXA2 is formed through the sequential metab- mune cell activity. Nonsteroidal anti-inflammatory drugs olism of arachidonic acid by COX and TX synthase (4). In aqueous by guest on October 1, 2021 (NSAIDs) inhibit COX and consequently block synthesis of all solution, TXA2 has a very short t1/2 and is rapidly hydrolyzed to prostanoids (4). Because NSAIDs are potent anti-inflammatory form the stable, physiologically inactive metabolite TXB2. The agents, it has been inferred that prostanoids have dominant actions actions of TXA2 are mediated through binding to cell surface re- to promote inflammation. However, individual prostanoids may ceptors. TX receptors (by convention designated TP for TX-pros- have distinct and even opposing effects on inflammatory and im- tanoid receptors) have been cloned from human, rat, and mouse mune responses. For example, anti-inflammatory actions of PGE2 and, like other prostanoid receptors, they belong to the superfamily have been documented in a number of systems (2, 3, 5Ð10). This of G protein-coupled receptors (11Ð14). TP receptors are ex- suggests that the actions of prostanoids in immune and inflamma- pressed in a number of tissues, including thymus, lung, kidney, tory responses are complex. Precise identification of the prostanoid spleen, and placenta (11Ð14). Although pharmacological studies metabolites that promote inflammation and stimulate immune re- had suggested heterogeneity of function and agonist binding char- sponses will clarify the role of the COX pathway in disease patho- acteristics (15Ð17), only a single TP receptor gene has been iden- genesis. Because NSAIDs have a number of untoward actions re- tified, and our analysis of mice in which the TP receptor gene was disrupted by gene targeting is consistent with the existence of only *Division of Nephrology, Duke University and Durham Veterans Affairs Medical one TP receptor gene (18). Centers, Durham, NC 27705; and †Department of Medicine, University of North TXA2 acting through the TP receptor has a number of biologic Carolina, Chapel Hill, NC 27514 actions that may be relevant to its role in the pathogenesis of im- Received for publication January 21, 2003. Accepted for publication October 6, 2003. mune injury. First, it is a potent vasoconstrictor and platelet ag- The costs of publication of this article were defrayed in part by the payment of page gregant (1, 19). Second, TX directly stimulates biosynthesis of charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. extracellular matrix proteins (20) and may also alter their metab- 1 This work was supported by the Research Service of the Department of Veterans olism (21), thereby promoting fibrosis and scarring. Finally, phar- Affairs and National Institutes of Health Grants AI001389 and DK38103. macological studies have suggested that TXA2 may enhance cer- 2 D.W.T. and P.N.R. contributed equally to this work. tain lymphocyte and macrophage functions, and thus may also 3 Address correspondence and reprint requests to Dr. Thomas M. Coffman, Building have direct immunomodulatory effects (22Ð25). However, the na- 6/Nephrology (111I), Veterans Affairs Medical Center, 508 Fulton Street, Durham, ture and mechanisms of these putative effects of TXA in immune NC 27705. E-mail address: [email protected] 2 responses are not clear. We have used TP receptor-deficient mice 4 Abbreviations used in this paper: TX, thromboxane; COX, cyclooxygenase; MIF, macrophage migration inhibitory factor; MIP, macrophage-inflammatory protein; to examine the contribution of TP receptors to the regulation of NSAID, nonsteroidal anti-inflammatory drug; TP, TX-prostanoid; TX, thromboxane. cellular immune responses. Our studies suggest that activation of

TP receptors promotes T cell proliferation, and these actions con- removed by panning using a polyvalent anti-mouse Ig (Sigma-Aldrich). tribute to immune-mediated tissue injury. Nonadherent cells were gently removed and passed through sterilized nylon wool columns (Polysciences, Warrington, PA; 108 cells/g nylon wool) to remove macrophages. The purity of the resultant cells was measured by Materials and Methods flow cytometry using markers for T lymphocytes (CD3), B lymphocytes Animals (B220), NK cells (NK1.1; all Abs from BD PharMingen), and macrophages (F4/80; Serotec, Raleigh, NC). Generally, T cells represented Ͼ85Ð Mice lacking TP receptors were generated by gene targeting, as previously 90% of eluted cells. The resulting cells were then counted and plated in reported (18). To obviate any confounding effects of background genes in round-bottom 96-well plates at the indicated ratios with irradiated alloge- our studies, the TP mutation was backcrossed onto C57BL/6 and BALB/c neic stimulator splenocytes, using 3.5 ϫ 105 stimulator cells/well. After backgrounds for more than six generations. All assays of cellular immunity 3 ϩ ϩ Ϫ Ϫ incubation for 3 or 4 days at 37¡C with 5% CO ,[H]thymidine incorpo- were performed with TP / and TP / cells from both genetic back- 2 ration was determined, as described above. grounds, and findings were similar independent of genetic background. The ϩ ϩ Ϫ Ϫ In some experiments, pure populations of TP / and TP / splenic T genotype of individual mice was determined by Southern blotting, as de- cells were isolated using a Pan T Cell Isolation Kit (Miltenyi Biotec, Au- scribed (18). All mice were bred and maintained in the American Associ- burn, CA). Purity of the cell population was confirmed by fluorocytometry ation for the Accreditation of Laboratory Animal Care-accredited animal and averaged 97%. The enriched populations of T cells were added to wells facility of the Durham Veterans Affairs Medical Center, according to Na- coated with anti-CD3 Ab (BD Biosciences, Bedford, MA) or were stimu- tional Institutes of Health guidelines. lated with 6.25 ng/ml PMA and 500 mg/ml ionomycin. After incubation for 3 Mitogen stimulation of lymphocytes 18hat37¡C with 5% CO2,[H]thymidine incorporation was determined, as described above. Splenocyte suspensions were prepared from wild-type and TP-deficient mice by gently grinding the spleen between glass slides. The cells were Expression of T cell surface markers ϳ ϫ then centrifuged at 900 g to obtain a cell pellet. The pellet was re- ϩ/ϩ Ϫ/Ϫ

ϭ Downloaded from suspended in sterile RBC lysis buffer (150 mM NH Cl, 10 mM KHCO , Splenocytes from C57B6 TP and TP mice (n 5 for each group) 4 3 ␮ 130 mM EDTA) and incubated for 4 min at room temperature. Four mil- were stimulated with 1 g/ml of anti-CD3 Ab, as described above. At liliters of medium were added to stop the lysis reaction, and the cells were baseline and 3, 6, 24, and 48 h after stimulation, cells were washed twice washed three times with PBS. A total of 2 ϫ 105 cells was added to in FACS buffer (Dubecco’s PBS, 2% FCS, and sodium azide) and incu- ␮ individual wells of a 96-well plate, and the mitogens, PHA (Sigma-Aldrich, bated with anti-mouse CD16/CD32 (mouse Fc block, 1 g/million cells) St. Louis, MO) or anti-CD3 mAb (BD PharMingen, Lexington, KY), were for 15 min to reduce nonspeciﬁc binding. The cells were then stained for added at the ﬁnal concentrations indicated. The cells were cultured for 3 30 min with one of the following Abs: PE-labeled anti-CD25, anti-CD28, anti-CD69, and anti-CD154 along with a mix of FITC-labeled anti-CD4

days at 37¡C in a humidified incubator containing 5% CO2, and then 0.5 http://www.jimmunol.org/ ␮Ci of [3H]thymidine was added to each well. Following an additional 18 h and anti-CD8. For detection of CD152, cells were first labeled with the ␮ of incubation, [3H]thymidine incorporation was assessed by harvesting anti-CD4/anti-CD8 mix for 30 min, then permeabilized with 250 l Cyto- cells onto a glass fiber filtermat using an automated Tomtec Cell Harvester fix/Cytoperm solution. After 20 min, PE-labeled anti-CD152 Ab was added (Wallac/PerkinElmer, Gaithersburg, MD). Filter-bound radioactivity was and, after a 30-min incubation, was washed twice in PermWash solution. measured using a scintillation counter. Within each experiment, individual Cells were analyzed in a FACS scanner and analyzed for the intensity and conditions were examined in triplicate or quadruplicate samples. For each percentage of T cells (FITC CD4, CD8 gate) expressing each marker. All mitogen, a minimum of three experiments was performed (9Ð12 observa- Abs used in these studies were purchased from BD PharMingen. tions) with cells from each background (C57BL/6 and BALB/c). RNase protection assays Mixed lymphocyte response Total cellular RNA was extracted from TPϩ/ϩ and TPϪ/Ϫ lymphocytes Primary one-way MLR was performed, as described previously (26). Sin- following stimulation by anti-CD3 Ab or in MLR with the RNeasy kit by guest on October 1, 2021 gle-cell suspensions of responder splenocytes were reconstituted at various (Qiagen, Valencia, CA), according to manufacturer’s instructions, and was concentrations and were mixed with 4 ϫ 105 irradiated stimulator spleno- stored in RNase-free water at Ϫ70¡C. To detect chemokine and cytokine cytes at the indicated ratios. A total of 50 ␮l of each cell suspension was mRNA, commercially available multiprobe template sets (Riboquant; BD 32 added to individual wells of a 96-well plate. In some experiments, MLR PharMingen) were labeled with [␣- P]UTP (PerkinElmer), according to manufacturer’s instructions, and then diluted to a concentration of 300,000 was assessed in the presence of pharmacological agents that affect TXA2 synthesis or signaling, including: U46619 (TP agonist; Cayman Chemicals, cpm/␮l of hybridization buffer. All reagents used in probe synthesis were Ann Arbor, MI), SQ 29,548 (TP antagonist; Cayman Chemicals), and car- obtained from BD PharMingen (In Vitro Transcription Kit, catalogue. boxyheptyl imidazole (TX synthase inhibitor; Sapphire Bioscience, Sid- 45004K). RNA samples were thawed on ice and 5Ð10 ␮g aliquoted for ney, Australia). Plates were incubated at 37¡C in a humidified incubator studies. RNA samples were completely dried on a vacuum evaporator cen- trifuge without heat and solubilized in 8 ␮l of hybridization buffer by containing 5% CO2. After varying times of incubation, the plates were pulsed with 0.5 ␮Ci/well of [3H]thymidine. Following an additional 18-h gently vortexing for 3 min. Samples were mixed with 2 ␮l of the diluted incubation, the cells were harvested, as described above, and the cell-as- probe and transferred to a hybridization oven set at 90¡C. The temperature sociated counts were determined by scintillation counting. The values were was immediately turned down to 56¡C, and samples were allowed to hy- expressed as specific cpm (counts from wells containing responders and bridize with the probe for 12Ð16 h. The following probe sets were used: stimulators minus the average value of wells containing responders alone). MCK1 (IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-␥), MCK2b Within each experiment, individual conditions were examined in triplicate (IL-12, IL-10, IL-1, IL-18, IL-6, IFN-␥, macrophage migration inhibitory or quadruplicate samples. factor (MIF)), MCK3b (TNF-␤, lymphotoxin ␤, TNF-␣, IL-6, IFN-␥, IFN-␤, TGF-␤, MIF), and MCK5 (lymphotactin, RANTES, eotaxin, mac- TXB2 generation by cultured lymphocytes rophage-inflammatory protein-1␤ (MIP-1␤), MIP-1␣, MIP-2, IFN-␥-inducible protein-10, monocyte chemoattractant protein-1, T cell activation- To determine whether TX was generated during anti-CD3 stimulation of T 3). RNase protection assays were performed using the RNase Protection ϫ 6 cells, splenocyte suspensions were prepared and 3.5 10 cells were Assay Kit (BD PharMingen; catalogue 45014K) and following the protocol placed in each well of 12-well plates with 1 ml of complete medium with suggested by the manufacturer. Briefly, RNase-protected samples were re- 10% FCS. Anti-CD3 Ab was added in concentrations ranging from 0 to 10 moved from the oven and subjected to sequential digestion with RNase and mg/ml. After 72 h, the cultures were centrifuged and the supernatants were Ϫ proteinase K. After treatment with chloroform:isoamyl alcohol, the aque- removed and stored at 70¡C. Concentrations of TXB2, the stable metab- ous phase was removed and RNA precipitated with 4 M ammonium acetate olite of TXA2, were later measured by RIA, as described previously (27). and 100% ethanol, and samples were incubated for 30 min at Ϫ70¡C. RNA Within each experiment, the effect of each concentration of Ab was tested was then pelleted, washed with 90% ethanol, air dried, and resuspended in in triplicate cultures in three independent experiments. TXB2 generation 5 ␮lof1ϫ loading buffer. Samples were heat blocked (90¡C) for 3 min, was determined by subtracting background levels from the unstimulated then run on acrylamide gels. Gels were covered with Saran wrap and dried cultures that did not receive anti-CD3 Ab and dividing by cell number. under vacuum at 80¡C for 45 min. The dried gels were placed on film in T cell enrichment a cassette with an intensifying screen and developed at Ϫ70¡C. The exposure time ranged from 2 h (for the housekeeping genes) to 5 days (for Splenocyte suspensions were washed in PBS, and contaminating RBC faint bands). Films were scanned and bands were analyzed as a ratio of were lysed by incubating cells in cold (4¡C) PBS containing 0.1 mM target RNA/GAPDH control using the Scion Image for Windows program. Ϯ Na2EDTA, 0.15 M NH4Cl, and 1.0 mM KHCO3. B lymphocytes were Data were expressed as mean arbitrary units SD. We performed a total The Journal of Immunology 6391 of 10 experiments using male and female mice, on both C57B6 and BALB/c backgrounds, as responders (total ϭ 21 wild-type and 21 TPϪ/Ϫ animals). Mouse heart transplantation Heterotopic cardiac transplants in mice were performed, as described previously (26). Recipient BALB/c (H-2d)TPϩ/ϩ and TPϪ/Ϫ mice were anes- thetized with isoflurane and prepared by separation of the aorta and vena cava between the renal vessels and the bifurcation of the iliac arteries. The donor heart was harvested from an MHC-disparate TPϩ/ϩ C57BL/6 (H-2b) mouse, and an end-to-end anastomosis was created between the recipient aorta and the ascending aorta from the donor heart. A similar anastomosis was created between recipient vena cava and the superior vena cava of the donor heart. The total ischemia time averaged 15 min and did not vary between the groups. Surgical mortality of the recipients was less than 10%. Allograft survival was monitored by direct palpation of the transplanted heartbeat through the abdominal wall, and graft failure was defined as the FIGURE 2. Reduced response to anti-CD3 Ab in TP-deficient lympho- cessation of palpable heartbeat. cytes. Anti-CD3 Ab was added to single-cell suspensions of splenocytes In a separate group of animals, hearts from wild-type C57BL/6 mice Ⅺ Ϫ/Ϫ f were transplanted into BALB/c TPϩ/ϩ (n ϭ 6) and TPϪ/Ϫ (n ϭ 9) recip- from either wild-type ( )orTP mice ( ) in concentrations ranging ␮ 3 ients, and all transplant recipients were treated with subtherapeutic doses from 0.1 to 10 g/ml. Proliferation was assessed as [ H]thymidine incor- (20 mg/kg by i.p. injection) of cyclosporin A beginning on the day of poration. Data are depicted in cpm as specific cell-associated counts ϩ/ϩ p Ͻ 0.002 vs TP .) Downloaded from ,ء) .(transplant and continuing for 7 days. Allograft survival was monitored, as (ϫ1000 described above. To evaluate allograft histopathology, heart transplants from additional animals (n ϭ 9) were removed on day 7 after transplantation, fixed in 10% buffered Formalin, sectioned, and stained with H&E, eration of wild-type BALB/c cells and that the intensity of prolif- and the slides were reviewed by a pathologist (P. Ruiz), who was masked eration was dose proportional over a range of concentrations from to the experimental groups. The severity of rejection, interstitial infiltrates, ␮ myocyte injury, and vascular abnormalities were each graded separately 0.6 to 5 g/ml. In TP-deficient BALB/c splenocytes, PHA also using a semiquantitative scale, in which 0 was no abnormality, and 1, 2, caused a similar dose-dependent increase in proliferation. How- and 3 represented mild, moderate, and severe abnormalities, as described ever, at each concentration of PHA, proliferation was 20Ð45% http://www.jimmunol.org/ previously (7). lower in TP-deficient compared with wild-type splenocytes. Sim- The reverse experiment was also performed. Hearts from TPϩ/ϩ (n ϭ 7) Ϫ Ϫ ilar findings were observed with the C57BL/6 line (data not or TP / (n ϭ 9) C57BL/6 mice were transplanted into wild-type BALB/c animals; recipients were treated with 20 mg/kg of cyclosporin A; and al- shown). ϩ ϩ Ϫ Ϫ lograft survival was determined, as described above. We next compared proliferation of TP / and TP / BALB/c cells using a more specific T cell mitogen, anti-CD3 Ab. As shown Statistical analysis in Fig. 2, anti-CD3 Ab induced a brisk proliferative response in The values for each parameter within a group are expressed as the mean Ϯ wild-type splenocytes, and over a range from 0.1 to 10 ␮g/ml the SEM. For comparisons between TPϩ/ϩ and TPϪ/Ϫ groups, statistical sig-

magnitude of the response was proportional to the Ab concentra- by guest on October 1, 2021 nificance was assessed using an unpaired t test for normally distributed tion. A similar dose-dependent increase in proliferation was ob- data. A paired t test was used for comparisons within groups. For non- parametric analyses, a Mann-Whitney U test was used. served in TP-deficient cells, but, as in the PHA experiments, the magnitude of the proliferative response was attenuated by 20Ð25% Results in the anti-CD3-treated TPϪ/Ϫ splenocytes compared with wild- ϩ ϩ To examine whether the absence of TP receptors influences lym- type cells. A similar difference was seen between C57BL/6 TP / Ϫ Ϫ phocyte functions, we first compared proliferation of TPϩ/ϩ and and TP / cells (data not shown). To confirm that generation of TPϪ/Ϫ splenocytes following exposure with the nonspecific mito- TX is augmented in this setting, we measured concentrations of gen PHA. Fig. 1 shows that PHA markedly stimulated the prolif- TXB2, the stable metabolite of TXA2, in supernatants of bulk cultures of wild-type splenocytes. Results from a representative experiment are depicted in Fig. 3. Compared with cultures that re-

ceived no mitogen, TXB2 generation was augmented in the cultures that were exposed to anti-CD3 Ab (68 Ϯ 3 pg/ml/106 cells

FIGURE 1. PHA-induced proliferation is attenuated in TP-deﬁcient FIGURE 3. TX generation by cultured splenocytes. Bulk cultures of lymphocytes. PHA was added to single-cell suspensions of splenocytes splenic lymphocytes were stimulated with anti-CD3 Ab in concentrations ϩ/ϩ Ⅺ Ϫ/Ϫ f ␮ from either TP ( )orTP mice ( ) in concentrations ranging from ranging from 0 to 10 g/ml. After 24 h, TXB2 concentrations in the su- ␮ 3 0.6 to 5 g/ml. Proliferation was assessed as [ H]thymidine incorporation. pernatant were measured by RIA. TXB2 generation was augmented in the p ϭ 0.012 vs 0 mg/ml; ¤, p Ͻ 0.0001 ,ء) .anti-CD3 Ab-stimulated cultures ,ء) .(Data are depicted in cpm as speciﬁc cell-associated counts (ϫ1000 p Ͻ 0.002 vs TPϩ/ϩ.) vs 0 mg/ml.) 6392 TX RECEPTORS AND CELLULAR IMMUNITY

with 10 mg/ml anti-CD3 Ab vs 2 Ϯ 2 pg/ml/106 cells in controls; p Ͻ 0.0001). TXB2 generation was similarly augmented in stimulated TP-deficient splenocytes (data not shown). To examine whether TP receptors contribute to proliferation in a model cellular immune response, we measured alloantigen-induced proliferation in a one-way MLR. After 5 days in culture with irradiated, allogeneic stimulators, proliferative responses were significantly blunted in TP receptor-deficient cells compared with controls, across a range of stimulator cell concentrations (Fig. 4). Depending on the responder to stimulator ratio, proliferation by TP-deficient cells was reduced by 35Ð80%. This was not simply a difference in the kinetics of the response because proliferation by TP receptor-deficient cells was also reduced after 3, 4, and 6 days FIGURE 5. Effects of pharmacological inhibitors on proliferation of in MLR (data not shown). In contrast, there was no difference in wild-type lymphocytes in MLR. One-way MLR was performed using wild- ϩ/ϩ Ϫ/Ϫ the level of proliferation in reverse MLR using TP or TP type responder cells in the presence of medium alone, the NSAID indo- cells as stimulators (data not shown). When purified T cells were methacin, the TP receptor antagonist SQ 29,548, or the TX synthase in- p Ͻ ,ء ;used as responders, significantly less proliferation was once again hibitor carboxyheptyl imidizole (CI). (‡, p Ͻ 0.005 vs media alone observed in the T cells lacking TP receptors compared with con- 0.01 vs media alone.) trols (6141 Ϯ 567 cpm-TPϩ/ϩ vs 3822 Ϯ 363 cpm-TPϪ/Ϫ; p ϭ 0.0065). Thus, stimulation of the TP receptor occurs as a part of Downloaded from

the cellular alloimmune response, and activation of the TP receptor Ϫ/Ϫ promotes lymphocyte proliferation. Similar to the other in vitro TP cells. As before, the presence of the TX synthase inhibitor ϩ/ϩ signiﬁcantly attenuated proliferation of wild-type cells in the assays of cellular immunity, robust differences between TP and ϩ/ϩ Ϫ/Ϫ MLR. This inhibition could be completely reversed in TP TP cells were observed in MLR using both the C57BL/6 and cells ␮ BALB/c lines (data not shown). by coadministration of 3 M of the TP agonist U46619 along with

Ϫ/Ϫ the TX synthase inhibitor. The apparent discrepancy between the http://www.jimmunol.org/ To confirm that the defect in proliferation of TP cells was due to the absence of TP receptor signaling and not to a subtle concentration of the agonist U46619 that was required to reverse the actions of the inhibitor (3 ␮M) and the concentrations of TXB developmental abnormality in the TP receptor-deficient mice, one- 2 way MLR experiments were performed in the presence of phar- that were measured in the supernatants (pg/ml) described above is most likely due to the relatively low affinity of U46619 for the TP macological agents that alter the synthesis or activity of TXA .As 2 receptor (K Ϸ300 nM) compared with the natural ligand TXA shown in Fig. 5, treatment of wild-type splenocytes with the TP d 2 (K ϷpM). receptor antagonist SQ 29,548 caused potent inhibition of alloan- d As we had observed previously, proliferation of TPϪ/Ϫ respond- tigen-induced proliferation. Administration of the TX synthase in- ϩ/ϩ hibitor (carboxyheptyl imidazole) caused a virtually identical re- ers in MLR was significantly lower than TP controls. However, by guest on October 1, 2021 duction in the proliferative response. In contract indomethacin, an in contrast to the wild-type cells, exposure to the TX synthase inhibitor did not further reduce proliferation of TP-deficient cells. NSAID that inhibits synthesis of all prostanoids, including TXA2, significantly enhanced proliferation in the MLR, highlighting the Likewise, administration of the TP agonist along with the TX syn- complexity of the actions of the COX pathway on immune thase inhibitor did not affect proliferation of the TP-deficient cells. responses. To investigate whether attenuated adaptive immune responses in In additional experiments depicted in Fig. 6, the specificity of TP deficiency were associated with altered expression of key sur- the pharmacological effects of TX inhibition was further explored face proteins on T cells, we compared expression of a range of ϩ/ϩ markers, including CD25, CD28, CD69, CD152, and CD154, in by comparing responses to TX synthase inhibition in TP and

FIGURE 6. TX synthase inhibitor and TP agonist alter proliferation of FIGURE 4. Proliferation in MLR is reduced when responder cells lack TPϩ/ϩ, but not TPϪ/Ϫ responders in MLR. One-way MLR was performed TP receptors. MLR was performed using splenocytes from (H-2d)TPϩ/ϩ using splenocyte suspensions from TPϩ/ϩ (left)orTPϪ/Ϫ (right) mice. and TPϪ/Ϫ mice as responders and irradiated splenocytes from (H-2b) mice MLR was performed with medium alone (Ⅺ), with the TX synthase in- as stimulators. Proliferation, measured as [3H]thymidine incorporation in hibitor carboxyheptyl imidazole (f), or with the TX synthase inhibitor ϩ cell-speciﬁc cpm, is signiﬁcantly reduced in splenocytes from TPϪ/Ϫ mice 3 ␮M TP agonist U46619 (u). (‡, p Ͻ 0.001 vs TPϩ/ϩ media alone or p Ͻ 0.001 vs TPϩ/ϩ ,ء ;f) compared with TPϩ/ϩ controls (Ⅺ) across a range of stimulator con- TPϩ/ϩ with carboxyheptyl imidazole and U46619) (.p Ͻ 0.001 vs TPϩ/ϩ.) media alone ,ء) .centrations The Journal of Immunology 6393

conducted using purified populations (Ͼ97%) of T cells. As shown in Fig. 7, proliferation of highly purified TP-deficient T cells stimulated with anti-CD3 Ab was reduced by Ͼ25% compared with wild-type controls. This was similar to our previous results using mixed splenocytes (Fig. 2). By contrast, exposure of TPϩ/ϩ and TPϪ/Ϫ cells to PMA with ionomycin caused brisk proliferation in both groups. By delivering this maximal, Ag-independent calcium signal, the differences in the response between TP-deficient and wild-type cells that we had seen with other mitogens were abolished. To determine whether the actions of TP receptors to promote T cell proliferation that we observed in vitro were relevant to im- FIGURE 7. Proliferation of highly enriched T cells stimulated by anti- mune responses in the intact animal, we turned to a well-charac- CD3 Ab or PMA and ionomycin. Highly enriched (Ͼ95% pure) popula- terized model of heterotopic cardiac transplantation. In these stud- ϩ/ϩ Ϫ/Ϫ tions of T cells were isolated from TP (Ⅺ) and TP (f) mice. The ies, we determined graft survival when the donor, the recipient, or cells were stimulated with anti-CD3 Ab on coated plates (left) or a com- both donor and recipient were TP deficient. In this aggressive bination of the phorbol ester PMA and the ionophore ionomycin. Prolif- model of allograft rejection, TPϩ/ϩ (H-2b) hearts transplanted into eration measured as [3H]thymidine incorporation was determined 24 h TPϩ/ϩ (H-2d) recipients were rejected rapidly, and the mean time later. to graft failure was 9 Ϯ 1 days. Survival of TPϩ/ϩ hearts in TP- deficient recipients (8 Ϯ 1 days) was virtually identical with Downloaded from anti-CD3-stimulated TPϩ/ϩ and TPϪ/Ϫ lymphocytes by cytoflu- controls. orometry. Expression of these proteins was not significantly al- Although our in vitro studies indicated a significant role for TP tered by TP deficiency, and there were no significant differences receptors to promote cellular alloimmune responses, the absence of between wild-type and TP-deficient T cells in the intensity or the TP receptors on recipient immune cells alone was not sufficient to proportion of cells expressing these markers at baseline and 3, 6, prolong allograft survival in this aggressive model of rejection. In 12, and 48 h following TCR cross-linking (data not shown). a further attempt to uncover a contribution of these actions of TP http://www.jimmunol.org/ Next, we performed RNase protection assays to detect cytokine receptor activation to an inflammatory response in vivo, we per- mRNA in TPϩ/ϩ and TPϪ/Ϫ lymphocytes during anti-CD3 stim- formed an additional transplant experiment. In this study, hearts ulation and MLRs. After several independent experiments, we from wild-type donors were transplanted into MHC-disparate re- could not find consistent differences in mRNA expression for: cipients that were wild type or TP deficient. Both groups of recip- IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-␥, IL-12, ients were then treated with a subtherapeutic dose of cyclosporin A IL-10, IL-1, IL-18, MIF, TNF-␤, lymphotoxin ␤, TNF-␣, IFN-␤, (20 mg/kg/day). As depicted in Fig. 8, following treatment with TGF-␤, lymphotactin, RANTES, eotaxin, MIP-1␤, MIP-1␣, low dose cyclosporine, allograft survival was significantly pro- MIP-2, IFN-␥-inducible protein-10, monocyte chemoattractant longed in the recipients lacking TP receptors (16 Ϯ 2 days) com- by guest on October 1, 2021 protein-1, and T cell activation-3 between wild-type and TP pared with controls (12 Ϯ 1; p ϭ 0.008). However, in the converse knockout animals. experiment, the absence of TP receptors in donor tissue did not

In a number of cell types, the TP receptor is linked to G␣q and significantly enhance survival of allografts transplanted into wild- signals through phospholipase C and intracellular calcium (28Ð type recipients (13 Ϯ 1vs12Ϯ 1 days; p Ͼ 0.15). 30). To determine whether alterations in calcium signaling might There was a significant attenuation of the pathological severity be responsible for attenuated proliferative responses observed in of rejection in the TP-deficient recipients, corresponding with the TP-deficient lymphocytes, we tested responses to PMA plus iono- improvement in graft survival that was observed in this group. This mycin. Moreover, to clearly document that these effects of TP was reflected by a reduction in the overall acute rejection score receptors are due to direct effects on T cells, these studies were (1.3 Ϯ 0.3 vs 2.4 Ϯ 0.2; p ϭ 0.003), in interstitial inflammatory cell infiltrates (1.2 Ϯ 0.2 vs 2.5 Ϯ 0.3; p ϭ 0.007), and in perivas- cular injury (1.2 Ϯ 0.2 vs 2.25 Ϯ 0.25; p ϭ 0.001). There was also a tendency toward reduced myocyte injury in the allografts transplanted into TP-deficient recipients (1.2 Ϯ 0.2 vs 2.4 Ϯ 0.4; p ϭ 0.1). Thus, the absence of TP receptors on immune cells of the recipients is sufficient to attenuate the intensity and severity of many of the key features of graft injury in this model.

Discussion The actions of COX metabolites of arachidonic acid in inflammation and immune responses are complex (3). These lipid mediators can evoke the cardinal features of inflammation (31) and may also influence inflammation by regulating the activities of immune and inflammatory cells (2, 3, 5Ð10). The overall impact of the COX pathway on cellular immune responses is determined by the profile

Ϫ Ϫ of prostanoid synthesis within the microenvironment along with FIGURE 8. Prolonged cardiac allograft survival in TP / recipients treated with low doses of cyclosporine. Heterotopic cardiac allografts were the repertoire of prostanoid receptors that is expressed by a par- placed in TPϩ/ϩ (f)orTPϪ/Ϫ (F) mice. Recipient animals were treated ticular immune cell population. In this regard, T cells are known to with cyclosporin A (20 mg/kg) from day 0 to 7 posttransplant. Cyclospor- express several classes of prostanoid receptors, including EP2 and Ϫ/Ϫ ine caused a significant prolongation of survival in the TP group com- EP4 receptors for PGE2 and the TP receptor for TXA2 (1, 3, 12). pared with controls (16 Ϯ 2vs12Ϯ 1 days, p ϭ 0.01). These receptors are coupled to different intracellular signaling 6394 TX RECEPTORS AND CELLULAR IMMUNITY pathways, and therefore might be expected to have different effects illustrated in Fig. 5, there was a stark contrast between the effects on cellular function. EP2 and EP4 receptors activate adenylate of the COX inhibitor, which actually enhanced proliferation, and cyclase (1, 12), and accumulation of cAMP is generally associated the TX inhibitors, which attenuated the MLR. Thus, modulation of with inhibition of T cell functions. By contrast, TP receptors cou- an inflammatory response may differ substantially when the ac- ple to phospholipase C and intracellular calcium (1, 12), signals tions of a specific prostanoid are inhibited compared with global that tend to promote immune cell activation. COX inhibition with an NSAID. Pharmacological studies have suggested a role for TP receptors Our finding that TP receptors expressed on T cells promote cel- in regulating cellular immunity. For example, TX synthase inhib- lular immune responses is reminiscent of recently described im- itors have been shown to diminish lymphocyte proliferation and T munoregulatory actions of the AT1 receptor for angiotensin II (26). cell cytotoxicity in vitro (22Ð24). However, interpretation of stud- We previously demonstrated that AT1 receptors stimulate T cell ies using TX synthase inhibitors is problematic because when TX proliferation spontaneously and during the course of MLR. The synthase is inhibited, PG endoperoxides such as PGH2 may accu- magnitude and character of the AT1 effects are very similar to mulate (32). These compounds can act as agonists at the TP re- those that we are reporting now for TP receptors. Because the AT1 ceptor and thus attenuate efficacy. Moreover, accumulated PGH2 and TP receptors may use G␣q and G␣13 (28Ð30, 33, 34) proteins may be used as substrate for synthesis of other prostanoids. This for signaling, it is possible that their immunomodulatory actions shunting of endoperoxide substrate to increase synthesis of other are mediated by common distal signals that are linked to these G prostanoids such as PGE2, which inhibits T cell responses (7), may proteins. Our studies indicate that alterations in TCR-dependent therefore produce cellular actions that are unrelated to inhibition of calcium signaling are critical to the actions of TP receptors to TX synthesis. regulate T cell proliferation, as exposure to ionomycin and PMA, Our studies using genetically altered mice unequivocally iden- which trigger an exaggerated, Ag-independent calcium signal, res- Downloaded from tify a role for TX in regulation of cellular immune responses. We cues the attenuated responsiveness of TP-deficient cells.

find that TXA2, acting through the TP receptor, augments the vigor Enhanced production of TX has been implicated in the patho- of mitogen-induced proliferation. This effect is apparent with a genesis of various immunological diseases. A role for TXA2 in the nonspecific mitogen such as PHA or with anti-CD3 Ab, a T cell- pathogenesis of autoimmune disease was suggested by Patrono et specific mitogen. To determine whether TP receptors stimulate al. (35), who found that urinary excretion of TX metabolites was lymphocyte proliferation in a more complex response, we used the enhanced in patients with active lupus nephritis. In these patients, http://www.jimmunol.org/

MLR as a model of the cellular activation by alloantigens. The there was an inverse correlation between urinary TXB2 excretion MLR is designed to mimic the conditions that might occur in a and creatinine clearance. In another study of patients with biopsy- transplanted organ when recipient immune cells are activated by proven lupus nephritis, infusions of a specific TX receptor antag- recognition of foreign MHC Ags expressed on the donor tissue. onist increased glomerular filtration rate and renal plasma flow by When the responder cell population was derived from mice that 25% (36). Kelley et al. (37) first demonstrated enhanced produc- lack TP receptors, we found that proliferative responses in MLR tion of TX in kidneys from autoimmune mice. In these murine were substantially attenuated. Proliferation was also reduced when models, treatment with TX antagonists preserved renal function, purified T cells from TPϪ/Ϫ mice were used as responders. A reduced glomerular capillary immune complex deposits, and less- by guest on October 1, 2021 similar attenuation was observed when highly enriched T cells ened glomerular inflammation (27, 38). A similar role for TXA2 were stimulated with anti-CD3 Ab. has been demonstrated in transplant rejection. Foegh et al. (39)

Along with its direct effects to influence T cell functions, TXA2 first demonstrated that excretion of TX metabolites was enhanced may also influence the maturation and development of the T cell during episodes of acute rejection in human renal allograft recip- repertoire in the thymus. TP receptors are expressed at high levels ients. Subsequent studies from several laboratories demonstrated in the thymus, most prominently in immature thymocyte popula- increased production of TX in animal models of rejection (40, 41) tions (25). Stimulation of TP receptors on these cells induces pro- and showed that TX inhibitors could prolong graft survival and grammed cell death, suggesting that TP receptors on thymocytes improve graft function (40, 42). might play a role in selection of maturing T lymphocytes (25). In pathological conditions such as autoimmune disease and However, in our previous analysis of TP-deficient mice, we found transplant rejection, there are several pathways that could be used no significant alteration in the size, histopathology, or cellular con- by TXA2 to promote tissue injury, including actions on vascular stitution of the thymus or other lymphoid organs (18). Nonethe- and procoagulant systems in the target organ, stimulation of proin- less, to ensure that the differences that we observed in the cellular flammatory cytokine release from mononuclear cells (43), and by responses of TP-deficient mice were not due to a subtle develop- potentiating cellular immune responses through the mechanisms mental defect caused by the absence of TP receptors in the thymus, that we have described in this work. To begin to distinguish the we performed MLR experiments using wild-type cells in the pres- relative contributions of these pathways and to determine whether ence of a TX receptor antagonist or synthase inhibitor. Inhibition the actions of TP receptors to promote in vitro cellular immunity of TX synthesis or blockade of TP receptors similarly diminished are apparent in vivo, we used a model of cardiac allograft rejection allospecific proliferation in wild-type mice. The attenuation in which donor and recipient are completely mismatched at the achieved by pharmacological blockade, ϳ50% of normal, was in MHC locus, resulting in a very aggressive acute cellular rejection the range observed in the genetic experiments. The more exagger- response to the allograft (26). In unmodified rejection, we found ated defect seen in TP-deficient cells may reflect the complete that the absence of TP receptors on recipient tissues was not suf- absence of TP receptors compared with the more limited inhibition ficient to prolong graft survival. In contrast, if recipients are treated that can be achieved pharmacologically. Because the extent of the with subtherapeutic doses of cyclosporine, graft survival is pro- antiproliferative effect was similar with synthase inhibition and longed compared with wild-type controls when TP receptors are receptor blockade and proliferation was restored with TP agonist, absent only on recipient immune cells. The prolonged graft sur- the contribution of endoperoxide shunting to the actions of the TX vival observed in TP-deficient recipients is associated with signif- synthase inhibitor appears to be negligible in this circumstance. icant amelioration in the severity of histopathological manifesta- Furthermore, the absence of any effect of TX synthase inhibition in tions of rejection. This suggests that a contribution of TP-mediated the TPϪ/Ϫ cells confirms pharmacological specificity. Finally, as actions to promote cellular immunity can be uncovered when the The Journal of Immunology 6395 rejection response is attenuated. Moreover, these findings also im- 18. Thomas, D., R. Mannon, P. Mannon, A. Latour, J. Oliver, M. Hoffman, ply potential additive or cooperative actions of TP blockade with O. Smithies, B. Koller, and T. Coffman. 1998. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2. J. Clin. calcineuriun inhibition. 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Vertessen, H. Deckmyn, and J. Vermylen. 1985. Effects of receptors expressed on lymphocytes can modify the nature of a thromboxane A2 on lymphocyte proliferation. Cell. Immunol. 90:458. 23. Leung, K., and E. Mihich. 1980. Prostaglandin modulation of development of cellular immune response. Stimulation of TP receptors promotes cell-mediated immunity in culture. Nature 288:597. lymphocyte activation. Thus, TP receptors, along with their sig- 24. Ruiz, P., L. Rey, R. Spurney, T. Coffman, and A. Viciana. 1992. Thromboxane naling pathways, represent potentially useful therapeutic targets in augmentation of alloreactive T cell function. Transplantation 54:498. 25. Ushikubi, F., Y. Aiba, K. Nakamura, T. Namba, M. Hirata, O. Mazda, disorders such as transplant rejection and autoimmune disease. Y. Katsura, and S. Narumiya. 1993. Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis. J. Exp. Med. 178:1825. Acknowledgments 26. 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