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The Journal of Immunology, 2003, 171: 6389-6395.
Copyright © 2003 by The American Association of Immunologists

Proinflammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses 1

Dennis W. Thomas2,*, Paulo N. Rocha2,*, Chandra Nataraj*, Lisa A. Robinson*, Robert F. Spurney*, Beverly H. Koller{dagger} and Thomas M. Coffman3,*

* Division of Nephrology, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC 27705; and {dagger} Department of Medicine, University of North Carolina, Chapel Hill, NC 27514


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins and thromboxanes (TX) 4 that are produced through the cyclooxygenase (COX) pathway of arachidonic acid metabolism influence many biological processes (1, 2), including inflammation and immune responses (3). During inflammation, prostanoids may affect disease pathogenesis by modulating tissue responses and/or by directly regulating immune cell activity. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX and consequently block synthesis of all prostanoids (4). Because NSAIDs are potent anti-inflammatory agents, it has been inferred that prostanoids have dominant actions to promote inflammation. However, individual prostanoids may have distinct and even opposing effects on inflammatory and immune responses. For example, anti-inflammatory actions of PGE2 have been documented in a number of systems (2, 3, 5, 6, 7, 8, 9, 10). This suggests that the actions of prostanoids in immune and inflammatory responses are complex. Precise identification of the prostanoid metabolites that promote inflammation and stimulate immune responses will clarify the role of the COX pathway in disease pathogenesis. Because NSAIDs have a number of untoward actions related to broad-based COX inhibition in tissues such as the gastrointestinal tract and the kidney, precise identification of proinflammatory prostanoids provides a template for the development of more specific anti-inflammatory therapies.

TXA2 is one prostanoid with actions that appear to be primarily proinflammatory. TXA2 is formed through the sequential metabolism of arachidonic acid by COX and TX synthase (4). In aqueous solution, TXA2 has a very short t1/2 and is rapidly hydrolyzed to form the stable, physiologically inactive metabolite TXB2. The actions of TXA2 are mediated through binding to cell surface receptors. TX receptors (by convention designated TP for TX-prostanoid receptors) have been cloned from human, rat, and mouse and, like other prostanoid receptors, they belong to the superfamily of G protein-coupled receptors (11, 12, 13, 14). TP receptors are expressed in a number of tissues, including thymus, lung, kidney, spleen, and placenta (11, 12, 13, 14). Although pharmacological studies had suggested heterogeneity of function and agonist binding characteristics (15, 16, 17), only a single TP receptor gene has been identified, and our analysis of mice in which the TP receptor gene was disrupted by gene targeting is consistent with the existence of only one TP receptor gene (18).

TXA2 acting through the TP receptor has a number of biologic actions that may be relevant to its role in the pathogenesis of immune injury. First, it is a potent vasoconstrictor and platelet aggregant (1, 19). Second, TX directly stimulates biosynthesis of extracellular matrix proteins (20) and may also alter their metabolism (21), thereby promoting fibrosis and scarring. Finally, pharmacological studies have suggested that TXA2 may enhance certain lymphocyte and macrophage functions, and thus may also have direct immunomodulatory effects (22, 23, 24, 25). However, the nature and mechanisms of these putative effects of TXA2 in immune responses are not clear. We have used TP receptor-deficient mice to examine the contribution of TP receptors to the regulation of cellular immune responses. Our studies suggest that activation of TP receptors promotes T cell proliferation, and these actions contribute to immune-mediated tissue injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Mice lacking TP receptors were generated by gene targeting, as previously reported (18). To obviate any confounding effects of background genes in our studies, the TP mutation was backcrossed onto C57BL/6 and BALB/c backgrounds for more than six generations. All assays of cellular immunity were performed with TP+/+ and TP-/- cells from both genetic backgrounds, and findings were similar independent of genetic background. The genotype of individual mice was determined by Southern blotting, as described (18). All mice were bred and maintained in the American Association for the Accreditation of Laboratory Animal Care-accredited animal facility of the Durham Veterans Affairs Medical Center, according to National Institutes of Health guidelines.

Mitogen stimulation of lymphocytes

Splenocyte suspensions were prepared from wild-type and TP-deficient mice by gently grinding the spleen between glass slides. The cells were then centrifuged at ~900 x g to obtain a cell pellet. The pellet was resuspended in sterile RBC lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 130 mM EDTA) and incubated for 4 min at room temperature. Four milliliters of medium were added to stop the lysis reaction, and the cells were washed three times with PBS. A total of 2 x 105 cells was added to individual wells of a 96-well plate, and the mitogens, PHA (Sigma-Aldrich, St. Louis, MO) or anti-CD3 mAb (BD PharMingen, Lexington, KY), were added at the final concentrations indicated. The cells were cultured for 3 days at 37°C in a humidified incubator containing 5% CO2, and then 0.5 µCi of [3H]thymidine was added to each well. Following an additional 18 h of incubation, [3H]thymidine incorporation was assessed by harvesting cells onto a glass fiber filtermat using an automated Tomtec Cell Harvester (Wallac/PerkinElmer, Gaithersburg, MD). Filter-bound radioactivity was measured using a scintillation counter. Within each experiment, individual conditions were examined in triplicate or quadruplicate samples. For each mitogen, a minimum of three experiments was performed (9–12 observations) with cells from each background (C57BL/6 and BALB/c).

Mixed lymphocyte response

Primary one-way MLR was performed, as described previously (26). Single-cell suspensions of responder splenocytes were reconstituted at various concentrations and were mixed with 4 x 105 irradiated stimulator splenocytes at the indicated ratios. A total of 50 µl of each cell suspension was added to individual wells of a 96-well plate. In some experiments, MLR was assessed in the presence of pharmacological agents that affect TXA2 synthesis or signaling, including: U46619 (TP agonist; Cayman Chemicals, Ann Arbor, MI), SQ 29,548 (TP antagonist; Cayman Chemicals), and carboxyheptyl imidazole (TX synthase inhibitor; Sapphire Bioscience, Sidney, Australia). Plates were incubated at 37°C in a humidified incubator containing 5% CO2. After varying times of incubation, the plates were pulsed with 0.5 µCi/well of [3H]thymidine. Following an additional 18-h incubation, the cells were harvested, as described above, and the cell-associated counts were determined by scintillation counting. The values were expressed as specific cpm (counts from wells containing responders and stimulators minus the average value of wells containing responders alone). Within each experiment, individual conditions were examined in triplicate or quadruplicate samples.

TXB2 generation by cultured lymphocytes

To determine whether TX was generated during anti-CD3 stimulation of T cells, splenocyte suspensions were prepared and 3.5 x 106 cells were placed in each well of 12-well plates with 1 ml of complete medium with 10% FCS. Anti-CD3 Ab was added in concentrations ranging from 0 to 10 mg/ml. After 72 h, the cultures were centrifuged and the supernatants were removed and stored at -70°C. Concentrations of TXB2, the stable metabolite of TXA2, were later measured by RIA, as described previously (27). Within each experiment, the effect of each concentration of Ab was tested in triplicate cultures in three independent experiments. TXB2 generation was determined by subtracting background levels from the unstimulated cultures that did not receive anti-CD3 Ab and dividing by cell number.

T cell enrichment

Splenocyte suspensions were washed in PBS, and contaminating RBC were lysed by incubating cells in cold (4°C) PBS containing 0.1 mM Na2EDTA, 0.15 M NH4Cl, and 1.0 mM KHCO3. B lymphocytes were removed by panning using a polyvalent anti-mouse Ig (Sigma-Aldrich). 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 flow cytometry using markers for T lymphocytes (CD3), B lymphocytes (B220), NK cells (NK1.1; all Abs from BD PharMingen), and macrophages (F4/80; Serotec, Raleigh, NC). Generally, T cells represented >85–90% of eluted cells. The resulting cells were then counted and plated in round-bottom 96-well plates at the indicated ratios with irradiated allogeneic stimulator splenocytes, using 3.5 x 105 stimulator cells/well. After incubation for 3 or 4 days at 37°C with 5% CO2, [3H]thymidine incorporation was determined, as described above.

In some experiments, pure populations of TP+/+ and TP-/- splenic T cells were isolated using a Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). Purity of the cell population was confirmed by fluorocytometry and averaged 97%. The enriched populations of T cells were added to wells coated with anti-CD3 Ab (BD Biosciences, Bedford, MA) or were stimulated with 6.25 ng/ml PMA and 500 mg/ml ionomycin. After incubation for 18 h at 37°C with 5% CO2, [3H]thymidine incorporation was determined, as described above.

Expression of T cell surface markers

Splenocytes from C57B6 TP+/+ and TP-/- mice (n = 5 for each group) were stimulated with 1 µg/ml of anti-CD3 Ab, as described above. At baseline and 3, 6, 24, and 48 h after stimulation, cells were washed twice in FACS buffer (Dubecco’s PBS, 2% FCS, and sodium azide) and incubated with anti-mouse CD16/CD32 (mouse Fc block, 1 µg/million cells) for 15 min to reduce nonspecific binding. The cells were then stained for 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 and anti-CD8. For detection of CD152, cells were first labeled with the anti-CD4/anti-CD8 mix for 30 min, then permeabilized with 250 µl Cytofix/Cytoperm solution. After 20 min, PE-labeled anti-CD152 Ab was added and, after a 30-min incubation, was washed twice in PermWash solution. Cells were analyzed in a FACS scanner and analyzed for the intensity and percentage of T cells (FITC CD4, CD8 gate) expressing each marker. All Abs used in these studies were purchased from BD PharMingen.

RNase protection assays

Total cellular RNA was extracted from TP+/+ and TP-/- lymphocytes following stimulation by anti-CD3 Ab or in MLR with the RNeasy kit (Qiagen, Valencia, CA), according to manufacturer’s instructions, and was stored in RNase-free water at -70°C. To detect chemokine and cytokine mRNA, commercially available multiprobe template sets (Riboquant; BD PharMingen) were labeled with [{alpha}-32P]UTP (PerkinElmer), according to manufacturer’s instructions, and then diluted to a concentration of 300,000 cpm/µl of hybridization buffer. All reagents used in probe synthesis were obtained from BD PharMingen (In Vitro Transcription Kit, catalogue. 45004K). RNA samples were thawed on ice and 5–10 µg aliquoted for studies. RNA samples were completely dried on a vacuum evaporator centrifuge without heat and solubilized in 8 µl of hybridization buffer by gently vortexing for 3 min. Samples were mixed with 2 µl of the diluted probe and transferred to a hybridization oven set at 90°C. The temperature was immediately turned down to 56°C, and samples were allowed to hybridize with the probe for 12–16 h. The following probe sets were used: MCK1 (IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-{gamma}), MCK2b (IL-12, IL-10, IL-1, IL-18, IL-6, IFN-{gamma}, macrophage migration inhibitory factor (MIF)), MCK3b (TNF-{beta}, lymphotoxin {beta}, TNF-{alpha}, IL-6, IFN-{gamma}, IFN-{beta}, TGF-{beta}, MIF), and MCK5 (lymphotactin, RANTES, eotaxin, macrophage-inflammatory protein-1{beta} (MIP-1{beta}), MIP-1{alpha}, MIP-2, IFN-{gamma}-inducible protein-10, monocyte chemoattractant protein-1, T cell activation-3). RNase protection assays were performed using the RNase Protection Assay Kit (BD PharMingen; catalogue 45014K) and following the protocol suggested by the manufacturer. Briefly, RNase-protected samples were removed from the oven and subjected to sequential digestion with RNase and proteinase K. After treatment with chloroform:isoamyl alcohol, the aqueous phase was removed and RNA precipitated with 4 M ammonium acetate and 100% ethanol, and samples were incubated for 30 min at -70°C. RNA was then pelleted, washed with 90% ethanol, air dried, and resuspended in 5 µl of 1x loading buffer. Samples were heat blocked (90°C) for 3 min, then run on acrylamide gels. Gels were covered with Saran wrap and dried under vacuum at 80°C for 45 min. The dried gels were placed on film in a cassette with an intensifying screen and developed at -70°C. The exposure time ranged from 2 h (for the housekeeping genes) to 5 days (for faint bands). Films were scanned and bands were analyzed as a ratio of target RNA/GAPDH control using the Scion Image for Windows program. Data were expressed as mean arbitrary units ± SD. We performed a total of 10 experiments using male and female mice, on both C57B6 and BALB/c backgrounds, as responders (total = 21 wild-type and 21 TP-/- animals).

Mouse heart transplantation

Heterotopic cardiac transplants in mice were performed, as described previously (26). Recipient BALB/c (H-2d) TP+/+ and TP-/- mice were anesthetized 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 TP+/+ 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 cessation of palpable heartbeat.

In a separate group of animals, hearts from wild-type C57BL/6 mice were transplanted into BALB/c TP+/+ (n = 6) and TP-/- (n = 9) recipients, and all transplant recipients were treated with subtherapeutic doses (20 mg/kg by i.p. injection) of cyclosporin A beginning on the day of transplant and continuing for 7 days. Allograft survival was monitored, as described above. To evaluate allograft histopathology, heart transplants from additional animals (n = 9) were removed on day 7 after transplantation, fixed in 10% buffered Formalin, sectioned, and stained with H&E, and the slides were reviewed by a pathologist (P. Ruiz), who was masked to the experimental groups. The severity of rejection, interstitial infiltrates, myocyte injury, and vascular abnormalities were each graded separately using a semiquantitative scale, in which 0 was no abnormality, and 1, 2, and 3 represented mild, moderate, and severe abnormalities, as described previously (7).

The reverse experiment was also performed. Hearts from TP+/+ (n = 7) or TP-/- (n = 9) C57BL/6 mice were transplanted into wild-type BALB/c animals; recipients were treated with 20 mg/kg of cyclosporin A; and allograft survival was determined, as described above.

Statistical analysis

The values for each parameter within a group are expressed as the mean ± SEM. For comparisons between TP+/+ and TP-/- groups, statistical significance was assessed using an unpaired t test for normally distributed data. A paired t test was used for comparisons within groups. For nonparametric analyses, a Mann-Whitney U test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine whether the absence of TP receptors influences lymphocyte functions, we first compared proliferation of TP+/+ and TP-/- splenocytes following exposure with the nonspecific mitogen PHA. Fig. 1 shows that PHA markedly stimulated the proliferation of wild-type BALB/c cells and that the intensity of proliferation was dose proportional over a range of concentrations from 0.6 to 5 µg/ml. In TP-deficient BALB/c splenocytes, PHA also caused a similar dose-dependent increase in proliferation. However, at each concentration of PHA, proliferation was 20–45% lower in TP-deficient compared with wild-type splenocytes. Similar findings were observed with the C57BL/6 line (data not shown).



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  		FIGURE 1. PHA-induced proliferation is attenuated in TP-deficient lymphocytes. PHA was added to single-cell suspensions of splenocytes from either TP+/+ ({square}) or TP-/- mice ({blacksquare}) in concentrations ranging from 0.6 to 5 µg/ml. Proliferation was assessed as [3H]thymidine incorporation. Data are depicted in cpm as specific cell-associated counts (x1000). (*, p < 0.002 vs TP+/+.)

 
We next compared proliferation of TP+/+ and TP-/- BALB/c cells using a more specific T cell mitogen, anti-CD3 Ab. As shown in Fig. 2, anti-CD3 Ab induced a brisk proliferative response in wild-type splenocytes, and over a range from 0.1 to 10 µg/ml the magnitude of the response was proportional to the Ab concentration. A similar dose-dependent increase in proliferation was observed in TP-deficient cells, but, as in the PHA experiments, the magnitude of the proliferative response was attenuated by 20–25% in the anti-CD3-treated TP-/- splenocytes compared with wild-type cells. A similar difference was seen between C57BL/6 TP+/+ and TP-/- cells (data not shown). To confirm that generation of TX is augmented in this setting, we measured concentrations of 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 received no mitogen, TXB2 generation was augmented in the cultures that were exposed to anti-CD3 Ab (68 ± 3 pg/ml/106 cells with 10 mg/ml anti-CD3 Ab vs 2 ± 2 pg/ml/106 cells in controls; p < 0.0001). TXB2 generation was similarly augmented in stimulated TP-deficient splenocytes (data not shown).



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  		FIGURE 2. Reduced response to anti-CD3 Ab in TP-deficient lymphocytes. Anti-CD3 Ab was added to single-cell suspensions of splenocytes from either wild-type ({square}) or TP-/- mice ({blacksquare}) in concentrations ranging from 0.1 to 10 µg/ml. Proliferation was assessed as [3H]thymidine incorporation. Data are depicted in cpm as specific cell-associated counts (x1000). (*, p < 0.002 vs TP+/+.)

 


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  		FIGURE 3. TX generation by cultured splenocytes. Bulk cultures of splenic lymphocytes were stimulated with anti-CD3 Ab in concentrations ranging from 0 to 10 µg/ml. After 24 h, TXB2 concentrations in the supernatant were measured by RIA. TXB2 generation was augmented in the anti-CD3 Ab-stimulated cultures. (*, p = 0.012 vs 0 mg/ml; §, p < 0.0001 vs 0 mg/ml.)

 
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 35–80%. 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 in MLR (data not shown). In contrast, there was no difference in the level of proliferation in reverse MLR using TP+/+ or TP-/- cells as stimulators (data not shown). When purified T cells were used as responders, significantly less proliferation was once again observed in the T cells lacking TP receptors compared with controls (6141 ± 567 cpm-TP+/+ vs 3822 ± 363 cpm-TP-/-; p = 0.0065). Thus, stimulation of the TP receptor occurs as a part of the cellular alloimmune response, and activation of the TP receptor promotes lymphocyte proliferation. Similar to the other in vitro assays of cellular immunity, robust differences between TP+/+ and TP-/- cells were observed in MLR using both the C57BL/6 and BALB/c lines (data not shown).



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  		FIGURE 4. Proliferation in MLR is reduced when responder cells lack TP receptors. MLR was performed using splenocytes from (H-2d) TP+/+ and TP-/- mice as responders and irradiated splenocytes from (H-2b) mice as stimulators. Proliferation, measured as [3H]thymidine incorporation in cell-specific cpm, is significantly reduced in splenocytes from TP-/- mice ({blacksquare}) compared with TP+/+ controls ({square}) across a range of stimulator concentrations. (*, p < 0.001 vs TP+/+.)

 
To confirm that the defect in proliferation of TP-/- cells was due to the absence of TP receptor signaling and not to a subtle developmental abnormality in the TP receptor-deficient mice, one-way MLR experiments were performed in the presence of pharmacological agents that alter the synthesis or activity of TXA2. As shown in Fig. 5, treatment of wild-type splenocytes with the TP receptor antagonist SQ 29,548 caused potent inhibition of alloantigen-induced proliferation. Administration of the TX synthase inhibitor (carboxyheptyl imidazole) caused a virtually identical reduction in the proliferative response. In contract indomethacin, an NSAID that inhibits synthesis of all prostanoids, including TXA2, significantly enhanced proliferation in the MLR, highlighting the complexity of the actions of the COX pathway on immune responses.



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  		FIGURE 5. Effects of pharmacological inhibitors on proliferation of wild-type lymphocytes in MLR. One-way MLR was performed using wild-type responder cells in the presence of medium alone, the NSAID indomethacin, the TP receptor antagonist SQ 29,548, or the TX synthase inhibitor carboxyheptyl imidizole (CI). ({ddagger}, p < 0.005 vs media alone; *, p < 0.01 vs media alone.)

 
In additional experiments depicted in Fig. 6, the specificity of the pharmacological effects of TX inhibition was further explored by comparing responses to TX synthase inhibition in TP+/+ and TP-/- cells. As before, the presence of the TX synthase inhibitor significantly attenuated proliferation of wild-type cells in the MLR. This inhibition could be completely reversed in TP+/+ cells by coadministration of 3 µM of the TP agonist U46619 along with the TX synthase inhibitor. The apparent discrepancy between the concentration of the agonist U46619 that was required to reverse the actions of the inhibitor (3 µM) and the concentrations of TXB2 that were measured in the supernatants (pg/ml) described above is most likely due to the relatively low affinity of U46619 for the TP receptor (Kd{approx}300 nM) compared with the natural ligand TXA2 (Kd{approx}pM).



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  		FIGURE 6. TX synthase inhibitor and TP agonist alter proliferation of TP+/+, but not TP-/- responders in MLR. One-way MLR was performed using splenocyte suspensions from TP+/+ (left) or TP-/- (right) mice. MLR was performed with medium alone ({square}), with the TX synthase inhibitor carboxyheptyl imidazole ({blacksquare}), or with the TX synthase inhibitor + 3 µM TP agonist U46619 (). ({ddagger}, p < 0.001 vs TP+/+ media alone or TP+/+ with carboxyheptyl imidazole and U46619; *, p < 0.001 vs TP+/+ media alone.)

 
As we had observed previously, proliferation of TP-/- responders in MLR was significantly lower than TP+/+ controls. However, in contrast to the wild-type cells, exposure to the TX synthase inhibitor did not further reduce proliferation of TP-deficient cells. Likewise, administration of the TP agonist along with the TX synthase inhibitor did not affect proliferation of the TP-deficient cells.

To investigate whether attenuated adaptive immune responses in TP deficiency were associated with altered expression of key surface proteins on T cells, we compared expression of a range of markers, including CD25, CD28, CD69, CD152, and CD154, in anti-CD3-stimulated TP+/+ and TP-/- lymphocytes by cytofluorometry. Expression of these proteins was not significantly altered by TP deficiency, and there were no significant differences between wild-type and TP-deficient T cells in the intensity or the proportion of cells expressing these markers at baseline and 3, 6, 12, and 48 h following TCR cross-linking (data not shown).

Next, we performed RNase protection assays to detect cytokine mRNA in TP+/+ and TP-/- lymphocytes during anti-CD3 stimulation and MLRs. After several independent experiments, we could not find consistent differences in mRNA expression for: IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-{gamma}, IL-12, IL-10, IL-1, IL-18, MIF, TNF-{beta}, lymphotoxin {beta}, TNF-{alpha}, IFN-{beta}, TGF-{beta}, lymphotactin, RANTES, eotaxin, MIP-1{beta}, MIP-1{alpha}, MIP-2, IFN-{gamma}-inducible protein-10, monocyte chemoattractant protein-1, and T cell activation-3 between wild-type and TP knockout animals.

In a number of cell types, the TP receptor is linked to G{alpha}q and signals through phospholipase C and intracellular calcium (28, 29, 30). To determine whether alterations in calcium signaling might be responsible for attenuated proliferative responses observed in TP-deficient lymphocytes, we tested responses to PMA plus ionomycin. Moreover, to clearly document that these effects of TP receptors are due to direct effects on T cells, these studies were conducted using purified populations (>97%) of T cells. As shown in Fig. 7, proliferation of highly purified TP-deficient T cells stimulated with anti-CD3 Ab was reduced by >25% compared with wild-type controls. This was similar to our previous results using mixed splenocytes (Fig. 2). By contrast, exposure of TP+/+ and TP-/- 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.



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  		FIGURE 7. Proliferation of highly enriched T cells stimulated by anti-CD3 Ab or PMA and ionomycin. Highly enriched (>95% pure) populations of T cells were isolated from TP+/+ ({square}) and TP-/- ({blacksquare}) mice. The cells were stimulated with anti-CD3 Ab on coated plates (left) or a combination of the phorbol ester PMA and the ionophore ionomycin. Proliferation measured as [3H]thymidine incorporation was determined 24 h later.

 
To determine whether the actions of TP receptors to promote T cell proliferation that we observed in vitro were relevant to immune responses in the intact animal, we turned to a well-characterized model of heterotopic cardiac transplantation. In these studies, we determined graft survival when the donor, the recipient, or both donor and recipient were TP deficient. In this aggressive model of allograft rejection, TP+/+ (H-2b) hearts transplanted into TP+/+ (H-2d) recipients were rejected rapidly, and the mean time to graft failure was 9 ± 1 days. Survival of TP+/+ hearts in TP-deficient recipients (8 ± 1 days) was virtually identical with controls.

Although our in vitro studies indicated a significant role for TP receptors to promote cellular alloimmune responses, the absence of TP receptors on recipient immune cells alone was not sufficient to prolong allograft survival in this aggressive model of rejection. In a further attempt to uncover a contribution of these actions of TP receptor activation to an inflammatory response in vivo, we performed an additional transplant experiment. In this study, hearts from wild-type donors were transplanted into MHC-disparate recipients that were wild type or TP deficient. Both groups of recipients were then treated with a subtherapeutic dose of cyclosporin A (20 mg/kg/day). As depicted in Fig. 8, following treatment with low dose cyclosporine, allograft survival was significantly prolonged in the recipients lacking TP receptors (16 ± 2 days) compared with controls (12 ± 1; p = 0.008). However, in the converse experiment, the absence of TP receptors in donor tissue did not significantly enhance survival of allografts transplanted into wild-type recipients (13 ± 1 vs 12 ± 1 days; p > 0.15).



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  		FIGURE 8. Prolonged cardiac allograft survival in TP-/- recipients treated with low doses of cyclosporine. Heterotopic cardiac allografts were placed in TP+/+ ({blacksquare}) or TP-/- (•) mice. Recipient animals were treated with cyclosporin A (20 mg/kg) from day 0 to 7 posttransplant. Cyclosporine caused a significant prolongation of survival in the TP-/- group compared with controls (16 ± 2 vs 12 ± 1 days, p = 0.01).

 
There was a significant attenuation of the pathological severity of rejection in the TP-deficient recipients, corresponding with the improvement in graft survival that was observed in this group. This was reflected by a reduction in the overall acute rejection score (1.3 ± 0.3 vs 2.4 ± 0.2; p = 0.003), in interstitial inflammatory cell infiltrates (1.2 ± 0.2 vs 2.5 ± 0.3; p = 0.007), and in perivascular injury (1.2 ± 0.2 vs 2.25 ± 0.25; p = 0.001). There was also a tendency toward reduced myocyte injury in the allografts transplanted into TP-deficient recipients (1.2 ± 0.2 vs 2.4 ± 0.4; p = 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 5, 6, 7, 8, 9, 10). The overall impact of the COX pathway on cellular immune responses is determined by the profile of prostanoid synthesis within the microenvironment along with the repertoire of prostanoid receptors that is expressed by a particular immune cell population. In this regard, T cells are known to express several classes of prostanoid receptors, including EP2 and EP4 receptors for PGE2 and the TP receptor for TXA2 (1, 3, 12). These receptors are coupled to different intracellular signaling pathways, and therefore might be expected to have different effects on cellular function. EP2 and EP4 receptors activate adenylate cyclase (1, 12), and accumulation of cAMP is generally associated with inhibition of T cell functions. By contrast, TP receptors couple to phospholipase C and intracellular calcium (1, 12), signals that tend to promote immune cell activation.

Pharmacological studies have suggested a role for TP receptors in regulating cellular immunity. For example, TX synthase inhibitors have been shown to diminish lymphocyte proliferation and T cell cytotoxicity in vitro (22, 23, 24). However, interpretation of studies using TX synthase inhibitors is problematic because when TX synthase is inhibited, PG endoperoxides such as PGH2 may accumulate (32). These compounds can act as agonists at the TP receptor and thus attenuate efficacy. Moreover, accumulated PGH2 may be used as substrate for synthesis of other prostanoids. This shunting of endoperoxide substrate to increase synthesis of other prostanoids such as PGE2, which inhibits T cell responses (7), may therefore produce cellular actions that are unrelated to inhibition of TX synthesis.

Our studies using genetically altered mice unequivocally identify a role for TX in regulation of cellular immune responses. We find that TXA2, acting through the TP receptor, augments the vigor of mitogen-induced proliferation. This effect is apparent with a nonspecific mitogen such as PHA or with anti-CD3 Ab, a T cell-specific mitogen. To determine whether TP receptors stimulate lymphocyte proliferation in a more complex response, we used the MLR as a model of the cellular activation by alloantigens. The MLR is designed to mimic the conditions that might occur in a transplanted organ when recipient immune cells are activated by recognition of foreign MHC Ags expressed on the donor tissue. When the responder cell population was derived from mice that lack TP receptors, we found that proliferative responses in MLR were substantially attenuated. Proliferation was also reduced when purified T cells from TP-/- mice were used as responders. A similar attenuation was observed when highly enriched T cells were stimulated with anti-CD3 Ab.

Along with its direct effects to influence T cell functions, TXA2 may also influence the maturation and development of the T cell repertoire in the thymus. TP receptors are expressed at high levels in the thymus, most prominently in immature thymocyte populations (25). Stimulation of TP receptors on these cells induces programmed cell death, suggesting that TP receptors on thymocytes might play a role in selection of maturing T lymphocytes (25). However, in our previous analysis of TP-deficient mice, we found no significant alteration in the size, histopathology, or cellular constitution of the thymus or other lymphoid organs (18). Nonetheless, to ensure that the differences that we observed in the cellular responses of TP-deficient mice were not due to a subtle developmental defect caused by the absence of TP receptors in the thymus, we performed MLR experiments using wild-type cells in the presence of a TX receptor antagonist or synthase inhibitor. Inhibition of TX synthesis or blockade of TP receptors similarly diminished allospecific proliferation in wild-type mice. The attenuation achieved by pharmacological blockade, ~50% of normal, was in the range observed in the genetic experiments. The more exaggerated defect seen in TP-deficient cells may reflect the complete absence of TP receptors compared with the more limited inhibition that can be achieved pharmacologically. Because the extent of the antiproliferative effect was similar with synthase inhibition and receptor blockade and proliferation was restored with TP agonist, the contribution of endoperoxide shunting to the actions of the TX synthase inhibitor appears to be negligible in this circumstance. Furthermore, the absence of any effect of TX synthase inhibition in the TP-/- cells confirms pharmacological specificity. Finally, as illustrated in Fig. 5, there was a stark contrast between the effects of the COX inhibitor, which actually enhanced proliferation, and the TX inhibitors, which attenuated the MLR. Thus, modulation of an inflammatory response may differ substantially when the actions of a specific prostanoid are inhibited compared with global COX inhibition with an NSAID.

Our finding that TP receptors expressed on T cells promote cellular immune responses is reminiscent of recently described immunoregulatory actions of the AT1 receptor for angiotensin II (26). We previously demonstrated that AT1 receptors stimulate T cell proliferation spontaneously and during the course of MLR. The magnitude and character of the AT1 effects are very similar to those that we are reporting now for TP receptors. Because the AT1 and TP receptors may use G{alpha}q and G{alpha}13 (28, 29, 30, 33, 34) proteins for signaling, it is possible that their immunomodulatory actions are mediated by common distal signals that are linked to these G proteins. Our studies indicate that alterations in TCR-dependent calcium signaling are critical to the actions of TP receptors to regulate T cell proliferation, as exposure to ionomycin and PMA, which trigger an exaggerated, Ag-independent calcium signal, rescues the attenuated responsiveness of TP-deficient cells.

Enhanced production of TX has been implicated in the pathogenesis of various immunological diseases. A role for TXA2 in the pathogenesis of autoimmune disease was suggested by Patrono et al. (35), who found that urinary excretion of TX metabolites was enhanced in patients with active lupus nephritis. In these patients, there was an inverse correlation between urinary TXB2 excretion and creatinine clearance. In another study of patients with biopsy-proven lupus nephritis, infusions of a specific TX receptor antagonist increased glomerular filtration rate and renal plasma flow by 25% (36). Kelley et al. (37) first demonstrated enhanced production of TX in kidneys from autoimmune mice. In these murine models, treatment with TX antagonists preserved renal function, reduced glomerular capillary immune complex deposits, and lessened glomerular inflammation (27, 38). A similar role for TXA2 has been demonstrated in transplant rejection. Foegh et al. (39) first demonstrated that excretion of TX metabolites was enhanced during episodes of acute rejection in human renal allograft recipients. Subsequent studies from several laboratories demonstrated increased production of TX in animal models of rejection (40, 41) and showed that TX inhibitors could prolong graft survival and improve graft function (40, 42).

In pathological conditions such as autoimmune disease and transplant rejection, there are several pathways that could be used by TXA2 to promote tissue injury, including actions on vascular and procoagulant systems in the target organ, stimulation of proinflammatory cytokine release from mononuclear cells (43), and by potentiating cellular immune responses through the mechanisms that we have described in this work. To begin to distinguish the relative contributions of these pathways and to determine whether the actions of TP receptors to promote in vitro cellular immunity are apparent in vivo, we used a model of cardiac allograft rejection in which donor and recipient are completely mismatched at the MHC locus, resulting in a very aggressive acute cellular rejection response to the allograft (26). In unmodified rejection, we found that the absence of TP receptors on recipient tissues was not sufficient to prolong graft survival. In contrast, if recipients are treated with subtherapeutic doses of cyclosporine, graft survival is prolonged compared with wild-type controls when TP receptors are absent only on recipient immune cells. The prolonged graft survival observed in TP-deficient recipients is associated with significant amelioration in the severity of histopathological manifestations of rejection. This suggests that a contribution of TP-mediated actions to promote cellular immunity can be uncovered when the rejection response is attenuated. Moreover, these findings also imply potential additive or cooperative actions of TP blockade with calcineuriun inhibition. Although we suggest that some portion of the beneficial effects of TP deficiency on graft survival is due to attenuated T cell responses, this may not be the sole mechanism. A contribution of reduced proinflammatory actions on recipient macrophages and monocytes may also play a role. Dissecting the individual contributions of these interrelated mechanisms will be an interesting area for future study.

Taken together, these studies indicate that G protein-coupled TP receptors expressed on lymphocytes can modify the nature of a cellular immune response. Stimulation of TP receptors promotes lymphocyte activation. Thus, TP receptors, along with their signaling pathways, represent potentially useful therapeutic targets in disorders such as transplant rejection and autoimmune disease.


   Acknowledgments
 
We thank Kamie Snow, Pat Flannery, and Jody Tucker for expert technical assistance, and Norma Turner for secretarial and administrative help.


   Footnotes
 
1 This work was supported by the Research Service of the Department of Veterans Affairs and National Institutes of Health Grants AI001389 and DK38103. Back

2 D.W.T. and P.N.R. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Thomas M. Coffman, Building 6/Nephrology (111I), Veterans Affairs Medical Center, 508 Fulton Street, Durham, NC 27705. E-mail address: tcoffman{at}acpub.duke.edu Back

4 Abbreviations used in this paper: TX, thromboxane; COX, cyclooxygenase; MIF, macrophage migration inhibitory factor; MIP, macrophage-inflammatory protein; NSAID, nonsteroidal anti-inflammatory drug; TP, TX-prostanoid; TX, thromboxane. Back

Received for publication January 21, 2003. Accepted for publication October 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Narumiya, S., Y. Sugimoto, F. Ushikubi. 1999. Prostanoid receptors: structures, properties, and functions. Physiol. Rev. 79:1193.[Abstract/Free Full Text]
  2. Goetzl, E., S. An, W. Smith. 1995. Diversity and specificity of the eicosanoid mediators of normal physiology and disease. FASEB J. 9:1051.[Abstract]
  3. Tilley, S., T. Coffman, B. Koller. 2001. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J. Clin. Invest. 108:15.[Medline]
  4. Smith, W. L.. 1992. Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol. 263:F181.
  5. Goodwin, J., A. Bankhurst, R. Messner. 1977. Suppression of human T-cell mitogenesis by prostaglandin: existence of a prostaglandin-producing suppressor cell. J. Exp. Med. 146:1719.[Abstract/Free Full Text]
  6. Minakuchi, R., M. Wacholtz, L. Davis, P. Lipsky. 1990. Delineation of the mechanism of inhibition of human T cell activation by PGE2. J. Immunol. 145:2616.[Abstract]
  7. Nataraj, C., D. Thomas, S. Tilley, M. Nguyen, R. Mannon, B. Koller, T. Coffman. 2001. Receptors for prostaglandin E2 that regulate cellular immune responses in the mouse. J. Clin. Invest. 108:1229.[Medline]
  8. Scales, W., S. Chensue, I. Otterness, S. Kunkel. 1989. Regulation of monokine gene expression: prostaglandin E2 suppresses tumor necrosis factor but not interleukin-1{alpha} or {beta} m-RNA and cell associated bioactivity. J. Leukocyte Biol. 45:416.[Abstract]
  9. Snyder, D. S., D. I. Beller, E. R. Unanue. 1982. Prostaglandins modulate macrophage Ia expression. Nature 299:163.[Medline]
  10. Van der Pouw Kraan, T., L. Boeije, R. Smeenk, J. Wijdenes, L. Aarden. 1995. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J. Exp. Med. 181:775.[Abstract/Free Full Text]
  11. Abe, T., K. Takeuchi, N. Takahashi, E. Tsutsumi, Y. Taniyama, K. Abe. 1995. Rat kidney thromboxane receptor: molecular cloning, signal transduction, and intrarenal expression localization. J. Clin. Invest. 96:657.
  12. Coleman, R. A., W. L. Smith, S. Narumiya. 1994. VIII. International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol. Rev. 46:205.[Medline]
  13. Hirata, M., Y. Hayashi, F. Ushikubi, Y. Yokota, R. Kageyama, S. Nakanishi, S. Narumiya. 1991. Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature 349:617.[Medline]
  14. Namba, T., Y. Sugimoto, M. Hirata, Y. Hayashi, A. Honda, A. Watabe, M. Negishi, A. Ichikawa, S. Narumiya. 1992. Mouse thromboxane A2 receptor: cDNA cloning, expression and Northern blot analysis. Biochem. Biophys. Res. Commun. 184:1197.[Medline]
  15. Furci, L., D. Fitzgerald, G. Fitzgerald. 1991. Heterogeneity of prostaglandin contraction and platelet aggregation. J. Pharmacol. Exp. Ther. 258:74.[Abstract/Free Full Text]
  16. Mais, D. E., D. L. Saussy, A. Chaikhouni, P. J. Kochel, D. R. Knapp, N. Hamanaka, P. V. Halushka. 1985. Pharmacologic characterization of human and canine thromboxane A2/prostaglandin H2 receptors in platelets and blood vessels: evidence for different receptors. J. Pharmacol. Exp. Ther. 233:418.[Abstract/Free Full Text]
  17. Mais, D., D. DeHoll, J. Sightler, P. Halushka. 1988. Different pharmacologic activities for 13-azapinane thromboxane A2 analogs in platelet and blood vessels. Eur. J. Pharmacol. 148:309.[Medline]
  18. Thomas, D., R. Mannon, P. Mannon, A. Latour, J. Oliver, M. Hoffman, O. Smithies, B. Koller, T. Coffman. 1998. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2. J. Clin. Invest. 102:1994.[Medline]
  19. FitzGerald, G. A., C. Healy, J. Daugherty. 1987. Thromboxane A2 biosynthesis in human disease. Fed. Proc. 46:154.[Medline]
  20. Bruggeman, L. A., E. A. Horigan, S. Horikoshi, P. E. Ray, P. E. Klotman. 1991. Thromboxane stimulates synthesis of extracellular matrix proteins in vitro. Am. J. Physiol. 261:F488.
  21. Coffman, T., R. Spurney, R. Mannon, R. Levenson. 1998. Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells. Am. J. Physiol. 275:F262.
  22. Ceuppens, J. L., S. Vertessen, H. Deckmyn, J. Vermylen. 1985. Effects of thromboxane A2 on lymphocyte proliferation. Cell. Immunol. 90:458.[Medline]
  23. Leung, K., E. Mihich. 1980. Prostaglandin modulation of development of cell-mediated immunity in culture. Nature 288:597.[Medline]
  24. Ruiz, P., L. Rey, R. Spurney, T. Coffman, A. Viciana. 1992. Thromboxane augmentation of alloreactive T cell function. Transplantation 54:498.[Medline]
  25. Ushikubi, F., Y. Aiba, K. Nakamura, T. Namba, M. Hirata, O. Mazda, Y. Katsura, S. Narumiya. 1993. Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis. J. Exp. Med. 178:1825.[Abstract/Free Full Text]
  26. Nataraj, C., M. Oliverio, R. Mannon, P. Mannon, L. Audoly, L. A. C. Amuchastegui, O. Smithies, T. Coffman. 1999. Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. J. Clin. Invest. 104:1693.[Medline]
  27. Spurney, R. F., P. Y. Fan, P. Ruiz, F. Sanfilippo, D. S. Pisetsky, T. M. Coffman. 1992. Thromboxane receptor blockade reduces renal injury in murine lupus nephritis. Kidney Int. 41:973.[Medline]
  28. Djellas, Y., J. Manganello, K. Antonakis, G. Le Breton. 1999. Identification of G{alpha}13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors. J. Biol. Chem. 274:14325.[Abstract/Free Full Text]
  29. Kinsella, B., D. O’Mahony, G. Fitzgerald. 1997. The human thromboxane A2 receptor {alpha} isoform (TP{alpha}) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2{alpha}. J. Pharmacol. Exp. Ther. 281:957.[Abstract/Free Full Text]
  30. Knezevic, I., C. Borg, G. C. Le Breton. 1993. Identification of Gq as one of the G-proteins which copurify with human platelet thromboxane A2/prostaglandin H2 receptors. J. Biol. Chem. 268:26011.[Abstract/Free Full Text]
  31. Williams, T., J. Morley. 1973. Prostaglandins as potentiators of increased vascular permeability in inflammation. Nature 246:215.[Medline]
  32. Oates, J., G. Fitzgerald, R. Branch, E. Jackson, H. Knapp, L. Roberts. 1988. Clinical implications of prostaglandin and thromboxane A2 formation. N. Engl. J. Med. 319:689.[Medline]
  33. Macrez-Lepretre, N., F. Kalkbrenner, J.-L. Morel, G. Schultz, J. Mironneau. 1997. G protein heterotrimer G{alpha}13{beta}1{gamma}3 couples the angiotensin AT1A receptor to increases in cytoplasmic calcium in rat portal vein myocytes. J. Biol. Chem. 272:10095.[Abstract/Free Full Text]
  34. Ushio-Fukai, M., K. Griendling, M. Akers, P. Lyons, R. Alexander. 1998. Temporal dispersion of activation of phospholipase C-{beta}1 and -{gamma} isoforms by angiotensin II in vascular smooth muscle cells: role of {alpha}q/11, {alpha}12, and {beta}{gamma} G protein subunits. J. Biol. Chem. 273:19772.[Abstract/Free Full Text]
  35. Patrono, C., G. Ciabattoni, G. Remuzzi. 1985. Functional significance of renal prostacyclin and thromboxane A2 production in patients with systemic lupus erythematosus. J. Clin. Invest. 76:1011.
  36. Pierucci, A., B. Simonetti, G. Pecci. 1989. Improvement of renal function with selective thromboxane antagonism in lupus nephritis. N. Engl. J. Med. 320:421.[Abstract]
  37. Kelley, V. E., S. Sneve, S. Musinski. 1986. Increased renal thromboxane production in murine lupus nephritis. J. Clin. Invest. 77:252.
  38. Salvati, P., E. Lamberti, R. Ferrario, R. G. Ferrario, G. Scampini, F. Pugliese, P. Barsotti, C. Patrono. 1995. Long-term thromboxane-synthase inhibition prolongs survival in murine lupus nephritis. Kidney Int. 47:1168.[Medline]
  39. Foegh, M. L., J. F. Winchester, M. Zmudka, G. B. Helfrich, P. W. Ramwell, G. E. Schreiner. 1982. Aspirin inhibition of thromboxane release in thrombosis and renal transplant rejection. Lancet 1:48.
  40. Coffman, T. M., W. E. Yarger, P. E. Klotman. 1985. Functional role of thromboxane production by acutely rejecting renal allografts in rats. J. Clin. Invest. 75:1242.
  41. Khirabadi, B. S., M. L. Foegh, P. W. Ramwell. 1985. Urine immunoreactive thromboxane B2 in rat cardiac allograft rejection. Transplantation 39:6.[Medline]
  42. Coffman, T. M., P. Ruiz, F. Sanfilippo, P. E. Klotman. 1989. Chronic thromboxane inhibition preserves function of rejecting rat renal allografts. Kidney Int. 35:24.[Medline]
  43. Caughey, G. E., M. Pouliot, L. G. Cleland, M. J. James. 1997. Regulation of tumor necrosis factor-{alpha} and IL-1{beta} synthesis by thromboxane A2 in nonadherent human monocytes. J. Immunol. 158:351.[Abstract]



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