6390 TX RECEPTORS AND CELLULAR IMMUNITY
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 ny-
lon 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 macro-
phages (F4/80; Serotec, Raleigh, NC). Generally, T cells represented 85–
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 3.5 105 stimulator cells/well. After
backgrounds for more than six generations. All assays of cellular immunity
incubation for 3 or 4 days at 37°C with 5% CO2, [3H]thymidine incorpo-
were performed with TP / and TP / cells from both genetic back-
ration was determined, as described above.
grounds, and ﬁndings were similar independent of genetic background. The
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
Mitogen stimulation of lymphocytes 18 h at 37°C with 5% CO2, [3H]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
then centrifuged at 900 g to obtain a cell pellet. The pellet was re-
suspended in sterile RBC lysis buffer (150 mM NH4Cl, 10 mM KHCO3, Splenocytes from C57B6 TP / and TP / mice (n 5 for each group)
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 2 105 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,
days at 37°C in a humidiﬁed incubator containing 5% CO2, and then 0.5 anti-CD69, and anti-CD154 along with a mix of FITC-labeled anti-CD4
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 (9 –12 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 TP / and TP / 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
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 4 105 irradiated stimulator spleno- stored in RNase-free water at 70°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
added to individual wells of a 96-well plate. In some experiments, MLR PharMingen) were labeled with [ -32P]UTP (PerkinElmer), according to
was assessed in the presence of pharmacological agents that affect TXA2 manufacturer’s instructions, and then diluted to a concentration of 300,000
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 5–10 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-
containing 5% CO2. After varying times of incubation, the plates were trifuge without heat and solubilized in 8 l of hybridization buffer by
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 12–16 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- -in-
ducible 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
cells, splenocyte suspensions were prepared and 3.5 106 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 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 70°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 l of 1 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 70°C. The ex-
posure 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.
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 21 wild-type and 21 TP /
Mouse heart transplantation
Heterotopic cardiac transplants in mice were performed, as described pre-
viously (26). Recipient BALB/c (H-2d) TP / and TP / 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 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 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
were transplanted into BALB/c TP / (n 6) and TP / (n 9) recip- from either wild-type ( ) or TP / mice (f) in concentrations ranging
ients, and all transplant recipients were treated with subtherapeutic doses from 0.1 to 10 g/ml. Proliferation was assessed as [3H]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
transplant and continuing for 7 days. Allograft survival was monitored, as ( 1000). ( , p 0.002 vs TP / .)
described above. To evaluate allograft histopathology, heart transplants
from additional animals (n 9) were removed on day 7 after transplan-
tation, 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 20 – 45%
previously (7). lower in TP-deﬁcient compared with wild-type splenocytes. Sim-
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
ilar ﬁndings were observed with the C57BL/6 line (data not
animals; recipients were treated with 20 mg/kg of cyclosporin A; and al- shown).
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
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 wild-type splenocytes, and over a range from 0.1 to 10 g/ml the
SEM. For comparisons between TP / and TP / groups, statistical sig- magnitude of the response was proportional to the Ab concentra-
niﬁcance was assessed using an unpaired t test for normally distributed
data. A paired t test was used for comparisons within groups. For non-
tion. A similar dose-dependent increase in proliferation was ob-
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 20 –25%
Results in the anti-CD3-treated TP / splenocytes compared with wild-
To examine whether the absence of TP receptors inﬂuences lym- type cells. A similar difference was seen between C57BL/6 TP /
phocyte functions, we ﬁrst compared proliferation of TP / and and TP / cells (data not shown). To conﬁrm that generation of
TP / 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 cul-
tures of wild-type splenocytes. Results from a representative ex-
periment 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 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
from either TP / ( ) or TP / mice (f) in concentrations ranging from ranging from 0 to 10 g/ml. After 24 h, TXB2 concentrations in the su-
0.6 to 5 g/ml. Proliferation was assessed as [3H]thymidine incorporation. pernatant were measured by RIA. TXB2 generation was augmented in the
Data are depicted in cpm as speciﬁc cell-associated counts ( 1000). ( , anti-CD3 Ab-stimulated cultures. ( , p 0.012 vs 0 mg/ml; §, p 0.0001
p 0.002 vs TP / .) vs 0 mg/ml.)
6392 TX RECEPTORS AND CELLULAR IMMUNITY
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 stim-
ulated TP-deﬁcient splenocytes (data not shown).
To examine whether TP receptors contribute to proliferation in
a model cellular immune response, we measured alloantigen-in-
duced proliferation in a one-way MLR. After 5 days in culture with
irradiated, allogeneic stimulators, proliferative responses were sig-
niﬁcantly 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 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-
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-
used as responders, signiﬁcantly less proliferation was once again hibitor carboxyheptyl imidizole (CI). (‡, p 0.005 vs media alone; , p
observed in the T cells lacking TP receptors compared with con- 0.01 vs media alone.)
trols (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 TP / cells. As before, the presence of the TX synthase inhibitor
assays of cellular immunity, robust differences between TP / and signiﬁcantly attenuated proliferation of wild-type cells in the
TP / cells were observed in MLR using both the C57BL/6 and MLR. This inhibition could be completely reversed in TP / cells
BALB/c lines (data not shown). by coadministration of 3 M of the TP agonist U46619 along with
To conﬁrm that the defect in proliferation of TP / cells was the TX synthase inhibitor. The apparent discrepancy between the
due to the absence of TP receptor signaling and not to a subtle concentration of the agonist U46619 that was required to reverse
developmental abnormality in the TP receptor-deﬁcient mice, one- the actions of the inhibitor (3 M) and the concentrations of TXB2
way MLR experiments were performed in the presence of phar- that were measured in the supernatants (pg/ml) described above is
macological agents that alter the synthesis or activity of TXA2. As most likely due to the relatively low afﬁnity of U46619 for the TP
shown in Fig. 5, treatment of wild-type splenocytes with the TP receptor (Kd 300 nM) compared with the natural ligand TXA2
receptor antagonist SQ 29,548 caused potent inhibition of alloan- (Kd pM).
tigen-induced proliferation. Administration of the TX synthase in- As we had observed previously, proliferation of TP / respond-
hibitor (carboxyheptyl imidazole) caused a virtually identical re- ers in MLR was signiﬁcantly lower than TP / controls. However,
duction in the proliferative response. In contract indomethacin, an in contrast to the wild-type cells, exposure to the TX synthase
NSAID that inhibits synthesis of all prostanoids, including TXA2, inhibitor did not further reduce proliferation of TP-deﬁcient cells.
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
by comparing responses to TX synthase inhibition in TP / and markers, including CD25, CD28, CD69, CD152, and CD154, in
FIGURE 6. TX synthase inhibitor and TP agonist alter proliferation of
FIGURE 4. Proliferation in MLR is reduced when responder cells lack TP / , but not TP / responders in MLR. One-way MLR was performed
TP receptors. MLR was performed using splenocytes from (H-2d) TP / using splenocyte suspensions from TP / (left) or TP / (right) mice.
and TP / mice as responders and irradiated splenocytes from (H-2b) mice MLR was performed with medium alone ( ), with the TX synthase in-
as stimulators. Proliferation, measured as [3H]thymidine incorporation in hibitor carboxyheptyl imidazole (f), or with the TX synthase inhibitor
cell-speciﬁc cpm, is signiﬁcantly reduced in splenocytes from TP / mice 3 M TP agonist U46619 (u). (‡, p 0.001 vs TP / media alone or
(f) compared with TP / controls ( ) across a range of stimulator con- TP / with carboxyheptyl imidazole and U46619; , p 0.001 vs TP /
centrations. ( , p 0.001 vs TP / .) media alone.)
The Journal of Immunology 6393
conducted using puriﬁed populations ( 97%) of T cells. As shown
in Fig. 7, proliferation of highly puriﬁed TP-deﬁcient T cells stim-
ulated 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
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 ( 95% pure) popula- terized model of heterotopic cardiac transplantation. In these stud-
tions of T cells were isolated from TP / ( ) 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, TP / (H-2b) hearts transplanted into
eration measured as [3H]thymidine incorporation was determined 24 h
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
anti-CD3-stimulated TP / and TP / 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
Next, we performed RNase protection assays to detect cytokine receptor activation to an inﬂammatory response in vivo, we per-
mRNA in TP / and TP / 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 2 days) com-
protein-1, and T cell activation-3 between wild-type and TP pared with controls (12 1; p 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 (28 – type recipients (13 1 vs 12 1 days; p 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 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 perivas-
cular 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 trans-
planted 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.
The actions of COX metabolites of arachidonic acid in inﬂamma-
tion 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, 5–10). The overall impact of the COX
pathway on cellular immune responses is determined by the proﬁle
of prostanoid synthesis within the microenvironment along with
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 TP / (f) or TP / (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
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 2 vs 12 1 days, p 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 (22–24). 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 (28 –30, 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-
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,
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 TP / mice were used as responders. A reduced glomerular capillary immune complex deposits, and less-
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, 50% 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 TP / 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,
O. Smithies, B. Koller, and T. Coffman. 1998. Coagulation defects and altered
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Acknowledgments 26. Nataraj, C., M. Oliverio, R. Mannon, P. Mannon, L. Audoly, L. A. C. Amuchastegui,
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assistance, and Norma Turner for secretarial and administrative help. responses through a calcineurin-dependent pathway. J. Clin. Invest. 104:1693.
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