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Chemoattractant by Human Endothelial Cells Is Cyclosporin A-Resistant and Promotes T Cell Adhesion: Implications for Cyclosporin A-Resistant Immune Inflammation1
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Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697; and
Department of Molecular Sciences, Pfizer Inc., Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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chemoattractant (I-TAC) is a recently
discovered member of the CXC chemokine family. It is a potent T cell
chemoattractant expressed by IFN-
-treated astrocytes, monocytes,
keratinocytes, bronchial epithelial cells, and neutrophils. In this
study, we show that I-TAC is also expressed by IFN--treated
endothelial cells (EC), both at the mRNA and protein levels. Induction
of the I-TAC message is rapid and sustained over 24 h. TNF-
does not induce I-TAC mRNA alone, but does act
synergistically with IFN-. Blocking Abs to I-TAC, or to its
receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating
that the expressed protein is functional. Finally, the expression of
I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A,
suggesting that I-TAC may contribute to the chronic immune inflammation
characteristic of graft arteriosclerosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines belong to a superfamily of chemotactic cytokines that play a general role in leukocyte recirculation throughout the body and specifically in the migration of cells into inflammatory sites. Cells expressing the appropriate chemokine receptor migrate along a concentration gradient established by the inflamed tissue. Migration of specific cells across the endothelium, and activation of integrins, most likely involves chemokines tethered to the surface of EC via interactions with extracellular matrix proteins (2, 3). Tethering of chemokines helps maintain high local concentrations in the presence of continuous blood flow through the vessel. Through the expression of specific chemokines, EC play an active role in selectively recruiting immune cells to an inflammatory site and thus help to shape the nature of the immune response.
Different chemokine families attract different subsets of leukocytes
(4, 5, 6). The two main families are the CC and CXC
chemokines. In general, the CC chemokines attract monocytes,
eosinophils, basophils, and T cells. The CXC family can be split into
the ELR and the non-ELR CXC chemokines. Chemokines containing the ELR
motif (glutamate, leucine, and arginine) are chemotactic for
neutrophils, whereas the non-ELR CXC chemokines are chemotactic for T
cells. In particular, three non-ELR CXC members, IFN--inducible
protein-10 (IP-10), monokine induced by IFN- (Mig), and
IFN-inducible T cell chemoattractant (I-TAC), are all induced by
IFN- in certain cells and are chemotactic for T cells, in particular
Th1 memory T cells (7, 8). The specificity of these
chemokines for activated T cells is attributed to their ability to all
bind a single receptor, CXCR3, which is exclusively expressed by
effector T cells (9). The most recently discovered of
these CXC chemokines, I-TAC, was identified in human astrocytes treated
with cytokines (10). I-TAC has also been independently
isolated and named ßR-1 (11) or IP-9 (12).
I-TAC is most similar at the amino acid level to IP-10
(
40%), is chemotactic for activated memory T cells, and binds with
higher affinity to CXCR3 than do either IP-10 or Mig.
I-TAC mRNA is expressed not only by IFN--treated
astrocytes but also by IFN--treated monocytes (10),
bronchial epithelial cells (13), neutrophils
(14), and keratinocytes (12).
There is now considerable evidence that EC not only actively recruit T
cells into sites of inflammation but also present Ag and provide
costimulation (15, 16, 17, 18). Interestingly, cytokine production
by EC-activated T cells is resistant to the immunosuppressive drug
cyclosporin A (CsA) (19, 20). We have previously suggested
that this ability of EC may be important in stimulating the chronic
immune inflammation often seen in the coronary arteries of transplanted
hearts (21). In a search for genes expressed by EC during
their interaction with activated T cells in the presence of CsA, we
found that EC express I-TAC and that this expression is inducible by
IFN- and is resistant to suppression by CsA. Furthermore, we show
that EC-derived I-TAC is functional, in that blocking Ab to I-TAC or to
its receptor, CXCR3, inhibits the adhesion of activated T cells to EC
monolayers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant IFN- and TNF- were obtained from BioSource
International (Camarillo, CA). Abs to I-TAC, both polyclonal and
monoclonal, and the synthesized I-TAC protein were generated at Pfizer
(Groton, CT). The anti-CXCR3 mAb (clone 1C6) was obtained from
PharMingen (La Jolla, CA). The control Ab, HB64, was isolated from the
hybridoma clone obtained from the American Type Culture Collection
(Manassas, VA).
Cell isolation and culture
HUVEC were isolated from umbilical veins and cultured as described previously (22) on 1% gelatin-coated tissue culture plastic or on 5 µg/ml fibronectin (Collaborative Biomedical Products, Bedford, MA)-coated glass chamber slides (Lab-tek; Nalge Nunc International, Naperville, IL) in medium 199 (M199) supplemented with 20% FBS, antibiotics (all from Life Technologies, Grand Island, NY), endothelial cell growth supplement (Collaborative Biomedical Products), and heparin (Sigma, St. Louis, MO). Human capillary endothelial cells (HUCE) were isolated and cultured as described previously (23). Smooth muscle cells (SMC) were cultured from umbilical artery explants grown in DMEM with 10% FBS and antibiotics. Skin fibroblasts were purchased from American Type Culture Collection and were grown in the same medium as the SMC. PBMC were isolated by centrifugation of whole blood over lymphocyte separation medium (Organon Tecknika, Durham, NC). PBLs were obtained by elutriation and were a gift from Dr. Andrea Tenner (Irvine, CA).
Subtractive hybridization
Subtractive hybridization was conducted using the PCR-select cDNA subtraction kit from Clontech (Palo Alto, CA). The tester population of cells consisted of HUVEC cultured with PBMC, 5 µg/ml PHA, and 300 ng/ml CsA (both from Sigma) in RPMI 1640 supplemented with 10% FBS and antibiotics for 18 h at 37°C and 5% CO2. The driver population consisted of separate dishes of HUVEC, PBMC incubated with 5 µg/ml PHA, and PBMC incubated with 25 ng/ml PMA and 1 µg/ml ionomycin (both from Calbiochem, San Diego, CA) for 18 h at 37°C and 5% CO2. Total RNA was isolated using Trizol reagent (Life Technologies, Grand Island, NY), and the total RNA from each driver cell population was pooled. Poly(A)+ RNA was isolated using the Micro Poly(A)Pure mRNA kit from Ambion (Woodward, TX). The cDNA was made from the poly(A)+ RNA and subtractive hybridization was performed according to the manufacturer’s protocol. Nested PCR products were cloned into pBluescript (Stratagene, La Jolla, CA), sequenced using Thermosequenase (Amersham, Arlington Heights, IL), and analyzed by BLAST against GenBank.
RNA analysis
Cells were grown until confluent on 100-mm dishes and treated
with IFN- and/or TNF- and/or CsA, at the concentration indicated,
for 12–24 h. Where used, 25 x 106 PBMC
were cultured with 5 µg/ml PHA or 25 ng/ml PMA and 1 µg/ml
ionomycin for 18 h. Total RNA was isolated using Trizol reagent
(Life Technologies). For Northern blot analysis, 20–30 µg RNA was
electrophoresed on a 1.2% agarose-formaldehyde gel and then
transferred to a nylon membrane using a Turboblotter (Schleicher &
Schuell, Keene, NH). Blots were preincubated in hybridization solution
(50% formamide, 1% SDS, 5x SSC, 5x Denhardt’s solution, and 250
µg/ml denatured salmon sperm DNA) for 1 h at 42°C and then
hybridized at 42°C overnight in hybridization solution with 1 x
106 cpm/ml of a
[-32P]dATP-radiolabeled DNA probe, prepared
by random priming a cDNA fragment of the chemokine gene or a 1.4-kb
fragment of the GAPDH gene. Blots were exposed to
autoradiography film for 5–24 h or were analyzed on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Protein analysis
Cells were incubated in M199 supplemented with 20% FBS,
antibiotics, and 1000 U/ml IFN- for 20 h. Proteins were
isolated using a TCA precipitation protocol as described elsewhere
(24). Cells were washed twice in TBS-EDTA (20 mM Tris-HCl
(pH 7.4), 155 mM NaCl, and 1 mM EDTA) at room temperature. Cells were
lysed at room temperature by incubation for 10 min in cell lysis
solution (2% SDS, 5 mM EDTA, 5 mM EGTA, 20 mM Tris-HCl (pH 7.4), and
1.6 mg/ml protease inhibitor cocktail (Roche, Indianapolis, IN)).
Proteins were precipitated out of the cell lysates with 100% TCA and
pellets were washed with 2.5% TCA, neutralized with 3 M Tris, and
diluted with water. Twenty microliters of the sample was diluted with
2 x sample buffer (62.5 mM Tris (pH 6.8), 10% glycerol, 2% SDS,
1% bromophenol blue, and 2.5% 2-ME), separated on a 12% Tris-glycine
gel for 1 h at 100 V, and transferred to a polyvinylidene
difluoride membrane. As a reference, chemically synthesized I-TAC
protein (50 ng) was run in parallel. I-TAC protein was detected using a
rabbit anti-human I-TAC polyclonal Ab, goat anti-rabbit
HRP-conjugated secondary Ab (Bio-Rad, Hercules, CA), and enhanced
chemiluminescence detection reagents (Amersham). Blots were exposed to
autoradiography film for 5 min.
Adhesion assays
HUVEC were grown on glass chamber slides (Lab-tek) coated with 5 µg/ml fibronectin. Where indicated, EC and PBMC were preincubated in 5 µg/ml anti-I-TAC, anti-CXCR3, or control Ab for 15 min at 37°C. To allow visualization, the PBMC were also preincubated with 2 µM FITC dye (CellTracker Green CMFDA; Molecular Probes, Eugene, OR) for 30 min at 37°C. After addition of PBMC, PHA (5 µg/ml), and Abs (to a final concentration of 5 µg/ml), cultures were allowed to incubate for 20 h at 37°C. Nonadherent cells were washed off in M199 and remaining cells were fixed in 4% paraformaldehyde for 10 min. Slides were mounted and viewed using a Zeiss Axiophot (Oberkochen, Germany). Total numbers of adherent cells were determined by counting fluorescent cells in randomly chosen fields. Data from five fields were pooled. Approximately 25% of the input cells adhered to the EC monolayers incubated with control (HB64) Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify genes expressed by EC during their interaction with activated T cells, we performed a differential subtraction/hybridization screen. CsA was included in the media to allow identification of genes resistant to immunosuppression. I-TAC was found to be differentially expressed in the tester cDNA (EC + T cells) compared with the driver cDNA (EC or T cells alone). The cDNAs present after subtractive hybridization were cloned into pBluescript and 96 colonies were screened. Of the 96 colonies, 54% (52/96) had cDNA inserts that matched the DNA sequence of I-TAC and were hybridized with an I-TAC DNA probe. The cDNA inserts covered 85% of the I-TAC cDNA sequence.
I-TAC mRNA is expressed by IFN--treated HUVEC
To determine which cells in the tester population, EC or PBMC,
were expressing the I-TAC gene, we performed Northern blot
analysis. Total RNA was isolated from EC cultured with or without
IFN- (1000 U/ml), EC cultured with PHA-activated PBMC, and PBMC
cultured alone. As shown in Fig. 1
, I-TAC
mRNA was detectable in EC treated with IFN- (lane
2) and EC cultured with activated T cells (lane
6), but not in resting EC (lane 1). This concurs
with previous reports (10, 13, 14) that I-TAC expression,
like other non-ELR CXC chemokine expression, is inducible by IFN-.
In EC/T cell cocultures, I-TAC is presumably induced by T cell-derived
IFN-. We and others have demonstrated the rapid induction of T cell
IFN- synthesis in this culture system (16, 25). Three
transcripts were expressed and their sizes were determined to be 4,
2.8, and 1.8 kb, respectively, based on the mobility of m.w. markers.
The smallest transcript was the most abundant. Previous reports show a
range of I-TAC transcript sizes in various cells from 1.4 to
4.5 kb (10), but again the 1.8-kb transcript predominates.
We did not detect expression of I-TAC mRNA in resting PBMC
(lane 3), PHA-activated PBMC (lane
4), or PMA/ionomycin-activated PBMC (lane
5).
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were obtained with RNA isolated from HUVEC
treated with IFN- for 18 h. To determine whether
I-TAC RNA could be induced in EC earlier than 18 h, we
isolated RNA from EC treated with 1000 U/ml IFN- for different times
up to 24 h. I-TAC mRNA transcripts could be detected as
early as 4 h, and levels continued to increase up to 24 h,
the latest time studied (Fig. 2). We also
examined the effect of CsA on the inducible expression of
I-TAC RNA. In agreement with the subtraction/hybridization
data, EC incubated with IFN- and CsA (300 ng/ml) synthesized
equivalent amounts of I-TAC mRNA as cells cultured in the
absence of CsA (Fig. 2). At this concentration of CsA, T cell IL-2
synthesis in the absence of EC costimulation is completely inhibited
(data not shown).
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and TNF- act synergistically to induce I-TAC
mRNA
Previous reports have suggested that TNF- and IL-1ß can act
synergistically with IFN- to induce I-TAC message in some
cells. This is the case in neutrophils (14) and astrocytes
(10). However, no synergy was seen in bronchial epithelial
cells (13) or in monocytes (10). We addressed
this issue with HUVEC and found strong synergy. IFN- alone induced
moderate amounts of I-TAC mRNA in a dose-dependent manner
(Fig. 3). TNF- alone was ineffective.
A combination of 100 U/ml IFN- and 1, 10, or 100 ng/ml TNF- gave
a greater than 3-fold increase over the additive effect of IFN- and
TNF-, thus the two cytokines act synergistically in HUVEC.
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in HUCE and
skin fibroblasts
HUVEC represent large vessel EC and are used as a model system for
postcapillary venules. To determine whether I-TAC is also
expressed by capillary EC, we isolated RNA from HUCE. We also isolated
RNA from two stromal cell populations, SMC, and skin fibroblasts. Cells
were cultured with or without IFN- for 18 h and mRNA was
analyzed by Northern blot. I-TAC mRNA was detectable in both
HUCE and HUVEC when treated with IFN- (Fig. 4). Interestingly, more I-TAC
mRNA is expressed by HUCE than HUVEC. Somewhat lower levels were
expressed by IFN--treated fibroblasts and message was barely
detectable in SMC. EC and fibroblasts expressed identical sized
transcripts.
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-treated HUCE and fibroblasts, as in HUVEC, were equal
whether or not CsA was present (Fig. 4). Very little I-TAC
message was present in SMC. The expression IP-10,
Mig, and MCP-1 by these cells was also resistant
to suppression by CsA. Expression of I-TAC protein in EC
To determine whether I-TAC protein is expressed by HUVEC and HUCE,
we performed Western blot analysis on cell lysates from cells incubated
with and without IFN- for 20 h. HUVEC and HUCE treated with
IFN- expressed a protein that migrated in parallel with an 8-kDa
synthesized I-TAC protein (Fig. 5).
Interestingly, HUCE expressed more I-TAC protein than HUVEC, which
concurs with the levels of I-TAC mRNA expression observed in
EC (Fig. 4). I-TAC protein was not detectable in fibroblast or SMC cell
lysates.
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To determine whether the I-TAC expressed by EC functions in
lymphocyte recruitment, we first examined the ability of an
anti-CXCR3 Ab to block the adhesion of T cells to EC monolayers. EC
were cultured on fibronectin-coated chamber slides and FITC dye-loaded
PBMC were added along with anti-CXCR3 or control Ab. After 20
h of coculture in the presence of PHA, nonadherent cells were washed
off with M199, and adherent cells were fixed in 4% paraformaldehyde.
Previous experiments demonstrated that CXCR3 was detectable on
PHA-treated PBMC after 18–24 h (data not shown). Anti-CXCR3 Ab,
compared with a control Ab, reduced the adhesion of T cells to the EC
by an average of 30% (ANOVA, p < 0.001) (Table I). In early experiments, we counted both
adherent cells bound to the apical surface of the EC as well as cells
that had transmigrated to the basal side. We found no reproducible
difference in the outcome of the experiment using either count;
therefore, all results shown are pooled data. As well as I-TAC, CXCR3
is also the receptor for IP-10 and Mig, both of which are expressed by
EC. To determine the specific contribution of I-TAC to IFN--induced
T cell adhesion, we used neutralizing Ab to I-TAC. The degree of
blocking was somewhat variable, ranging from 6 to >40% with a mean of
22% (ANOVA, p < 0.05) (Table I), raising the
possibility that in different cultures IP-10 or Mig may be the
predominant CXC chemokine affecting T cell adhesion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Notably, the
induction of I-TAC was resistant to suppression by
CsA. During an immune response, EC are activated by inflammatory cytokines to express adhesion molecules which are necessary for the initial interaction of EC with T cells and other leukocytes (1). There is now mounting evidence that subsequent to their extravasation, T cells (specifically of the CD45RO+ memory phenotype) can recognize Ag on EC and receive sufficient costimulation from them to become fully activated (16, 17, 18). In the last few years, a more subtle role for EC in the extravasation process has been revealed. An important early step in recruitment of T cells is the up-regulation of integrin affinity. The tight binding of integrins to their Ig superfamily ligands is essential for firm adhesion of leukocytes to the endothelium and for the cessation of rolling. Evidence suggests that increased affinity is triggered by chemokines, often secreted by EC, and bound to their surface. Chemokines are generally chemotactic for subsets of leukocytes; IP-10, Mig, and I-TAC, for example, preferentially recruit activated memory T cells. EC, therefore, via secretion of specific chemokines may shape the subsequent immune response. We have previously suggested that triggering of T cell activation at different thresholds may also allow EC to affect the outcome of an antigenic challenge (16).
It has been proposed that chemokines bind to the surface of EC through
heparan-sulfate interactions (3, 27). For example, IL-8,
platelet factor 4 (PF4), macrophage-inflammatory protein-1ß, and
IP-10 have all been shown in vitro to bind to EC (2, 27, 28, 29, 30, 31). Given the similarities between I-TAC and IP-10, it
seems likely that I-TAC would also be secreted and captured at the EC
surface. However, we were not able to detect I-TAC protein either in
supernatants of activated EC or on the cell surface by
immunohistochemistry (data not shown). The failure to detect bound
I-TAC on cultured EC may be due to low sensitivity of the assay or,
alternatively, to masking of the recognition epitope as a result of
I-TAC binding to heparan sulfate or other surface proteins. We were
also unable to detect bound I-TAC on freshly prepared fragments of
umbilical vein treated with IFN- (data not shown), suggesting that
lack of detectable binding was not merely due to loss of binding sites
on passaged cells (3, 31).
To determine whether the I-TAC made by EC is functional, we performed T cell adhesion assays. We found that blocking CXCR3, the I-TAC receptor, decreased the adhesion of T cells to EC monolayers by an average of 30%. Because of the promiscuous binding of CXC chemokines by the CXCR3 receptor, this result does not distinguish between the effects of I-TAC, IP-10, and Mig. Blocking Abs to I-TAC also blocked adhesion, although the degree of blocking was more variable. There was a small but clear effect with the anti-I-TAC Ab, however, confirming that EC-derived I-TAC is, indeed, functional. The variability is likely due to natural variation in the balance of I-TAC, IP-10, and Mig expression in different combinations of EC and T cells from different donors, and highlights the functional redundancy commonly seen within chemokine families.
Given previous reports that CXCR3 is expressed exclusively on memory T cells, it is likely that the responding T cells in our cultures are also memory cells. I-TAC, therefore, acts quite differently to cytokines such as secondary lymphoid tissue chemokine and macrophage-inflammatory protein-3ß which, acting through CCR7, preferentially recruit naive T cells (32). Recruitment of memory T cells by EC would also be consistent with our previous data demonstrating activation of memory, but not naive, T cells by cultured EC (16).
It is interesting to note that the I-TAC gene was expressed
by EC under conditions in which EC provide CsA resistance to T cells.
We have previously shown that EC-derived signals render T cell IL-2
synthesis resistant to CsA, and we have suggested that this may be
important in the setting of graft arteriosclerosis, which appears to be
driven by chronic immune inflammation, involving EC activation of
CD4+ memory T cells (16, 33). This
process has the hallmarks of a Th1-like reaction. Activated
CD4+ T cells synthesize IFN-, which further
activates EC to express MHC class II molecules and chemokines such as
I-TAC. As a consequence, more Th1 memory T cells are recruited and
activated, reinforcing the response. EC also express other CXC
chemokines such as IP-10, IL-8, and Mig (34, 35, 36, 37, 38) and CC
chemokines such as MCP-1 (39), which may further amplify
the reaction. Our demonstration that the expression of
I-TAC, IP-10, MIG, and
MCP-1 (Fig. 4) is resistant to CsA is significant, as most
transplant patients are receiving the drug as part of their
immunosuppressant therapy. Interestingly, I-TAC induction is
also resistant to the immunosuppressive drug dexamethasone
(13). Our data, therefore, support previous studies which
suggest that EC-derived chemokines, such as I-TAC, may have important
roles to play in transplant rejection (40). I-TAC, and
potentially other CXC chemokines, may thus contribute to ongoing
CsA-resistant T cell activation by driving the recruitment of memory T
cells into the grafted tissue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher C. W. Hughes, Department of Molecular Biology and Biochemistry, 3205 Biological Sciences II, University of California, Irvine, CA 92697.
3 Abbreviations used in this paper: EC, endothelial cells; CsA, cyclosporin A; HUCE, human capillary endothelial cells; SMC, smooth muscle cells; I-TAC, IFN-inducible T cell chemoattractant; IP-10, IFN- inducible protein-10; Mig, monokine-induced by IFN-; CXCR3, CXC chemokine receptor 3; MCP-1, monocyte chemoattractant protein-1; M199, medium 199.
Received for publication September 30, 1999. Accepted for publication February 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chemoattractant (I-TAC): a novel non- ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009.. J. Biol. Chem. 271:22878. (MIG), IFN-inducible T cell chemoattractant (I-TAC), and IFN--inducible protein-10 (IP-10) chemokines by human neutrophils. J. Immunol. 162:4928. production. J. Immunol. 159:3247.[Abstract]
-interferon-induced protein (IP-10) in delayed immune responses in human skin. J. Exp. Med. 166:1098.-interferon-inducible cytokine (IP-10). J. Exp. Med. 166:1084., LPS and IL-1ß. Science 243:1467.This article has been cited by other articles:
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  S. Wang, B. Lu, P. Wood, and R. D. Lund Grafting of ARPE-19 and Schwann Cells to the Subretinal Space in RCS Rats Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2552 - 2560. [Abstract] [Full Text] [PDF]
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 V. Petkovic, C. Moghini, S. Paoletti, M. Uguccioni, and B. Gerber I-TAC/CXCL11 is a natural antagonist for CCR5 J. Leukoc. Biol., September 1, 2004; 76(3): 701 - 708. [Abstract] [Full Text] [PDF]
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 K. Mohan, Z. Ding, J. Hanly, and T. B. Issekutz IFN-{gamma}-Inducible T Cell {alpha} Chemoattractant Is a Potent Stimulator of Normal Human Blood T Lymphocyte Transendothelial Migration: Differential Regulation by IFN-{gamma} and TNF-{alpha} J. Immunol., June 15, 2002; 168(12): 6420 - 6428. [Abstract] [Full Text] [PDF]
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K. Singbartl, J. Thatte, M. L. Smith, K. Wethmar, K. Day, and K. Ley A CD2-Green Fluorescence Protein-Transgenic Mouse Reveals Very Late Antigen-4-Dependent CD8+ Lymphocyte Rolling in Inflamed Venules J. Immunol., June 15, 2001; 166(12): 7520 - 7526. [Abstract] [Full Text] [PDF]
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 W. W. Hancock, B. Lu, W. Gao, V. Csizmadia, K. Faia, J. A. King, S. T. Smiley, M. Ling, N. P. Gerard, and C. Gerard Requirement of the Chemokine Receptor CXCR3 for Acute Allograft Rejection J. Exp. Med., November 20, 2000; 192(10): 1515 - 1520. [Abstract] [Full Text] [PDF]
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