Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1102573 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;73:771-780.)
© 2003 by Society for Leukocyte Biology

Antibody-mediated blockade of the CXCR3 chemokine receptor results in diminished recruitment of T helper 1 cells into sites of inflammation

Jenny H. Xie*, Naomi Nomura*, Min Lu{dagger}, Shiow-Ling Chen{dagger}, Greg E. Koch{dagger}, Youmin Weng{dagger}, Raymond Rosa*, Jerry Di Salvo{dagger}, John Mudgett{dagger}, Laurence B. Peterson*, Linda S. Wicker{dagger} and Julie A. DeMartino{dagger}

* Departments of Pharmacology and
{dagger} Immunology & Rheumatology, Merck Research Laboratories, Rahway, New Jersey

Correspondence: Dr. Jenny H. Xie, R80Y-125, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail: Jenny_xie{at}merck.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Naïve T cells, when activated by specific antigen and cytokines, up-regulate adhesion molecules as well as chemokine receptors on their surface, which allows them to migrate to inflamed tissues. Human studies have shown that CXCR3 is one of the chemokine receptors that is induced during T cell activation. Moreover, CXCR3-positive T cells are enriched at inflammatory sites in patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this study, we use a mouse model of inflammation to demonstrate that CXCR3 is required for activated T cell transmigration to inflamed tissue. Using an anti- mCXCR3 antibody, we have shown that in vitro-differentiated T helper (Th) 1 and Th2 cells up-regulated CXCR3 upon stimulation with specific antigen/major histocompatibility complex. However, only Th1 cells, when adoptively transferred to syngeneic recipients, are efficiently recruited to the peritoneum in an adjuvant-induced peritonitis model. Furthermore, the neutralizing anti-mCXCR3 antibody profoundly inhibits the recruitment of Th1 cells to the inflamed peritoneum. Real-time, quantitative reverse transcriptase-polymerase chain reaction analysis demonstrates that the CXCR3 ligands, interferon (IFN)-inducible protein 10 (CXCL10) and IFN-inducible T cell {alpha} chemoattractant (CXCL11), are among the many chemokines induced in the adjuvant-treated peritoneum. The anti-mCXCR3 antibody is also effective in inhibiting a delayed-type hypersensitivity response, which is largely mediated by enhanced trafficking of activated T cells to peripheral inflammatory sites. Collectively, our results suggest that CXCR3 has a critical role in T cell transmigration to sites of inflammation and thus, may serve as a molecular target for anti-inflammatory therapies.

Key Words: Th1/Th2 • chemokines • cell trafficking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recruitment of leukocytes from the blood stream into areas of injury or infection is essential for host defense but may also contribute to the inflammatory complications of allergic and autoimmune diseases. The leukocyte extravasation involves distinct steps, including initial rolling controlled by members of the selectin family or by {alpha}4 integrins, firm interaction mediated by integrins, and subsequent transendothelial migration [1 2 3 ]. Chemokines produced by endothelial cells and local inflammatory cells are an essential component of the leukocyte recruitment. Chemokines have been shown to trigger firm adhesion of leukocytes to vascular endothelium under flow condition by enhancing integrin–ligand-binding affinity [4 5 6 ]. Recently, Alon and colleagues [7 ] suggested that apical endothelial chemokines under shear force promote T cell migration across the endothelial barrier, even in the absence of chemotactic gradients. Inappropriate activation of the chemokine network has been associated with a number of human inflammatory diseases [8 ]; however, knowledge on specific roles of individual chemokines in a particular disease is very limited.

In contrast to granulocytes and monocytes, which predominate in the acute phase in local inflammation, recruitment of T cells needs antigen and cytokine stimulation to acquire necessary adhesion molecules and chemokine receptors. CXCR3, whose ligands include interferon (IFN)-inducible protein 10 (IP-10; CXCL10), monokine induced by IFN-{gamma} (MIG; CXCL9), and IFN-inducible T cell {alpha} chemoattractant (I-TAC; CXCL11), is one of the G protein-coupled chemokine receptors that is induced as a result of T cell activation [9 , 10 ]. In humans, expression of CXCR3 is restricted primarily to cells of the lymphoid lineage, including activated T cells, natural killer cells, and a subset of peripheral blood B cells [9 10 11 ]. In vitro, CXCR3 surface expression can be induced on T cells from human peripheral blood by treatment with exogenous interleukin (IL)-2, anti-T cell receptor (TCR) antibodies, or other stimuli that mimic the normal T cell activation process [12 , 13 ]. CXCR3 has been characterized as a marker for T cells associated in vivo with human inflammatory reactions. Its expression appears particularly prominent on cells with a T helper cell type 1 (Th1) bias, such as those enriched in the synovial fluid of patients with rheumatoid arthritis or in perivascular infiltrates of patients with active multiple sclerosis [13 14 15 ]. Hancock and colleagues [16] demonstrated that mice made deficient in CXCR3 showed profound resistance to development of acute allograft rejection. Thus, a CXCR3 antagonist may prove to be a therapeutic agent in the treatment of autoimmune disorders and transplant rejection.

In this study, we use a TCR transgenic model to study CXCR3 expression and function on T cells following activation with specific antigen and cytokines. Using an adjuvant-induced peritonitis model, the effect of anti-CXCR3 on the migration of adoptively transferred, in vitro-activated T cells to the inflamed peritoneum has been evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
AND transgenic mice, expressing an {alpha}/ß TCR (V{alpha}11, Vß3) recognizing Pigeon cytochrome C (88-104), was first developed by S. Hedrick and coworkers [17 ]. The AND transgenic mice purchased from Jackson Laboratory (Bar Harbor, ME) were backcrossed to the B10.BR strain for five generations. These mice were further backcrossed to B10.BR mice for seven generation at Merck Research Laboratories (Rahway, NJ). C57BL/6 mice (Taconic, Germantown, NY) were used for delayed-type hypersensitivity (DTH) response. DO11.10 TCR transgenic mice [18 ] were purchased from Jackson Laboratory. All mice were maintained under viral antigen-free/specific pathogen-free conditions in microisolator cages.

Reagents
The Pigeon cytochrome C peptide [(NH2)-KAERADLIAYLKQATAK-OH] was prepared at Merck Research Laboratories as described previously [19 ]. The murine CXCR3 peptide, (NH2)-YLEVSERQVLDASDFAF-Orn-C-OH, representing N-terminal receptor residues 2–18 [20 21 22 ], was custom-synthesized and purified at PeptidoGenic Research and Co. (Livermore, CA). Murine IL-12, IL-4, and IL-2 and antimurine CCR5 antibody were purchased from BD PharMingen (San Diego, CA).

Rabbit antimurine CXCR3 antibody preparation
To generate the anti-mCXCR3 antibody, rabbits were immunized with the N-terminal peptide (residues 2–18) (NH2)-YLEVSERQVLDASDFAF-Orn-C-OH coupled to thyroglobulin as described previously [19 ]. The immune sera were affinity-purified by peptide antigen affinity chromatography (Bio Express Cell Culture Services, West Lebanon, NH). Endotoxin levels were determined to be <1 EU/ml sera. Anti-mCXCR3 antibody staining of murine-activated T cells can be blocked by the immunizing peptide [23 ].

In vitro differentiation of T cells
A protocol similar to that described previously [24 ] was used to polarize IL-4- and IFN-{gamma}-producing CD4+ T cells following activation of naïve splenic cells from AND TCR mice. Briefly, spleens were prepared from AND TCR-transgenic mice. Red blood cells pigeon cytochrome C were eliminated using acetate kinase lysing buffer (Eq. 79-0422DG, Life Technologies, Grand Island, NY). Dead cells were removed using high phosphate-buffered saline (PBS). The cells were resuspended in RPMI-1640 medium (Cellgro, Mediatec, Herndon, VA) supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 µg/ml gentamycin, and 10% fetal calf serum. T cell differentiation was induced by culturing splenic cells at 2 x 106/ml with 1 µM pigeon cytochrome C peptide plus 2 ng/ml IL-2 and with 10 ng/ml IL-12 for Th1 cells or with 20 ng/ml IL-4 for Th2 cells. Cultures were fed with fresh medium (supplemented with peptide and appropriate cytokines) at days 2 and 4 and were then harvested at day 6 for the subsequent adoptive-transfer study.

Cytoplasmic staining of cytokines
BD PharMingen’s Cytofix/Cytoperm kit was used for cytokine detection at the single-cell level. Briefly, in vitro-differentiated Th1 and Th2 cells were restimulated with 1 µM PCC peptide. Four h later, GolgiPlug (Brefeldin A) was added to the culture for another 12-h incubation. Cells were then fixed, permeabilized, and stained with fluorescein isothiocyanate (FITC)-labeled anti-IFN-{gamma} and antigen-presenting cell (APC)-labeled anti-IL-4 monoclonal antibodies (mAb), according to the manufacturer’s manual.

Adoptive transfers and complete Freund’s adjuvant (CFA) injection
In vitro-differentiated AND TCR T cells were harvested at day 6 and centrifuged over Ficoll-sodium metrizoate and were then incubated with 1 µM green tracker 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes, Eugene, OR), diluted in L-15 medium (Cellgro, Mediatec) with 5% fetal bovine serum (FBS) for 20 min at 37°C. Cells were centrifuged and incubated for an additional 30 min at 37°C in fresh L-15 medium with 5% FBS. Cells were then centrifuged and resuspended in PBS at 3 x 107/ml. Labeled cells (1.5x107) in 500 µl PBS were typically transferred intravenously (i.v.) into irradiated (750 rads) AND TCR mice. Twenty-four hours later, mice were given intraperitoneal (i.p.) injections of 500 µl PBS emulsified in CFA (Difco, Detoit, MI). Seventy-two hours after CFA injection, spleen and peritoneal cells were harvested and analyzed by flow cytometry.

Fluorescein-activated cell sorter (FACS) analysis
In vitro polarized Th1 and Th2 cells (1x106) were first incubated with 5 µg/ml anti-CD16/CD32 (BD PharMingen) to block Fc receptors. The cells were then stained with 2 µg affinity-purified rabbit anti-mouse CXCR3 antibody or with 2 µg control rabbit immunoglobulin G (IgG), followed by incubation with 1 µg phycoerythrin (PE)-conjugated donkey anti-rabbit IgG (ICN, Costa Mesa, CA). In adoptive-transfer studies, CMFDA-labeled cells were detected by direct fluorescence in the FL1 channel on the flow cytometer. In most cases, these spleen and peritoneal cells were also stained with PE or APC-labeled anti-CD4 antibody (BD PharMingen). Single- or two-color fluorescence analysis was done using a FACScan (Becton Dickinson, San Jose, CA) using propidium iodide and light-scatter signals to gate out dead cells and debris.

T cell chemotaxis
A modified Boyden chamber chemotaxis system (ChemoTxTM, NeuroProbe, Gaithersburg, MD), consisting of a 96-well microplate and a filter (6.0-mm diameter, 5-µ pore size), coated on the bottom with fibronectin (50 µl of a 10 µg/ml solution, then air-dried), was used for chemotaxis measurements. Briefly, aliquots of polarized murine CD4+ T cells were washed and resuspended at 1 x 107cells/ml in warm (37°C) Hanks’ balanced saline solution (HBSS)/bovine serum albumin [(BSA); HBSS without phenol red, calcium, or magnesium (Mediatec)+0.01% BSA] and loaded with Calcein-AM (Molecular Probes) at a concentration of 2 µM for 30 min at 37°C. The cells were washed again in HBSS/BSA and resuspended in RPMI/BSA [RPMI without phenol red (Mediatec)+0.5% BSA+1% dimethyl sulfoxide] to a concentration of 1 x 107cells/ml. To initiate the chemotaxis, chemokines were diluted in warm (37°C) RPMI/BSA and added in 29 µl to the bottom of the microplate before affixing the filter to the unit. Aliquots (50 µl) of the Calcein-loaded T cells were then added to the top of the filter over each individual well. The microplates were subsequently incubated for 4 h at 37°C. Remaining cells were suctioned off the top of the filter. The filter was rinsed with PBS and wiped with a rubber squeegee. The plate with filter intact was read in a CytofluorTM II fluorometer (PerSeptive Biosystems, Foster City, CA).

Binding assay
Binding of 125I-IP-10 (2200 Ci/mmol, typically 20 PM) in the presence of unlabeled ligands was initiated by adding 6-day-cultured Th1 cells or mCXCR3-transfected RBL-2H3 cells (75,000 cells/point) as described previously [12 ]. After incubation at room temperature for 30 min, the cells were filtered through GF/C filters treated with 0.33% polyethyleneimine, washed with buffer containing 25 mM HEPES, 0.02% NaN3, and 0.5 M NaCl, pH 7.2, and counted using the Top Count.

DTH model
C57 BL/6 mice were immunized i.v. with 1.5 x 106 sheep RBC (sRBC; Colorado Serum Co., Denver) in 0.25 ml Dulbecco’s PBS. Four days after immunization, the mice were challenged by a subplantar injection in the hind footpad with 5 x 108 sRBC in 0.05 ml PBS. The hind-foot volume was measured with a mercury plethysmograph to a marked spot over the lateral malleolus just before challenge. The magnitude of the DTH response was measured 24 h later, and the data were expressed as the change (in µl) of footpad volume. Anti-CXCR3 (200 µg) was dosed intravenously while indomethacin (3 mg/kg) was dosed orally at the time of challenge.

Isolation of RNA and real-time, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; Taqman)
Total RNA was extracted using Trizol reagent. mRNA was then prepared using an Oligotex mRNA mini kit (Qiagen, Valencia, CA), according to the manufacturer’s protocol. cDNA was synthesized from RT of mRNA and further PCR amplified on an ABI PRISM 7700 sequence detector using reagents and protocols provided by Applied Biosystems (Foster City, CA). Primers and probes were designed with the Primer ExpressTM program (PE Biosystems, Foster City, CA) and checked for a lack of homology with other genes. Probes for genes of interest were labeled with the reporter dye FAM (6-carboxyfluorescein) and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific probe with VICTM. Except for GAPDH, forward primers were used at 300 nM, reverse primers at 900 nM, and probes at 200 nM; both GAPDH primers were used at 100 nM and the GAPDH probe at 200 nM. For a number of genes, predeveloped reagents were purchased from PE Biosystems and were used at concentrations suggested by the manufacturer. To detect the possible genomic DNA contamination, a minus RTase control was performed on each mRNA sample. PCR reactions for each mRNA species were performed in duplicate and in the same well as the GAPDH reaction. Average gene-specific threshold cycle (CT) values were normalized by subtracting GAPDH-specific CT values [CT (FAM)–CT (VIC)={Delta}CT]. mRNA amplification in CFA-treated sample was determined relative to saline-treated sample. {Delta}CT value of saline-treated sample was subtracted from {Delta}CT value for CFA-treated sample [{Delta}CT (CFA-treated)–{Delta}CT (saline-treated)={Delta}{Delta}CT]. Amplification number was calculated with the Eq. 2-{Delta}{Delta}CT. GAPDH forward primer: 5'-TGCACCACCAACTGCTTAG-3'; reverse primer: 5'-GGATGCAGGGATGATGTTC-3'; probe: 5'-VIC-CAGAAGACTGTGGATGGCCCCTC-TAMRA. MIG forward primer: 5'-TGCACGATGCTCCTGCA-3'; reverse primer: 5'-AGGTCTTTGAGGGATTTGTAGTG-3'; probe: 5'-6FAM-CAGCACCAGCCGAGG CACGA-TAMRA. IP-10 forward primer: 5'-GCCGTCATTTTCTGCCTCA-3'; reverse primer: 5'-GTCCTTGCGAGAGGGATCC-3'; probe: 5'-6FAM-CCTGCTGGGTCTGAGTGGGACTCAA-TAMRA. I-TAC forward primer: 5'-GGGCGCTGTCTTTGCATC-3'; reverse primer: 5'-AAGCTTTCTCGATCTCTGCCAT-3'; probe: 5'-6FAM-CCCCGCGATGAAAGCCGTCAA-TAMRA. CCR2 forward primer: 5'-AACAGTGCCCAGTTTTCTATAGG-3'; reverse primer: 5'-CGAGACCTCTTGCTCCCCA-3'; probe: 5'-6FAM-ACAGCAGATCGAGTGAGCTCTACATTCACTCC-TAMRA. CCR4 forward primer: 5'-AAGAAGAACAGAGCAGTGCGC-3'; reverse primer: 5'-GCGACCAGAAGCCGAGG-3'; probe: 5'-6FAM-TGATCTTCGGCGTGGTGGTCCTCT-TAMRA. CXCR3 forward primer: 5'-GCTGCTGTCCAGTGGGTTTT-3'; reverse primer: 5'-AGTTGATGTTGAACAAGGCGC-3'; probe: 5'-FAM-CCCTGGCCTCTGCAAAGTGGCA-TAMRA. Monocyte-chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), tumor necrosis factor {alpha} (TNF-{alpha}), IL-1ß, IFN-{gamma}, IL-4, and CCR5 primer and probe mixtures were obtained from Applied Biosystems.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of CXCR3 expression on murine Th cell subsets upon antigen stimulation
We generated mouse Th1 and Th2 cells by stimulating splenocytes from AND TCR-transgenic mice with PCC peptide in the presence of IL-12 or IL-4, respectively (see Materials and Methods). This protocol results in polarized IFN-{gamma} producing Th1 and IL-4 producing Th2 cells as shown by cytoplasmic staining (Fig. 1A ). To assess the surface expression of CXCR3 upon antigen stimulation in these two T cell subsets, we used affinity-purified rabbit polyclonal anti-CXCR3 antibodies (see Materials and Methods) for flow cytometry. This antibody has been shown to stain murine CXCR3 specifically by using transfected cells and by blocking staining with the immunizing peptide [23 , 25 ]. Using this antibody, we found that naive splenocytes from AND TCR mice had no detectable CXCR3 on the surface (Fig. 1C) . Upon antigen stimulation, induction of CXCR3 was detected by day 4, and the induction occurred in Th1 and Th2 cultures (Fig. 1B) . A Th1 bias for CXCR3 expression was not observed in mouse as is seen in human [26 , 27 ]. In fact, we found Th2 cells showed a slightly but consistently higher level of CXCR3 by flow cytometry in three independent cultures (Fig. 1C) . CCR5, conversely, was not induced under Th2 condition (Fig. 1B) , showing a strong Th1 bias as expected.



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  		Figure 1. Cytokine and chemokine receptor expression on murine Th cells. Th1 and Th2 cells were generated as described in Materials and Methods. (A) Polarization of Th1 and Th2 cells was examined by cytoplasmic staining of anti-IFN-{gamma} FITC and anti-IL-4 APC. Rat IgG1 controls were included. One of two comparable experiments is shown. (B) T cells, stimulated with antigen and cytokines for 4 or 7 days, were stained with rabbit anti-mCXCR3 (shown in red) or with rabbit IgG (shown in black) followed by PE-conjugated anti-rabbit IgG or were stained with rat anti-mCCR5 (shown in red) or with rat IgG (shown in black) followed by PE-conjugated anti-rat IgG. The histogram shown represents cells gated on the CD4+ population. One of two comparable experiments is shown. (C) Naïve cells represent splenic cells freshly isolated from AND TCR-transgenic mice, and Th1 and Th2 cells represent AND splenic cells stimulated with antigen and appropriated cytokines for 6 days. These cells were stained with rabbit anti-mCXCR3 (shown in green) or with rabbit IgG (shown in purple) followed by PE-conjugated anti-rabbit IgG. The histogram shown represents cells gated on the CD4+ population. Shown in the table is the percentage of Th1 or Th2 cells that are CXCR3-positive from three independent Th1 and Th2 cultures.

 
We next examined the mRNA level of CXCR3 in our murine Th1 and Th2 cultures, as preferential expression of CXCR3 in human Th1 cells was more evident at the mRNA level [26 , 27 ]. By using real-time PCR analysis, we found the CXCR3 mRNA level in mouse Th1 cells was two- to threefold higher than that in Th2 cells (Table 1 ). With the same cDNA samples, the CCR5 level in Th1 cells was approximately tenfold higher than that in Th2 cells. Expression of IFN-{gamma} and IL-4 was also analyzed to monitor polarization of our Th1 and Th2 cultures (Table 1) . CCR4, as expected, was expressed more in Th2 cells. In conclusion, murine CXCR3 expression seems to be largely dependent on antigen stimulation with very little influence from cytokines. The slight discrepancy between mRNA level and surface expression of CXCR3 in Th1 and Th2 cultures is likely a result of receptor internalization, as all three CXCR3 ligands are expressed at a much higher level in Th1 cells, as shown by real-time PCR analysis (Table 1) . I-TAC has been shown to be the most potent inducer of CXCR3 internalization [28 ].


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  		Table 1. Cytokine, Chemokine, and Chemokine Receptor mRNA Expression in Murine Th1 and Th2 Cells by Taqman Analysis

 
In vitro chemotaxis of murine Th1 and Th2 cells in response to CXCR3 ligands
To evaluate whether Th1 and Th2 cells, both of which express CXCR3 as shown above, are responsive to CXCR3 ligands, we examined the chemotactic response of Th subsets to I-TAC, IP-10, or MIG. As shown in Figure 2 , CXCR3+ Th2 cells exhibited chemotaxis to all three CXCR3 ligands to a similar extent as CXCR3+ Th1 cells in vitro (Fig. 2) . In contrast, Th2 cells failed to respond to RANTES or MIP-1ß (Fig. 2) , which is consistent with lack of CCR5 receptor expression on their surface as shown in Figure 1B . In conclusion, Th1 and Th2 cells migrated in response to CXCR3 ligands with similar chemotaxis index, suggesting that the CXCR3 receptor on both Th cell subsets is functional in vitro.



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  		Figure 2. Migratory response of Th1 and Th2 cells to various chemokines. Migration was assessed using a modified Boyden chamber chemotaxis system as described in Materials and Methods. The chemotactic response of calcein-AM-loaded Th1 and Th2 cells was tested in response to IP-10, MIG, I-TAC, regulated on activation, normal T expressed and secreted (RANTES), and MIP-1ß. Experiments were performed in triplicates. The results are expressed as a migration index, which is defined as mean number of cells for test substances divided by mean number of cells for medium control. One of three representative experiments is shown.

 
In vivo Th cell migration to CFA-treated peritoneum
To evaluate migration of Th cells to an inflammatory site quantitatively, we have modified a previously published CFA-induced peritonitis model [29 ]. In vitro polarized Th1 cells generated from AND TCR-transgenic mice were labeled with green tracker CMFDA before adoptive transfer into irradiated AND TCR-transgenic mice. Irradiation was sublethal (750 rads), and within the 4-day period of the assay, transferred T cells retained green tracker and were detectable and able to be quantified. Recipients were then challenged with an i.p. injection with CFA, and the number of adoptively transferred cells was detected by direct fluorescence in the FL1 channel on the flow cytometer. As a result of irradiation to the recipients, donor T cells became a significant population in lymphoid organs as well as inflammatory sites, which was desirable for quantification. Antigen was not injected with CFA, and therefore, the number of Th1 cells detected in the peritoneum was only a result of Th1 cell trafficking. We tested this modified model by adoptively transferring different numbers of T cells ranging from 2 to 15 million. Three days after i.p. injection of CFA, the number of green tracker-positive cells recovered from peritoneum correlated with the number of cells transferred (Fig. 3 ). Peritoneal cells (18%) were green tracker-positive when 1.5 x 107 cells were transferred, and only 2.9% of peritoneal cells were green tracker-positive when 2 x 106 cells were transferred. We therefore routinely use 1.5 x 107 T cells for subsequent transfer studies. In conclusion, this model is sensitive for quantifying T cell migration to the inflamed peritoneum.



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  		Figure 3. Migration of transferred Th1 cells to irradiated, CFA-treated mouse peritoneum. Th1 or Th2 cells (up to 1.5x107) were adoptively transferred into irradiated AND TCR mice (n=2). After 24 h, mice were given i.p. injections of PBS–CFA. Three days later, peritoneal cells were collected and analyzed by flow cytometry. The numbers in the upper-right corner of each panel is the percentage of the total peritoneal cells that are green tracker-positive. One of two comparable experiments is shown. FSC-H, FSC-height.

 
Preferential Th1 recruitment to CFA-treated mouse peritoneum
In the model described previously using nonirradiated recipients, we found that Th2 cells entered the inflamed peritoneum much less efficiently as compared with Th1 cells [29 ]. Here, we compared Th1 and Th2 migration using the modified protocol described above. As shown in Table 2 , when saline was injected into the peritoneum, very little migration (<1%) to the peritoneum was observed for transferred Th1 or Th2 cells. When inflammation was created in the peritoneal cavity with CFA treatment, transferred Th1 cells migrated to the peritoneum much more efficiently than Th2 cells (Table 2) . Percent of peritoneal cells (22.7±1.8) was green tracker-positive when Th1 cells were transferred, and only 6.0 ± 1.0 percent of peritoneal cells was positive when Th2 cells were transferred (P<0.005). Irradiation did not change the profile of preferential recruitment of Th1 cells. These results also demonstrated that expression of CXCR3 does not necessary lead to transmigration of T cells to sites of inflammation in vivo. Slightly more Th2 than Th1 cells were found in the spleens of the recipients, presumably as a result of the fact that fewer Th2 cells migrated to the inflamed peritoneum.


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  		Table 2. Preferential Th1 Recruitment to CFA-Treated Mouse Peritoneum

 
Effects of CXCR3 neutralization on T cell migration
Previously, we as well as others [29 , 30 ] demonstrated that blocking the interaction of selectin with its ligands using anti-P- and -E-selectin antibodies can inhibit Th1 cell migration to inflammatory sites. Here, we explore the roles of chemokines and chemokine receptors and their interactions in the recruitment of Th cells to the inflamed peritoneum. We first examined cytokine and chemokine mRNA expression in 3-day, CFA-treated peritoneal cavity by Taqman analysis. Using mRNA levels of peritoneal cell from saline-treated mice as a baseline, we found a number of cytokines were up-regulated (Table 3 ), including proinflammatory cytokines, TNF-{alpha}, IL-1ß, and IFN-{gamma}. MCP-1/CCL2 (a ligand for CCR2) and MIP-1{alpha}/CCL3 (a ligand for CCR5) were also induced in addition to two CXCR3 ligands, IP-10 and I-TAC (Table 3) .


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  		Table 3. Cytokine and Chemokine Expression in Peritoneal Cells from CFA-Treated Mouse Peritoneum with or without Prior Th1 Cell Transfer

 
To examine whether blocking the interaction of a single chemokine receptor with its ligand(s) would have a significant effect on T cell migration to inflammatory sites in vivo, we used a neutralizing polyclonal anti-CXCR3 antibody in the CFA-induced T cell recruitment model described above. The anti-CXCR3 antibody was first tested in vitro and was shown to block the binding of radiolabeled IP-10 to CXCR3-transfected rat basophilic leukemia (RBL) cells as well as in vitro-polarized Th1 cells, with IC50 at 5 nM and 100 nM, respectively (Fig. 4 ). Th1 cells generated from AND TCR mice were transferred to irradiated AND TCR mice, and 400 µg anti-CXCR3 or control rabbit IgG was administered i.v. The number of recruited Th1 cells was calculated by multiplying the total number of peritoneal cells with the percentage of CD4+green tracker+ cells obtained from flow cytometry. In this representative experiment, numbers of recruited Th1 cells in the peritoneum were (3.2±0.40) x 105 for the rabbit IgG-treated group versus (0.67±0.086) x 105 for the anti-CXCR3 antibody-treated group (n=9; P=0.002; Fig. 5 ). Thus, the number of Th1 cells in the CFA-treated peritoneum was reduced by 79% with anti-CXCR3 antibody treatment. Slightly more Th1 cells were found in the spleens of the anti-CXCR3 antibody-treated group, presumably as a result of the fact that fewer Th1 cells are trafficking to the inflamed peritoneal cavity.



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  		Figure 4. Inhibition of binding of CXCR3-positive cells to radiolabeled IP-10 by anti-mCXCR3 antibody. mCXCR3-transfected RBL-2H3 cells or activated murine T cells (6-day Th1 culture) were incubated with 125I-labeled human IP-10 in the presence of various concentrations of rabbit anti-mCXCR3 antibody. Experiment was performed in triplicates. Also shown are antibody concentrations required for achieving 50% inhibition for binding of CXCR3-transfected RBL cells and Th1 cells to radiolabeled IP-10 (IC50). One of two comparable experiments is shown.

 


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  		Figure 5. Effects of CXCR3 neutralization on in vivo Th1 cell migration to inflamed peritoneum. In vitro-differentiated Th1 cells (1.5x107) were preincubated with 400 µg anti-CXCR3 antibody before transferring to irradiated AND TCR mice through i.v. injection. Twenty-four hours later, mice were given i.p. injections of PBS–CFA. Three days later, peritoneal cells were collected and analyzed by flow cytometry. Shown in the upper panel is the number of Th1 cells recruited in the peritoneum, as determined by the percentage of CD4+green tracker+ T cells multiplied by the number of total peritoneal cells by a hemocytometer. The error bars represent SEM from nine mice in each group (*, P<0.005). Shown in the lower panel is the percentage of splenic cells that are CD4+green tracker+ (n=9). One of two comparable experiments is shown.

 
Induction of CXCR3 on T lymphocytes with antigen stimulation in vivo
We have demonstrated that naive T cells stimulated with antigen in vitro up-regulate CXCR3 on their surface, which is necessary but not sufficient for entering an inflammatory site. To examine the induction of CXCR3 in vivo, we adoptively transferred naive DO11 T cells into BALB/c recipient mice. After 24 h, the mice were immunized by footpad injection with ovalbumin (OVA) and CFA. Draining popliteal lymph nodes were removed 3 days later, and cells were stained for CD4, DO11 TCR (KJ126), and anti-CXCR3. As expected, DO11 CD4+KJ126+ T cells expanded in lymph nodes in response to OVA stimulation [29 , 31 ]. Naïve T cells did not express CXCR3 on the surface (data not shown). Upon antigen stimulation, CXCR3 was up-regulated only on antigen-specific CD4+KJ126+ T cells, demonstrating again the importance of T cell receptor signaling for induction of CXCR3 (Fig. 6 ). In conclusion, T cells up-regulate CXCR3 on the surface after encountering antigen/antigen-presenting cells in the draining lymph nodes, which is required for subsequent homing to sites of inflammation.



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  		Figure 6. Induction of CXCR3 expression on antigen-activated T cells in vivo. Naive DO11 splenic cells (3.0x107) were adoptively transferred into BALB/c mice (n=4). After 24 h, mice were immunized with OVA and CFA by footpad injection. Draining lymph nodes were collected 3 days later and were stained with APC-conjugated anti-CD4 and FITC-labeled KJ126 and with rabbit anti-mouse CXCR3 antibody or with rabbit IgG followed by anti-rabbit Ig conjugated with PE. Shown here is the CXCR3 expression on CD4+KJ126+ and CD4+KJ126– cells in the draining lymph node from a representative mouse. One of two comparable experiments is shown.

 
Effects of CXCR3 neutralization in a DTH model
DTH is a T cell-mediated immune response, which includes the induction phase in the lymph nodes and subsequent migration of effector T cells and other leukocytes to the site of antigen injection. IP-10 has been previously shown to play a role in DTH responses [32 , 33 ]. Here, we examined whether anti-CXCR3 antibody treatment, which inhibited T cell migration to sites of inflammation as shown in Figure 5 , would result in a diminished DTH response. Our DTH model is typically done by immunizing mice with sRBC, i.v., and challenging the mice 3 days later with RBC by a subplantar injection in the hind footpad. The magnitude of the DTH response is measured 24 h later with a mercury plethysmograph. To examine the role of CXCR3 in the DTH response, anti-CXCR3 antibody (200 µg) was injected i.v. at the time of challenge, and the volume of footpad swelling was measured. As shown in Figure 7 , anti-CXCR3 antibody treatment at the time of challenge decreased the footpad swelling by 70%. The anti-inflammatory agent, indomethacin, decreased the footpad swelling by 79%. In conclusion, anti-CXCR3 is effective in suppressing a DTH response.



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  		Figure 7. Effects of CXCR3 neutralization on DTH reactions in vivo. C57 BL/6 mice were immunized i.v. with 1.5 x 106 sRBC in PBS. Four days after immunization, the mice were challenged by a subplantar injection in the hind footpad with 5 x 108 sRBC. Anti-CXCR3 (200 µg; i.v.) or 3 mg/kg indomethacin (p.o.) were dosed at the time of challenge. The magnitude of the DTH response was measured 24 h later. Shown here is the change (in µl) of footpad volume over baselines determined before challenge. The error bars represent SD from 10 mice in each group (*, P<0.005; **, P<0.0005). One of two comparable experiments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have examined the roles of CXCR3 in mediating Th cell migration to an inflammatory site and in generating a T cell-mediated immune response. Our data indicate that activation of naive CD4 cells with specific antigen leads to rapid up-regulation of CXCR3 in vitro and in vivo. The dependence of CXCR3 induction on T cell receptor signaling is nicely demonstrated in vivo, as CD4+KJ126- T cells that cannot bind the administered antigen fail to up-regulate CXCR3. The cytokine influence on CXCR3 expression is modest. We have found that the mRNA level of CXCR3 is slightly higher in the presence of IL-12, and surface expression of CXCR3 is slightly higher in the presence of IL-4. Based on these data, CXCR3 is an activation rather than a differentiation marker, at least in the murine system. This is different from CCR5, which is expressed following activation under Th1 but not Th2 culture conditions.

In the process of establishing an in vivo T cell recruitment model, we have tried directly injecting chemokines i.p. We found that the numbers of transferred Th1 cells that traffic to the peritoneum are highly variable among individual animals. The CFA-induced peritonitis model, conversely, has provided us consistent results, as CFA-induced, inflammatory responses are more localized and sustained. Here, we have further modified our CFA-induced peritonitis model [29 ] by treating recipient mice with a sublethal level of irradiation. By doing so, the donor T cells become a significant population in lymphoid organs and the inflamed peritoneum, which makes it suitable for quantification of T cell homing. A number of groups have reported that T cells undergo homeostasis-driven proliferation in the secondary lymphoid organs in a lymphopenic environment, which can be induced by irradiation [34 35 36 ]. In agreement with their finding, we found some of the donor T cells in the CFA-treated peritoneum contain diluted green tracker by day 6 after irradiation and Th1 cell transfer. This does not appear to be a problem, as we routinely carry out this recruitment model within 3–4 days, when the number of donor cells has not increased significantly. Even if some degree of T cell expansion occurs in secondary lymphoid organs, the amount of Th1 cells in the inflamed peritoneum is determined only by the degree of T cell recruitment, as no local antigen is present to generate a local T cell-proliferative response. Irradiation is also shown to induce adhesion molecules including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin on endothelial cells in vitro [37 , 38 ]. Therefore, irradiation may enhance the inflammatory response that is induced by CFA in our system. Nevertheless, a similar, preferential Th1 recruitment is observed in irradiated and nonirradiated mice [29 , 30 ].

The preferential Th1 recruitment apparently cannot be explained by CXCR3 expression. The levels of CXCR3 on Th2 cells are at least as high as those on Th1 cells, and CXCR3+ Th2 cells exhibit chemotaxis to CXCR3 ligands to the same degree as CXCR3+ Th1 cells in vitro. Furthermore, CXCR3 expression on Th2 cells is stable in vivo when transferred to recipients treated with CFA alone (data not shown). Previously, we and others [29 , 30 , 39 ] showed that Th1 cells express higher levels of functional selectin ligands than do Th2 cells. This contributes, at least in part, to the preferential Th1 recruitment to an inflammatory site and strongly supports a model in which transmigration is a multistep process that requires participation of chemokine receptors in conjunction with adhesion molecules, including the selectin family. Our results indicate that CXCR3 is not sufficient to mediate T cell transmigration to an inflammatory site, as demonstrated in the case of the Th2 cells examined in the current study. However, CXCR3 is necessary, as CXCR3 neutralization diminishes the migration of Th1 cells dramatically.

Given the redundancy of chemokine networks shown in vitro, the degree of inhibition of T cell migration to an inflammatory site by anti-CXCR3 antibody alone is impressive. In our system, CCR5 is expressed in Th1 cells, and its ligand (mRNA level) is also expressed in CFA-treated peritoneum. Chemokines are appreciated mainly for their roles in enhancing binding affinity of integrins and inducing diapedesis through chemokine gradient. Alon and colleagues [7], who suggested that under shear force, apical endothelial chemokines promote T cell migration across endothelial barriers even in the absence of chemotactic gradients, recently revealed an additional function of chemokines. Piali et al. [40] reported that anti-CXCR3 inhibits adherence of IL-2-treated T cells to TNF-{alpha}- and IFN-{gamma}-treated human umbilical vein endothelial cells (HUVEC) under flow condition. It is interesting that transmigration across HUVEC monolayers is not inhibited by treatment with anti-CXCR3 antibody [40 ]. Furthermore, our preliminary studies reveal that a small molecule CCR2 antagonist inhibits monocyte transmigration through HUVEC monolayers without affecting firm adhesion of monocyte on endothelium under flow conditions (unpublished results). Based on these findings, biological redundancy of chemokines in vivo may not be as confounding as previously predicted. It is possible that in vivo, the chemokine system acts through a coordinated and perhaps sequential chain of events, and temporal and spatial control mechanisms come into play. Thus, one single chemokine receptor may play a unique, irreplaceable role in a particular step of leukocyte transmigration. It is a challenge, however, to identify the essential chemokine receptor for a particular disease.

In this study, trafficking Th1 cells to inflamed peritoneum is greatly inhibited; however, not completely stopped by anti-CXCR3 antibody treatment. We recovered donor T cells from the spleen and peritoneum of anti-CXCR3-treated mice and incubated them with rabbit IgG or anti-CXCR3 antibody to measure CXCR3 expression by flow cytometry. No additional binding to anti-CXCR3 antibody was detected in donor Th1 cells from the spleen. In the peritoneum, however, we detected a small number of Th1 cells that could bind anti- CXCR3 antibody in vitro, indicating these cells have escaped from binding to the antibody in vivo, or CXCR3 receptors are down-regulated in these cells after transmigration [28 ]. The first possibility suggests that incomplete antibody coverage is one of reasons that some Th1 cells make their way to the inflamed peritoneum. However, CXCR3-independent Th1 cell trafficking to peritoneum is not excluded in this system. The potential roles of CCR5 in Th1 trafficking to sites of inflammation need to be addressed, as CCR5 is expressed in Th1 cells (Fig. 1) , and its ligands are induced by CFA (Table 3) .

In light of the above observation, it is likely that in the clinic, 100% inhibition of effector T cell trafficking may not be achieved with one antibody or a small molecule chemokine receptor antagonist. Small doses of classical, immunosuppressive agents, e.g., cyclosporine A, may be required in combination with a CXCR3 antagonist to achieve clinical efficacy. In this regard, CXCR3 has the advantage of being involved in T cell activation in addition to T cell trafficking. Hancock and his colleagues [16] reported that T cells from CXCR3-deficient mice have diminished allogeneic responses. Recent work from Taniguchi and colleagues [41] demonstrates that the anti-CXCR3 antibodies inhibit the proliferation of naïve CD8+ T cells to allogeneic APCs, possibly through down-regulation of CD25 expression. The dual roles of CXCR3 make it an attractive molecular target for clinical application. Anti-CXCR3 antibodies have been shown to prolong allograft survival in rodents [16]. However, the ability of the CXCR3 blockade to ameliorate pre-existing autoimmune diseases remains to be determined.


   ACKNOWLEDGEMENTS
 
We thank Paul Fischer at Merck Flow Cytometry Center for excellent technical support.


   FOOTNOTES
 
Current address of Linda S. Wicker: Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK.

Received November 20, 2002; revised February 27, 2003; accepted February 28, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Butcher, E. C., Picker, L. J. (1996) Lymphocyte homing and homeostasis Science 272,60-66[Abstract]
  2. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm Cell 76,301-314[CrossRef][Medline]
  3. von Andrian, U. H., Mackay, C. R. (2000) T-cell function and migration. Two sides of the same coin N. Engl. J. Med. 343,1020-1034[Free Full Text]
  4. Weber, C., Alon, R., Moser, B., Springer, T. A. (1996) Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis J. Cell Biol. 134,1063-1073[Abstract/Free Full Text]
  5. Lloyd, A. R., Oppenheim, J. J., Kelvin, D. J., Taub, D. D. (1996) Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins J. Immunol. 156,932-938[Abstract]
  6. Carr, M. W., Alon, R., Springer, T. A. (1996) The C-C chemokine MCP-1 differentially modulates the avidity of beta 1 and beta 2 integrins on T lymphocytes Immunity 4,179-187[CrossRef][Medline]
  7. Cinamon, G., Shinder, V., Alon, R. (2001) Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines Nat. Immunol. 2,515-522[CrossRef][Medline]
  8. Gerard, C., Rollins, B. J. (2001) Chemokines and disease Nat. Immunol. 2,108-115[CrossRef][Medline]
  9. Loetscher, M., Gerber, B., Loetscher, P., Jones, S. A., Piali, L., Clark-Lewis, I., Baggiolini, M., Moser, B. (1996) Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes J. Exp. Med. 184,963-969[Abstract/Free Full Text]
  10. Cole, K. E., Strick, C. A., Paradis, T. J., Ogborne, K. T., Loetscher, M., Gladue, R. P., Lin, W., Boyd, J. G., Moser, B., Wood, D. E., Sahagan, B. G., Neote, K. (1998) Interferon-inducible T cell alpha 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. 187,2009-2021[Abstract/Free Full Text]
  11. Jones, D., Benjamin, R. J., Shahsafaei, A., Dorfman, D. M. (2000) The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia Blood 95,627-632[Abstract/Free Full Text]
  12. Weng, Y., Siciliano, S. J., Waldburger, K. E., Sirotina-Meisher, A., Staruch, M. J., Daugherty, B. L., Gould, S. L., Springer, M. S., DeMartino, J. A. (1998) Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors J. Biol. Chem. 273,18288-18291[Abstract/Free Full Text]
  13. Ruth, J. H., Rottman, J. B., Katsche, K. J., Jr, Qin, S., Wu, L., LaRosa, P., Pope, R. M., Kock, A. E. (2001) Selective lymphocyte chemokine receptor expression in the rheumatoid joint Arthritis Rheum. 44,2750-2760[CrossRef][Medline]
  14. Qin, S., Rottman, J. B., Myers, P., Kassam, N., Weinblatt, M., Loetscher, M., Koch, A. E., Moser, B., Mackay, C. R. (1998) The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions J. Clin. Invest. 101,746-754[Medline]
  15. Sorensen, T. L., Tani, M., Jennsen, J., Pierce, V., Lucchinetti, C., Folcik, V. A., Qin, S., Rottman, J., Sellebjerg, F., Streiter, R. M., Frederiksen, J. L., Ransohoff, R. M. (1999) Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients J. Clin. Invest. 103,807-815[Medline]
  16. Hancock, W. W., Lu, B., Gao, W., Csizmadia, V., Faia, K., King, J. A., Smiley, S. T., Ling, M., Gerard, N. P., Gerard, C. (2000) Requirement of the chemokine receptor CXCR3 for acute allograft rejection J. Exp. Med. 192,1515-1520[Abstract/Free Full Text]
  17. Kaye, J., Hsu, M. L., Sauron, M. E., Jameson, S. C., Gascoigne, N. R., Hedrick, S. M. (1989) Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor Nature 341,746-749[CrossRef][Medline]
  18. Murphy, K. M., Heimberger, A. B., Loh, D. Y. (1990) Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo Science 250,1720-1723[Abstract/Free Full Text]
  19. Miller, D. K., Ayala, J. M., Egger, L. A., Raju, S. M., Yamin, T. T., Ding, G. J., Gaffney, E. P., Howard, A. D., Palyha, O. C., Rolando, A. M., et al (1993) Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells J. Biol. Chem. 268,18062-18069[Abstract/Free Full Text]
  20. Tamaru, M., Tominaga, Y., Yatsunami, K., Narumi, S. (1998) Cloning of the murine interferon-inducible protein 10 (IP-10) receptor and its specific expression in lymphoid organs Biochem. Biophys. Res. Commun. 251,41-48[CrossRef][Medline]
  21. Soto, H., Wang, W., Strieter, R. M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Hedrick, J., Zlotnik, A. (1998) The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3 Proc. Natl. Acad. Sci. USA 95,8205-8210[Abstract/Free Full Text]
  22. Lu, B., Humbles, A., Bota, D., Gerard, C., Moser, B., Soler, D., Luster, A. D., Gerard, N. P. (1999) Structure and function of the murine chemokine receptor CXCR3 Eur. J. Immunol. 29,3804-3812[CrossRef][Medline]
  23. Park, M. K., Amichay, D., Love, P., Wick, E., Liao, F., Grinberg, A., Rabin, R. L., Zhang, H. H., Gebeyehu, S., Wright, T. M., Iwasaki, A., Weng, Y., DeMartino, J. A., Elkins, K. L., Farber, J. M. (2002) The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo J. Immunol. 169,1433-1443[Abstract/Free Full Text]
  24. Nakamura, T., Kamogawa, Y., Bottomly, K., Flavell, R. A. (1997) Polarization of IL-4- and IFN-{gamma}-producing CD4+ cells following activation of naive CD4+ T cells J. Immunol. 158,1085-1094[Abstract]
  25. Boztug, K., Carson, M. J., Pham-Mitchell, N., Asensio, V. C., DeMartino, J., Campbell, I. L. (2002) Leukocyte infiltration, but not neurodegeneration, in the CNS of transgenic mice with astrocyte production of the CXC chemokine ligand 10 J. Immunol. 169,1505-1515[Abstract/Free Full Text]
  26. Bonecchi, R., Bianchi, G., Bordignon, P. P., D’Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A., Sinigaglia, F. (1998) Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s J. Exp. Med. 187,129-134[Abstract/Free Full Text]
  27. Sallusto, F., Lenig, D., Mackay, C. R., Lanzavecchia, A. (1998) Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes J. Exp. Med. 187,875-883[Abstract/Free Full Text]
  28. Sauty, A., Colvin, R. A., Wagner, L., Rochat, S., Spertini, F., Luster, A. D. (2001) CXCR3 internalization following T cell-endothelial cell contact: preferential role of IFN-inducible T cell alpha chemoattractant (CXCL11) J. Immunol. 167,7084-7093[Abstract/Free Full Text]
  29. Xie, H., Lim, Y. C., Luscinskas, F. W., Lichtman, A. H. (1999) Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells J. Exp. Med. 189,1765-1776[Abstract/Free Full Text]
  30. Austrup, F., Vestweber, D., Borges, E., Lohning, M., Brauer, R., Herz, U., Renz, H., Hallmann, R., Scheffold, A., Radbruch, A., Hamann, A. (1997) P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues Nature 385,81-83[CrossRef][Medline]
  31. Jenkins, M. K., Khoruts, A., Ingulli, E., Mueller, D. L., McSorley, S. J., Reinhardt, R. L., Itano, A., Pape, K. A. (2001) In vivo activation of antigen-specific CD4 T cells Annu. Rev. Immunol. 19,23-45[CrossRef][Medline]
  32. Kaplan, G., Luster, A. D., Hancock, G., Cohn, Z. A. (1987) The expression of a gamma interferon-induced protein (IP-10) in delayed immune responses in human skin J. Exp. Med. 166,1098-1108[Abstract/Free Full Text]
  33. Gautam, S., Battisto, J., Major, J. A., Armstrong, D., Stoler, M., Hamilton, T. A. (1994) Chemokine expression in trinitrochlorobenzene-mediated contact hypersensitivity J. Leukoc. Biol. 55,452-460[Abstract]
  34. Goldrath, A. W., Bogatzki, L. Y., Bevan, M. J. (2000) Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation J. Exp. Med. 192,557-564[Abstract/Free Full Text]
  35. Cho, B. K., Rao, V. P., Ge, Q., Eisen, H. N., Chen, J. (2000) Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells J. Exp. Med. 192,549-556[Abstract/Free Full Text]
  36. Gudmundsdottir, H., Turka, L. A. (2001) A closer look at homeostatic proliferation of cd4(+) t cells: costimulatory requirements and role in memory formation J. Immunol. 167,3699-3707[Abstract/Free Full Text]
  37. Son, E. W., Cho, C. K., Rhee, D. K., Pyo, S. (2001) Inhibition of gamma-irradiation induced adhesion molecules and NO production by alginate in human endothelial cells Arch. Pharm. Res. 24,466-471[Medline]
  38. Prabhakarpandian, B., Goetz, D. J., Swerlick, R. A., Chen, X., Kiani, M. F. (2001) Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation Microcirculation 8,355-364[CrossRef][Medline]
  39. Lim, Y. C., Xie, H., Come, C. E., Alexander, S. I., Grusby, M. J., Lichtman, A. H., Luscinskas, F. W. (2001) IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands J. Immunol. 167,4476-4484[Abstract/Free Full Text]
  40. Piali, L., Weber, C., LaRosa, G., Mackay, C. R., Springer, T. A., Clark-Lewis, I., Moser, B. (1998) The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig Eur. J. Immunol. 28,961-972[CrossRef][Medline]
  41. Ogasawara, K., Hida, S., Weng, Y., Saiura, A., Sato, K., Takayanagi, H., Sakaguchi, S., Yokochi, T., Kodama, T., Naitoh, M., DeMartino, J. A., Taniguchi, T. (2002) Requirement of the IFN-alpha/beta-induced CXCR3 chemokine signalling for CD8+ T cell activation Genes Cells 7,309-320[Abstract]



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