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The Journal of Immunology, 2002, 168: 6420-6428.
Copyright © 2002 by The American Association of Immunologists

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}1

Karkada Mohan*, Ziqiang Ding*, John Hanly{dagger},{ddagger} and Thomas B. Issekutz2,*,{dagger},{ddagger}

Departments of * Pediatrics, {dagger} Medicine, {ddagger} Microbiology/Immunology, and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that the CXC chemokine, IFN-{gamma}-inducible T cell {alpha} chemoattractant (I-TAC), was chemotactic for IL-2-activated human T lymphocytes, which express abundant CXCR3. However, because most memory T lymphocytes are also CXCR3+, the ability of I-TAC to promote the migration of normal human blood T cells across HUVEC monolayers in Transwell chambers was examined. I-TAC induced a marked (4- to 6-fold) increase in transendothelial migration (TEM) of T cells across unstimulated HUVEC from 5.6 to 28% of input T cells and was substantially more active than IFN-{gamma}-inducible protein-10, another CXCR3 ligand. I-TAC significantly enhanced TEM of T cells across TNF-{alpha}, but not across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-activated HUVEC. IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-activated HUVEC produced substantial amounts of I-TAC, in contrast to TNF-{alpha}-treated EC. Both CD4+ and CD8+ T cells migrated in response to I-TAC to a similar extent, while memory T cells migrated several fold better than naive T cells. Blockade of LFA-1 strongly inhibited I-TAC-induced T cell TEM across unstimulated HUVEC, and ~50–60% of the TEM across cytokine-activated HUVEC. However, blocking both LFA-1 and very late Ag-4 abolished I-TAC induced T cell TEM. In vivo significant levels of I-TAC were detected in arthritic synovial fluid. Thus, I-TAC is one of the most potent chemoattractants of normal human blood CD4 and CD8 T cell TEM and is likely a major mediator of blood memory T lymphocyte migration to inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extravasation and accumulation of leukocytes in the tissues is a hallmark of chronic inflammatory conditions. Lymphocyte trafficking, both during normal circulation and recruitment to sites of inflammation involves a multistep process, in which lymphocyte surface integrins, endothelial adhesion molecules, and chemokines produced in the local microenvironment play an essential role (1, 2, 3). There are ~50 human chemokines identified to date (4, 5, 6, 7), which, based on the number and arrangement of the first two cysteine residues in their structure, are subdivided into four groups, CXC ({alpha}), CC ({beta}), C ({gamma}), and CX3C ({delta}). Chemokines up-regulated at the site of inflammation by proinflammatory cytokines, such as IFN-{gamma} and TNF-{alpha}, are thought to directly recruit lymphocytes by interacting with their chemokine receptors (4, 5, 6). CXC chemokines which have been shown to act on T lymphocytes include IL-8, stromal cell-derived factor (SDF-1),3 IFN-{gamma}-inducible protein-10 (IP-10), monokine induced by IFN-{gamma} (Mig) and IFN-{gamma}-inducible T cell {alpha} chemoattractant (I-TAC). Among these chemokines, SDF-1 shows broad specificity for different subtypes of T cells, while IP-10, Mig, and I-TAC are reported to be selectively chemotactic for memory or IL-2-activated T cells (8, 9). Hence, these latter three chemokines are thought to be important in the process of T cell recruitment in inflammation.

CXCR3 is the only receptor on T cells for IP-10, Mig, and I-TAC, and the binding affinity for I-TAC is reported to be much higher than for IP-10 and Mig (9). Nevertheless, all three of these chemokines are known ligands for CXCR3+ activated/memory T lymphocytes (8, 9, 10). I-TAC mRNA has been found to be up-regulated in IFN-{gamma}-treated astrocytes, monocytes (9), bronchial epithelial cells (11), neutrophils (12), and keratinocytes (13). In addition, I-TAC was recently shown to be up-regulated in IFN-{gamma}-stimulated human endothelial cells (ECs), suggesting a role for this chemokine in T lymphocyte recruitment to sites of inflammation (14). The pathophysiological role for I-TAC is not fully understood, although its expression by ECs in atherosclerotic lesions has been shown to correlate with CXCR3+ T cell accumulation within the lesions (15). Moreover, marked enrichment of CXCR3+ cells in the synovium, over blood, of patients with rheumatoid arthritis (16), and increased levels of circulating as well as intralesional CXCR3+ cells in pateints with multiple sclerosis (17, 18) suggest a possible chemotactic role for CXC chemokines in inflammation. In addition, enhanced expression of I-TAC mRNA by IFN-{gamma}-stimulated bronchial epithelial cells (11) and in allergic skin reactions (19) suggest that this chemokine may play an important role in the recruitment of activated T cells to lesions in the tuberculoid lung and to delayed type hypersensitivity reactions in the skin. Recently, prolonged cardiac allograft survival has been reported in IP-10-/- mice, also suggesting a role for CXC chemokines in graft rejection (20).

Previously, it was shown that T cells activated for 7–15 days in the presence of IL-2 and/or PHA, migrated in chemotaxis assays in response to CXC chemokines, IP-10, Mig, and I-TAC (9, 10). However, it is not clear whether blood T lymphocytes, which are not stimulated with IL-2 or PHA, can chemotax to these CXC chemokines. Because most circulating memory T cells express CXCR3, it was important to determine whether these lymphocytes would migrate without long-term activation to CXC chemokines and especially to I-TAC, which has the highest binding affinity of these chemokines (9). Moreover, transendothelial migration (TEM) of T cells in response to I-TAC, especially across cytokine-stimulated EC, as would occur during inflammation, has not been previously examined.

In the present study, chemotaxis and TEM of freshly isolated human peripheral blood T cells, without IL-2 preactivation, was investigated in response to I-TAC and IP-10. These freshly isolated T cells demonstrated substantial chemotaxis and TEM in response to I-TAC. TEM was further enhanced by TNF-{alpha}, but in contrast to other chemokines, not IFN-{gamma} stimulation of ECs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Recombinant human I-TAC and IP-10 were obtained from PeproTech (Rocky Hill, NJ), TNF-{alpha} (specific activity = 5 x 107 U/mg) was from R&D Systems (Minneapolis, MN), and IFN-{gamma} (107 U/mg) was purchased from Genentech (South San Francisco, CA). Anti-CXCR3 (1C6) was a gift from Dr. P. Ponath (Leukocyte, Cambridge, MA). Affinity purified, rabbit polyclonal anti-I-TAC Ab, with or without biotin label, were purchased from PeproTech. Anti-CD45 mAb (4B2) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Anti-CD45RA (G1–15) was a kind gift of Dr. J. Ledbetter (Bristol-Meyers-Squibb, Seattle, WA). Anti-CD45RO (UCHL-1) was obtained from Immunotech (Westbrook, ME). Affinity isolated goat-anti-mouse Ig was purchased from DAKO (Glostrup, Denmark). Anti-CD4 (OKT4), anti-CD8 (OKT8), and anti-LFA-1{beta} (IB4) Abs were obtained from ATCC. Anti-very late Ag (VLA)-4 (HP2/1) was a generous gift from Dr. F. Sanchez-Madrid (Universidad Autonoma de Madrid, Madrid, Spain).

Preparation of lymphocytes

Lymphocytes were isolated from human peripheral blood from healthy donors by gradient centrifugation and passage over a nylon wool column. In brief, acid citrate dextrose-heparin anticoagulated blood was gently mixed with an equal volume of warm PBS, layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden), and centrifuged at 900 x g for 20 min. The PBMC on top of the Ficoll-Paque were collected and washed three times with Ca2+Mg2+ free Tyrode’s solution. The PBMC were resuspended in RPMI 1640 (RPMI) medium with 10% human platelet-poor plasma (PPP) and were applied onto a nylon wool column. After 60 min of incubation, the unbound T lymphocytes were eluted, washed, resuspended in fresh RPMI medium plus 10% PPP at 2 x 106 cells/ml, and cultured overnight in tissue culture flasks. The nonadherent cells contained >96% T cells, <3% B cells, and <0.1% monocytes by immunofluorescence staining, and were >98% viable by trypan blue dye exclusion. In some experiments, to study the effect of IL-2 activation, T cells were cultured in RPMI + 10% PPP, with or without 400 U/ml recombinant human IL-2 for 8 days. Cells were cultured at a concentration range of 1.5–2 x 106 cells/ml medium; and to maintain the cell numbers at this concentration, IL-2-supplemented cultures were diluted at 2- to 3-day intervals using fresh IL-2 containing medium.

In some studies, naive (CD45RA+), memory (CD45RA-), CD4+- or CD8+-enriched T cells were purified by using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, T cells were incubated with mAb to either CD45RA (biotinylated G1–15), or CD4 (OKT4) or CD8 (OKT8) at 50 µg/108 cells/ml in RPMI medium plus 10% FCS at 4°C for 30 min. The cells were then washed twice and resuspended in HEPES-buffered HBSS containing 10% FCS at 107 cells/90 µl medium. Streptavidin-conjugated- or goat anti-mouse Ig-conjugated magnetic beads as appropriate were added and incubated at 6–12°C for 15 min, mixing every 5 min. Finally, cells were washed once and passed through a column in a magnetic field and the flow through cells were collected as CD45RA-, CD4- cells, or CD8- cells. The purity was >97% for the CD45RA- cell population, and >99% for CD4+ and CD8+ T cells by immunofluorescence staining.

Unfractionated, memory, naive, CD4+, CD4- or CD8+, CD8- T cells were labeled by incubating 5 x 107 cells/ml in RPMI + 15 mM HEPES + 10% FCS with 50 µCi/ml Na251CrO4 (Amersham, Oakville, Canada) at 37°C for 45 min. Cells were washed three times with RPMI and resuspended in RPMI + 5 mg/ml human serum albumin (HSA) for chemotaxis and TEM assays. Depending on the assay, cells were left untreated or pretreated with 20 µg/ml of anti-CD45, anti-LFA-1, anti-VLA-4, or anti-LFA-1 plus anti-VLA-4 mAbs for 20 min at room temperature, and then added on top of the EC monolayers in the TEM assay without removing the mAbs. In some assays, T cells were also pretreated with 10 µg/ml anti-CXCR3 mAb (1C6) before being tested for TEM to I-TAC.

Isolation and culture of ECs

HUVEC were isolated by collagenase digestion as described (21). Briefly, human umbilical veins were flushed with Ringer’s Lactate, then incubated with 0.5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO) at 37°C for 30 min. Detached EC were collected, washed, and then cultured in gelatin-coated flasks (Nunc, Naperville, IL) in RPMI containing 20% FCS (HyClone Laboratories, Logan, UT), 25 µg/ml EC growth supplement (BD Labware, Bedford, MA), 45 µg/ml heparin, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Confluent HUVEC in the flasks were gently trypsinized and seeded onto polycarbonate Transwell filters of 6.5-mm diameter and 5-µm pore size (Costar Corning, Cambridge, MA). The Transwell filters were prepared by coating with 0.01% gelatin at 37°C overnight followed by 3 µg of human fibronectin (Life Technologies, Grand Island, NY) at 37°C for 3 h. Then, 1.2 x 104 HUVEC in 100 µl HUVEC medium were seeded onto each filter and 0.6 ml of the culture medium was added to each lower chamber beneath the filter. After 6 days of culture, the integrity of confluent HUVEC monolayers was assessed by microscopic observation and by measuring the permeability of the monolayer using 125I-albumin diffusion.

Measurement of lymphocyte chemotaxis to I-TAC

Percentage of T cells migrating in response to I-TAC across 5 µm pore size polycarbonate filters (6.5-mm diameter) was investigated in 24-well Transwell chambers (Costar Corning). 51Cr-labeled T lymphocytes were suspended at 2 x 106 cells/ml in RPMI plus 5 mg/ml HSA and 100 µl of the cell suspension was added to the upper chamber of each Transwell precoated with gelatin and fibronectin. Chemokines were added to the lower chamber at the indicated concentrations. The plates were incubated for 60 min at 37°C in 5% CO2. The migrated T cells in the lower chambers were collected and the extent of T cell migration was measured by gamma counting. Percentage of migration was calculated by dividing the radioactivity of cells in the lower chamber by the total input radioactivity of the labeled cells added to the upper chamber.

Measurement of lymphocyte TEM

HUVEC were left untreated or were stimulated by adding TNF-{alpha} (200 U/ml), IFN-{gamma} (200 U/ml), or IFN-{gamma} plus TNF-{alpha} (200 U/ml each) to the lower chamber of the Transwells for 18 h. The endothelial monolayers in the Transwell inserts were rinsed with RPMI, 100 µl labeled cells at 2 x 106 cells/ml in RPMI plus HSA were added to the HUVEC monolayers, and the inserts were transferred to new wells (lower chambers) of a 24-well plate containing 0.6 ml of fresh RPMI plus HSA and the indicated chemokine. The Transwells were then incubated at 37°C in 5% CO2. After 4 h, T cells which had migrated through the HUVEC monolayer into the lower chambers were recovered. The radioactivity in these samples was determined by gamma counting. The percentage of migrated cells was calculated as above. Spontaneous release of 51Cr from the labeled cells during the 4-h migration assay was <2%.

Immunofluorescence staining

Briefly, T cells were washed, resuspended in PBS plus 0.5% BSA, and incubated with 10 µg/ml mAb to CD4, CD8, CD45RA, CD45RO, and CXCR3 at 4°C for 30 min. Cells were washed twice and incubated with FITC-conjugated sheep anti-mouse IgG (Sigma-Aldrich). Finally, cells were washed, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry.

Measurement of lymphocyte adhesion

HUVEC were grown to confluence in gelatin-coated 96-well tissue culture plates. 51Cr-labeled T lymphocytes (2 x 105 cells in 100 µl RPMI plus 10% FCS) were added to triplicate wells with or without I-TAC (200 ng/ml). In some experiments, HUVEC was pretreated with 200 ng/ml I-TAC for various times before adding labeled T cells. The cells were allowed to adhere at 37°C for 60 min. Nonadherent cells were removed by four washes with warm RPMI. The bound T cells were lysed with 0.1 N NaOH, collected into tubes, and the radioactivity was measured by gamma counting. Percentage of cell adhesion was calculated by dividing the radioactivity of bound cells by the radioactivity of total input cells.

Measurement of I-TAC

Synovial fluids were obtained from the rheumatology clinic of the Queen Elizabeth II Hospital (Dalhousie University), and the cell-free supernatants were stored at -80°C until tested for I-TAC. To obtain endothelial-conditioned medium, HUVEC was grown to confluency in 35-mm tissue culture wells in HUVEC medium and the growth medium was replaced with 3 ml/well RPMI plus 5 mg/ml HSA just before stimulating cells using 200 U/ml IFN-{gamma} and/or TNF-{alpha} for 18 h. HUVEC supernatant was collected and tested for I-TAC secretion using a sandwich ELISA with the ELISA amplification system (Life Technologies). Briefly, 50 µl/well of 0.5 µg/ml rabbit polyclonal anti-human I-TAC Ab in carbonate buffer (pH 9.6) was coated overnight at 4°C to 96-well Maxi-Sorp plates (Nunc Immuno Plates, Roskilde, Denmark), washed twice with 0.01% Tween 20/TBS, and blocked with 2% BSA at 37°C for 2 h. After three washes, samples and known concentrations of recombinant human I-TAC were added and incubated overnight at 4°C. Biotin-labeled polyclonal anti-human I-TAC Ab at 0.5 µg/ml (50 µl/well) was added and incubated for 2 h at 37° C, followed by streptavidin-alkaline phosphotase for 30 min at room temperature. Each step was separated by four washes. Then, 50 µl of NADPH substrate was added for 45 min, followed by 50 µl of the amplifier solution containing alcohol dehydrogenase and diaphorase according to manufacturer’s instructions. OD was read at 490 nm after stopping the reaction with 0.3 N H2SO4 solution. The detection limit of this ELISA amplification system was ~4 pg/ml.

Statistical analysis

Data were expressed as either the mean ± SEM of multiple assays, or as the mean ± SD of replicate from a representative experiment. ANOVA and Student’s unpaired t test were used to compare the differences between means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of I-TAC on T cell chemotaxis and TEM

The migration of unstimulated blood T cells, either across gelatin and fibronectin-coated microporous Transwell membranes or across HUVEC monolayers grown on Transwell membranes to various concentrations of I-TAC in the lower chamber of the Transwells was tested. For comparison, migration to IP-10, which also binds to CXCR3, was also measured. As shown in Fig. 1GoA, I-TAC at 10 ng/ml had little effect on T cell migration, but increased T cell chemotaxis by 5-fold from a background of 4–20% migration at a concentration of 100–300 ng/ml. In contrast, IP-10 at 100 ng/ml induced only a small increase in T cell migration, and at 300 ng/ml induced only a 3-fold increase in T cell chemotaxis, which was still less than that with I-TAC.



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  		FIGURE 1. Dose-dependant effect of I-TAC and IP-10 on T cell chemotaxis (A) and TEM (B). 51Cr-labeled blood T cells were added to the upper chamber of Transwells having either gelatin and fibronectin-coated membranes (A) or membranes with confluent HUVEC monolayers (B). Chemokines were added in the lower chamber at the indicated concentrations and migration was allowed to proceed for either 1 h (chemotaxis) or for 4 h (TEM) at 37°C. C, The effect of I-TAC induced TEM in the presence or absence of a chemokine gradient was tested by adding 200 ng/ml I-TAC to either lower, upper, or both Transwell chambers. D, The effect of anti-CXCR3 mAb (1C6) treatment of T cells on chemotaxis and TEM is shown. The percentage of migration was calculated by dividing the radioactivity of migrated cells by the total radioactivity of the cells added to the upper chamber. Data are expressed as means ± SD of triplicate wells of four to six similar experiments.

 
I-TAC also induced the migration of T cells across HUVEC monolayers. As shown in Fig. 1GoB, I-TAC at 100–400 ng/ml induced a 4- to 5-fold increase in T cell TEM from 6% to 22–28%, while IP-10 induced only a small increase in T cell TEM, and even at 400 ng/ml, only induced a 4% increased migration above medium control. Because there seems to be a plateau in the TEM to I-TAC between 200 and 400 ng/ml, in subsequent studies 200 ng/ml was used. Blood T cells from >10 donors showed a range of migration from 15 to 28% in response to this concentration of I-TAC with backgrounds of 2–6%. This variation is likely to be related to donor differences in I-TAC responsive T cells in the circulation, but in all experiments a strong TEM to I-TAC was observed and a much greater response to I-TAC than to IP-10.

To determine whether the T cell TEM to I-TAC was chemotactic or due to increased chemokinesis, the effect of adding I-TAC in the upper chamber with the T cells, or in both upper and lower chambers was tested. As shown in Fig. 1GoC, T cell migration across the HUVEC to the lower chamber was completely lost when I-TAC was added to either the upper or both upper and lower chambers, which indicated that I-TAC induced TEM was chemotactic not chemokinetic for the T cells in this system. This also suggested that the effect of I-TAC was on the T cells and not the HUVEC.

I-TAC specifically binds to CXCR3 on T lymphocytes; therefore, the effect of treating T cells with anti-CXCR3 on T cell chemotaxis and TEM was determined. I-TAC-induced T cell chemotaxis was inhibited by 90% and TEM by 84% following anti-CXCR3 mAb treatment (Fig. 1GoD), suggesting that I-TAC induced migration of the blood lymphocyte was mediated through CXCR3.

Effect of I-TAC on chemotaxis and TEM of T cells cultured with or without exogenous IL-2

Previous reports had shown that T cells cultured with IL-2 for 8 or more days to induce T cell proliferation and activation chemotaxed to I-TAC. Therefore, the effect of culturing T cells with and without IL-2 for 8 days on T cell chemotaxis and TEM to I-TAC was examined. Up to 90% of the T cells cultured without IL-2 were recovered and there was a 3-fold increase in the T cells in the presence of IL-2. Both groups of T cells demonstrated higher spontaneous migration to medium alone than did freshly isolated T cells (Table IGo vs Fig. 1GoA). However, both sets of cultured T cells showed similar and significant (p < 0.05) levels of chemotaxis to I-TAC whether they were cultured with or without IL-2.


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  		Table I. Effect of culturing blood T cells with IL-2 on chemotaxis and TEM to I-TAC1

 
The migration of T cells cultured in the presence or absence of IL-2 across HUVEC was quite different (Table IGo). T cells cultured without IL-2 had a relatively low spontaneous TEM and a marked increase in migration to I-TAC similar to fresh T cells. Although T cells cultured with IL-2 demonstrated chemotaxis to I-TAC in the absence of HUVEC (Table IGo); in the presence of HUVEC, these T cells spontaneously migrated across the EC in large numbers and the addition of I-TAC did not increase this further. Interestingly, these IL-2-activated T cells also had an increased spontaneous adherence to the HUVEC (data not shown). Immunofluorescence staining of fresh and cultured T cells showed that there was an increase in the proportion of T cells with a memory phenotype after culture, and culturing the cells did not increase CXCR3 expression, but IL-2 treatment nearly doubled the percentage of cells expressing CXCR3 (Table IIGo).


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  		Table II. CXCR3 expression and phenotype of human T cells cultured with or without IL-21

 
I-TAC-induced chemotaxis of T cell subpopulations

To identify the phenotype of T cells migrating in response to I-TAC, T cells were separated into CD4, CD8, CD45RA+ (naive), and CD45RA- (memory) fractions, and their chemotaxis to I-TAC across bare membranes was determined (Fig. 2Go). CD4+ T cells demonstrated lower spontaneous migration than CD4- T cells, but both CD4+ and CD4- T cells had a strong chemotactic response to I-TAC (Fig. 2GoA). Similarly, when T cells were separated into CD8+ and CD8- populations, their migration to I-TAC was comparable (data not shown), not only between these two populations, but also to that seen with CD4- and CD4+ cells, respectively.



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  		FIGURE 2. Chemotaxis of blood T cell subpopulations in response to I-TAC. CD4+, CD4-, CD45RA+ naive, and CD45RA- memory T cell subsets were isolated as described in Materials and Methods. Cells were 51Cr-labeled and 2 x 105 cells were allowed to migrate for 1 h to 200 ng/ml I-TAC. Values shown are percentage of total input radiolabeled cells and represent means ± SD of one of two similar experiments done in triplicate. Two additional experiments using CD8+ and CD8- T cells showed similar results to that of CD4+, CD4- T cells shown here. +, p < 0.05 vs medium controls; #, p < 0.02 naive vs memory T cells.

 
Fractionation of T cells into naive and memory T cells demonstrated that memory-enriched T cells had a slightly greater spontaneous migration than naive T cells, and only memory but not naive T cell chemotaxis was markedly increased to I-TAC (Fig. 2GoB). Memory-enriched T cells migrated ~4-fold (5–19%) more to I-TAC than to the medium control, while there was no significant response by naive T cells. Thus, I-TAC induced the chemotaxis of both CD4 and CD8 memory, but not naive T cells.

Effect of cytokine activation of the endothelium on T cell migration to I-TAC

In inflammation, T cells migrate across cytokine-activated EC in response to chemokines. Therefore, the effect of treating the HUVEC with TNF-{alpha}, IFN-{gamma}, and both cytokines for 18 h on I-TAC-induced T cell TEM was determined. As shown in Fig. 3Go, T cell migration across HUVEC treated with TNF-{alpha}, IFN-{gamma}, or both cytokines was increased 4- to 5-fold from 4% to 15–23%. I-TAC significantly increased lymphocyte TEM across the TNF-{alpha}-stimulated EC from 15 to 30% of input T cells. The TEM to I-TAC across TNF-{alpha}-activated HUVEC was also significantly greater than observed across unstimulated EC in response to I-TAC, and appeared to be additive to that of either I-TAC alone or TNF-{alpha} activation alone. In striking contrast to TEM across TNF-{alpha}-treated EC, T cell migration to I-TAC across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-stimulated EC was similar to the migration seen in the absence of I-TAC across HUVEC treated with these cytokines. Similarly, migration to I-TAC across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-stimulated EC was not significantly higher than that to I-TAC alone across unstimulated endothelium. Thus, IFN-{gamma} treatment of the HUVEC reduced the response to added I-TAC and also prevented the increase in TEM to I-TAC across TNF-{alpha}-stimulated HUVEC. These findings show that T cell TEM to I-TAC is differentially regulated by IFN-{gamma} and TNF-{alpha} activation of the endothelium. Moreover, this effect was not caused by change in T cell adhesion to cytokine-stimulated HUVEC, as we did not observe any effect of I-TAC on T cell adhesion to IFN-{gamma} and/or TNF-{alpha}-stimulated endothelium (data not shown).



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  		FIGURE 3. Effect of endothelial activation by cytokines on T cell TEM in response to I-TAC. ECs were unstimulated or stimulated with 200 U/ml of the indicated cytokines for 18 h and labeled T cells were allowed to migrate to either medium control or in response to 200 ng/ml I-TAC in a 4-h TEM as outlined in Fig. 1Go. Values shown are percentage of total input radiolabeled cells. Pooled data from 10–18 independent experiments, using T cells and HUVEC from different donors, are expressed as means ± SEM. *, p < 0.001 vs medium control; #, p < 0.0001, migration to I-TAC across unstimulated vs TNF-{alpha}-stimulated HUVEC.

 
Migration of CD4 and CD8 T cells to I-TAC across normal and cytokine-activated endothelium

The effect of I-TAC on TEM of CD4 and CD8 T cells across normal and cytokine-stimulated EC was investigated. I-TAC significantly increased TEM of both CD4+ and CD4- T lymphocytes across unstimulated HUVEC (Fig. 4Go). Both CD4+ and CD4- T cells demonstrated an increase in TEM across IFN-{gamma} and/or TNF-{alpha}-treated HUVEC. I-TAC-stimulated TEM was enhanced across TNF-{alpha} but not IFN-{gamma} and IFN-{alpha} plus TNF-{alpha}-treated endothelium for both CD4+ and CD4- T cells. Similar results were obtained using CD8+ and CD8- T cells (data not shown).



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  		FIGURE 4. TEM of CD4+ (A) and CD4- (B) T cells in response to I-TAC. HUVEC monolayers were either unstimulated or stimulated with 200 U/ml of the indicated cytokines for 18 h. CD4+ or CD4- T cells were labeled with 51Cr and used for TEM as outlined in Fig. 1Go. The migrated cells were collected and the values are expressed as percentage of total input radiolabeled cells. Data from one of two independent experiments, performed in triplicate, were expressed as means ± SD. Two more experiments using positively selected CD8 T cells (CD8+ and CD8-) showed similar results to that of CD4+, CD4- T cells shown here. *, p < 0.001 and X, p < 0.01, I-TAC vs medium control.

 
Migration of memory and naive T cells to I-TAC across normal and cytokine-activated endothelium

I-TAC induced a marked increase in CD45RA- memory-enriched T cell TEM across unstimulated HUVEC, but had a relatively meager effect on CD45RA+ naive-enriched T cells (Fig. 5Go) similar to the chemotactic response to I-TAC. Cytokine treatment of HUVEC increased memory, but not naive T cell TEM. I-TAC further enhanced memory-enriched lymphocyte TEM across TNF-{alpha}, but not IFN-{gamma} or IFN-{alpha} plus TNF-{alpha}-treated HUVEC. Naive enriched T cell migration was only slightly increased across TNF-{alpha}-activated EC, and was much less than that of memory T cells to all stimuli. Thus, memory T cells, but not naive T cells, were responsible for the increased TEM to I-TAC across unstimulated HUVEC, and for the increased response across TNF-{alpha}-stimulated endothelium. Supporting this observation, freshly isolated memory T cells had nearly 10-fold higher proportion of cells expressing CXCR3 (33%) compared with naive T cells (3.5%).



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  		FIGURE 5. TEM of CD45RA- memory (A and C) and CD45RA+ naive (B and D) T cells in response to I-TAC. HUVEC monolayers were either unstimulated or stimulated with 200 U/ml of the indicated cytokines for 18 h. CD45RA+ or CD45RA- T cells were labeled with 51Cr and used for TEM as outlined in Fig. 1Go. Migration is expressed as the percentage of total input radiolabeled cells. Data from two independent experiments performed in triplicate are expressed as means ± SD. X, p < 0.01 vs medium control; #, p < 0.01 naive vs memory T cells.

 
Effect of cytokine stimulation of HUVEC on I-TAC secretion

Because cytokine stimulation of the HUVEC differentially regulated T cell TEM to I-TAC, the effect of cytokine treatment on the production of I-TAC by HUVEC was determined in EC-conditioned media. As shown in Table IIIGo, unstimulated and TNF-{alpha}-stimulated HUVEC produced no detectable amounts of I-TAC. However, treatment of the HUVEC with IFN-{gamma} induced a large amount of I-TAC to be produced by the EC and released into the medium. Stimulation of the HUVEC with both IFN-{gamma} and TNF-{alpha} also induced I-TAC secretion and caused an even greater (>60%) increase than IFN-{gamma} activation alone. Thus, IFN-{gamma}, but not TNF-{alpha}, induced I-TAC production by HUVEC, and TNF-{alpha} acted synergistically with IFN-{gamma} to augment I-TAC secretion.


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  		Table III. Effect of cytokine stimulation on I-TAC production by HUVEC1

 
The role of LFA-1 and VLA-4 in I-TAC-induced T cell TEM

To determine the cell adhesion molecules involved in T cell migration to I-TAC, the effect of function blocking mAbs to LFA-1 and VLA-4 on T lymphocyte TEM across unstimulated and cytokine-treated HUVEC was examined. I-TAC-induced TEM of >20% of the T cells across unstimulated, IFN-{gamma}, and IFN-{gamma} plus TNF-{alpha}-treated HUVEC, and >30% T cell migration across TNF-{alpha}-stimulated HUVEC (Fig. 6Go). Anti-LFA-1 mAb blocked ~80% of the migration across unstimulated HUVEC, anti-VLA-4 mAb had little effect, while combined blockade of both LFA-1 and VLA-4 inhibited ~90% of the lymphocyte migration to I-TAC. Migration across IFN-{gamma}-stimulated HUVEC was completely abolished by anti-LFA-1 mAb, and unaffected by VLA-4 blockade. T cell TEM to I-TAC across TNF-{alpha} and IFN-{gamma} plus TNF-{alpha} stimulated HUVEC was significantly, but only partially (50–60%), inhibited by anti-LFA-1 mAb. Blocking VLA-4 inhibited this migration by ~10%, and the combination of LFA-1 and VLA-4 blockade completely abolished T cell TEM to I-TAC across these activated endothelia. These findings suggested that I-TAC-induced TEM is completely dependent on LFA-1 and VLA-4.



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  		FIGURE 6. Effect of blocking LFA-1 and VLA-4 on I-TAC-induced T cell TEM. HUVEC monolayers were either left unstimulated (A), or stimulated with 200 U/ml of the indicated cytokines (B–D) for 18 h. Blood T cells were labeled with 51Cr and either left untreated or treated with mAb to either CD45, LFA-1, VLA-4, or both LFA-1 plus VLA-4, and their TEM was measured as in Fig. 1Go. Values shown are means ± SD from triplicate wells, and are representative of three to four similar experiments. *, p < 0.001 untreated vs mAb treated T cells.

 
Presence of I-TAC in synovial fluid of arthritic joints

To evaluate the potential relevance of I-TAC to inflammation in vivo, the concentration of I-TAC in the synovial fluid of arthritic joints from patients with rheumatoid and osteoarthritis was determined. The synovial fluid samples from rheumatoid patients had significantly higher leukocyte counts (11.7 ± 0.9 x 106/ml) than those of patients with osteoarthritis (0.6 ± 0.2 x 106/ml). As shown in Fig. 7Go, significant concentrations of I-TAC were present in all of the rheumatoid joint synovial fluids. The mean concentration of I-TAC was 735 pg/ml. In contrast, no I-TAC was detected (<4 pg/ml) in the synovial fluid of patients with osteoarthritis. This suggests that large amounts of I-TAC are produced during joint inflammation in rheumatoid arthritis; and that osteoarthritis is not associated with production of this chemokine. Moreover, I-TAC is present at a site of chronic inflammation in vivo.



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  		FIGURE 7. Measurement of the concentration of I-TAC in the synovial fluid of patients with arthritis. Synovial fluid was obtained from patients with osteoarthritis (n = 12) and rheumatoid arthritis (n = 15) and the concentration of I-TAC was determined by ELISA as described in Materials and Methods. Each data point represents the I-TAC concentration in an individual synovial fluid sample and the lines represent the mean ± SEM I-TAC in the rheumatoid arthritis samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of chemokines in lymphocyte extravasation has been established in recent years; however, the relative contribution of many chemokines, even chemokines binding to the same receptors, in lymphocyte migration is inadequately understood (4, 6, 7, 22, 23). The CXC chemokines, I-TAC, IP-10, and Mig all bind to CXCR3, and these chemokines have been shown to act in vitro on IL-2-activated T cells, on allogeneically activated T cells in MLR, and on thymocytes during lymphopoiesis (8, 9, 10, 16, 24, 25). Although exogenous IL-2 can enhance CXCR3 expression on T cells in culture, we and others have also found CXCR3 expressed on 35–40% of normal blood T cells (Table IIGo) (10, 16, 26). Therefore, we investigated the chemotaxis and TEM of freshly isolated blood T cells in response to I-TAC, which is the most potent of the CXCR3 ligands, and compared it to IP-10 (9).

This is the first study to demonstrate that I-TAC is a chemoattractant for freshly isolated normal human blood T cells. I-TAC was effective at inducing both chemotaxis and TEM at 100–300 ng/ml, which is similar to the concentrations found to be effective with several other inflammation-associated chemokines, including RANTES, macrophage-inflammatory protein (MIP)-1{alpha}, and monocyte chemoattractant protein-1 (MCP-1) (27, 28). In addition to stimulating T lymphocyte chemotaxis across bare membranes, this study also demonstrates that I-TAC is a very potent inducer of T cell TEM. Moreover, with the exception of SDF-1, I-TAC appeared to be among the strongest stimulators of human T cell TEM, which we have observed (28). Concentrations as low as 25 ng/ml stimulated significant TEM, suggesting that in vivo I-TAC may be an effective recruiter of normal blood T lymphocytes.

Cole et al. (9) were unable to show that I-TAC induced chemotaxis of unstimulated human T cells. This may be the result of the differences in the techniques used to measure and enumarate the T cells. Cole et al. (9) coated filters with collagen on the bottom surface and the cells that migrated to the lower surface of the filter were scored microscopically. In contrast, in our experiments the upper surface of Transwell membranes were coated with gelatin and fibronectin and radiolabeled T cells, which had migrated through the membrane in response to I-TAC, were quantified by gamma counting. Chemokines can enhance binding to fibronectin and collagen, so the coating on the Transwells may have facilitated chemotaxis to I-TAC. Unactivated T cells are less adhesive than IL-2-activated T cells to extracellular matrix proteins, such as the collagen on the underside of the filters. This may have resulted in better adherence and detection of the IL-2-activated T cells than the unstimulated lymphocytes. In any case, it appears that I-TAC under appropriate circumstances is a potent stimulator of unstimulated T cell chemotaxis.

Our results also indicated that I-TAC was having a specific chemotactic effect. I-TAC induced a concentration-dependent increase in lymphocyte TEM, and I-TAC-stimulated TEM was abolished in the absence of a concentration gradient for the chemokine. Blockade of CXCR3 also prevented I-TAC-stimulated chemotaxis and TEM, demonstrating that the effect of I-TAC on blood T cells was mediated through binding to CXCR3. This suggests that CXCR3 on blood lymphocytes is functional without prolonged lymphocyte activation.

Previous studies had shown that the percentage of T cells expressing CXCR3 increased after culture of the lymphocyte in the presence of exogenous IL-2 and/or PHA for 8–21 days (10, 16). Moreover, T cells cultured with IL-2 chemotaxed in response to I-TAC (9, 10). In the present study, lymphocytes cultured for 8 days with exogenous IL-2 also increased their expression of CXCR3 (Table IIGo) and I-TAC induced both chemotaxis as well as TEM of these cells (Table IGo). In addition, our results show that T cells maintained in culture without exogenous IL-2 migrated to I-TAC to a similar extent as lymphocytes incubated with IL-2, and the chemotactic response to I-TAC of the T cells cultured for 8 days was comparable to that observed with T cells freshly isolated from the blood. Thus, our studies suggest that I-TAC-stimulated migration of T cells does not require prolonged IL-2 preactivation of the T cells and demonstrate that there are significant numbers of I-TAC responsive T cells in blood. This finding suggests a role for I-TAC in the recruitment of unactivated circulating blood CXCR3+ T cells to inflammatory sites.

The T cells used in this study are considered "resting" T cells because they were freshly isolated from healthy donors, and the cells were not activated in vitro using cytokines or mitogens. These freshly isolated T cells lack activation markers and are CD25low and <3% CD69+ similar to those previously reported by others and found to express CXCR3 (16, 24). Because we found that I-TAC induced 12–25% of T cells to migrate almost all of the migration observed was by T cells lacking activation markers. Activation with IL-2 did not substantially increase the already considerable chemotaxis induced by I-TAC (Table IGo) under the conditions used in this study. Thus, blood T cells in the absence of cytokine or mitogen stimulation appear to be highly responsive to I-TAC.

Our studies demonstrate that I-TAC can stimulate blood T lymphocyte chemotaxis, and suggest that it acts on T cells to induce transmigration of endothelium in vitro. Although the chemotactic effect of I-TAC on blood T cells suggests that I-TAC-induced TEM is mediated by its actions on T cells, ECs have also been shown to express functional CXCR3 (29, 30, 31). However, I-TAC-induced T cell TEM does not appear to be due to an effect of I-TAC on the HUVEC. Addition of I-TAC in the upper chamber of the Transwell containing the EC monolayer did not result in T cell TEM, and a chemotactic gradient was required for TEM.

Previous studies have shown that CXCR3 is expressed on both CD4+ and CD8+ T lymphocytes of the memory phenotype, and on some naive CD8+ cells (16, 26). Our results show that I-TAC stimulates the chemotaxis and TEM of both freshly isolated blood CD4+ and CD8+ T cells, and induces the migration of only memory (CD45RA-) and not naive (CD45RA+) T lymphocytes. This demonstrates that CXCR3 is functional on both memory CD4+ and CD8+ T cells in the blood. In this respect, I-TAC resembles MCP-1, RANTES, and MIP-1{alpha} in inducing human blood memory T cell TEM (28).

During inflammation, EC are stimulated by cytokines such as TNF-{alpha} and IFN-{gamma} to up-regulate adhesion molecule expression, which promotes TEM and lymphocyte recruitment into the inflammatory site (3, 32, 33, 34, 35). Migration of T cells, monocytes, and neutrophils in response to chemokines has also been shown to be augmented across cytokine-activated endothelium (28, 36, 37). I-TAC-induced T cell TEM was markedly increased by TNF-{alpha} activation of HUVEC (Fig. 3Go). This is similar to the previous report with the chemokines, RANTES, MIP-1{alpha}, and SDF-1 (28). TEM to these latter chemokines was also augmented by IFN-{gamma} activation of HUVEC, while TEM to I-TAC did not increase further across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-activated endothelium. Furthermore, the increase in TEM across TNF-{alpha}, but not across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha}-stimulated HUVEC was observed for both CD4+ and CD8+ memory T cells. Therefore, I-TAC appears to be unusual among these inflammation-associated chemokines in that I-TAC-induced TEM is differentially regulated by IFN-{gamma} and TNF-{alpha} treatment of the endothelium.

ECs stimulated with cytokines can produce chemokines which can affect T cell TEM (28, 38, 39, 40). As previously shown, T cell TEM across IFN-{gamma}- and TNF-{alpha}-activated endothelium has been shown to be pertussis toxin sensitive (28). Chemokines present at the luminal (apical) surface of EC enhance leukocyte adhesion but not migration, while chemokines on the abluminal (basal) surface of EC induce TEM (41). This suggests that there is a requirement for a chemokine gradient for the T cell to transmigrate the endothelium. Our results demonstrate that treatment of the HUVEC with IFN-{gamma} and IFN-{gamma} plus TNF-{alpha}, but not TNF-{alpha} alone induces large amounts of I-TAC to be produced (Table IIIGo). In addition, IFN-{gamma}-activated EC can also produce IP-10 and Mig (14, 15, 42, 43). Because I-TAC-stimulated TEM is highly dependent on a chemokine gradient (Fig. 1GoC), one explanation for the lack of any increase in cell migration to I-TAC across IFN-{gamma} or IFN-{gamma} plus TNF-{alpha} activated HUVEC may be the disruption of the required chemotactic gradient.

Another explanation for the decreased migration to I-TAC across IFN-{gamma}-treated EC may relate to the proteoglycans on the surface of the EC, which can bind chemokines (42, 44, 45, 46). These proteoglycans are thought to produce a high local concentration of chemokines in vivo to induce rapid integrin activation on rolling leukocytes (47). Thus, I-TAC, IP-10, and Mig induced by IFN-{gamma} and IFN-{gamma} plus TNF-{alpha} treatment of the HUVEC is likely immobilized on the EC. These immobilized CXCR3+ ligands may also interfere with the chemotactic gradient or may contribute to desensitization and internalization of CXCR3+, as has been shown for some other chemokines (48, 49). It was also shown recently that surface-bound I-TAC, IP-10, and Mig on IFN-{gamma} activated, but not on resting EC, upon coculture, could rapidly induce T cell CXCR3 internalization (50). Experiments to be reported elsewhere by us also indicate that CXCR3 on T cells contributes together with other chemokine receptors to TEM across IFN-{gamma}-stimulated endothelium, further suggesting that IFN-{gamma} activated EC-derived I-TAC might play a role in desensitizing CXCR3+ T cells.

In the case of monocytes, MCP-1 produced by IL-1-activated EC has been shown to inhibit monocyte TEM in response to exogenous MCP-1 (36). Taken together, it appears that the chemokines produced by the endothelium in response to TNF-{alpha} and/or IFN-{gamma} may play an important role in differential regulation of T cell TEM induced by I-TAC. One thing which is not clear is why this effect is observed with I-TAC, but not with other chemokines, such as RANTES. Cytokine activation of HUVEC can induce substantial secretion of RANTES (our unpublished observation), but RANTES-induced TEM is enhanced across TNF-{alpha}- and IFN-{gamma}-treated EC (28). Further studies on EC chemokine production and T cell chemokine receptor modulation will be needed to understand these processes.

Our studies also examined the role of the major T lymphocyte integrins in I-TAC-induced TEM, because previous studies had shown that both LFA-1 and VLA-4 could mediate TEM, but the contribution of these integrins varied based on the cytokines and chemokines used to induce lymphocyte migration (51). I-TAC-induced TEM of T cells across unstimulated and IFN-{gamma}-stimulated EC was completely or almost completely abolished by blockade of LFA-1 (Fig. 6Go). In contrast, I-TAC stimulated T cell TEM across TNF-{alpha} and TNF-{alpha} plus IFN-{gamma} was partially inhibited by anti-LFA-1 and abolished when both VLA-4 and LFA-1 were blocked. Previous studies had shown that TEM induced by RANTES, MIP-1{alpha}, and SDF-1 across unstimulated and IFN-{gamma}-treated HUVEC was mediated by LFA-1, while migration across TNF-{alpha}-activated HUVEC stimulated by these chemokines, but not in the absence of the chemokines, was dependent on both LFA-1 and VLA-4 (51). TEM induced by I-TAC across unstimulated and TNF-{alpha}-activated HUVEC similarly appears to depend on these two integrins, suggesting that I-TAC increases not only LFA-1 but also VLA-4 function during TEM. It is also interesting to note that TEM across EC treated with both TNF-{alpha} and IFN-{gamma} was also mediated by these two integrins, even though this migration was not enhanced by I-TAC, suggesting that TEM across this activated endothelium is induced by endothelial-derived chemokines, probably in part CXCR3 ligands.

Our findings that I-TAC induces normal blood T lymphocyte TEM suggests that T cell recruitment to inflamed tissues may be stimulated by the action of I-TAC on resting blood T cells. To determine whether I-TAC may be contributing to T cell migration in vivo, the concentration of I-TAC in arthritic synovial fluid was examined. I-TAC was abundant in synovial fluid from joints of patients with rheumatoid arthritis, but not osteoarthritis (Fig. 7Go). The finding of substantial levels of I-TAC in RA synovial fluid further supports an in vivo role for I-TAC in recruiting T cells to inflammation.

A recent report has shown increased levels of IP-10 in synovial fluid (52), and suggests that CXCR3 ligands likely play an important role in RA. The studies here indicate that I-TAC may play a particularly important role in this T cell infiltration, because unlike IP-10, I-TAC can stimulate TEM by resting blood lymphocytes. Increased expression of CXCR3 on T cells in inflammatory reactions mediated by type 1 cytokines has been previously reported (15, 16, 18, 23, 43, 52, 53, 54, 55). Our results suggest that the CXCR3 on T cells in these inflammatory sites likely mediates TEM to the site, and may not be the result of the up-regulation of CXCR3 by T cells in response to the cytokines in the inflamed tissue. Future studies of the in vivo migration of CXCR3+ T cells are needed to determine the full role of this receptor in recruitment of T lymphocytes to inflammation.


   Footnotes
 
1 This work was supported by The Arthritis Society (Grant no. TAS89/0002) and a grant from the Canadian Institutes of Health Research (Grant no. MOP-42379). K.M. was supported by a Cancer Research and Education Nova Scotia trainee award with funding from Cancer Care Nova Scotia. Back

2 Address correspondence and reprint requests to Dr. Thomas B. Issekutz at the current address: Division of Immunology, Rheumatology and Infectious Diseases, Department of Pediatrics, Izaak Walton Killam Grace Health Center, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9. E-mail address: thomas.issekutz{at}dal.ca Back

3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; TEM, transendothelial migration; EC, endothelial cell; I-TAC, IFN-{gamma}-inducible T cell {alpha} chemoattractant; IP-10, IFN-{gamma}-inducible protein-10; Mig, monokine induced by IFN-{gamma}; VLA-4, very late Ag 4; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage inflammatory protein; PPP, platelet-poor plasma; HSA, human serum albumin. Back

Received for publication July 25, 2001. Accepted for publication April 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  2. Ebnet, K., E. P. Kaldjian, A. O. Anderson, S. Shaw. 1996. Orchestrated information transfer underlying leukocyte endothelial interactions. Annu. Rev. Immunol. 14:155.[Medline]
  3. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  4. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 392:565.[Medline]
  5. Moser, B., M. Loetscher, L. Piali, P. Loetscher. 1998. Lymphocyte responses to chemokines. Int. Rev. Immunol. 16:323.[Medline]
  6. Murdoch, C., A. Finn. 2000. Chemokine receptors and their role in inflammation and infectious diseases. Blood 95:3032.[Abstract/Free Full Text]
  7. Ward, S. G., J. Westwick. 1998. Chemokines: understanding their role in T-lymphocyte biology. Biochem. J. 333:457.
  8. Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L. Piali, I. Clark-Lewis, M. Baggiolini, B. Moser. 1996. Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184:963.[Abstract/Free Full Text]
  9. Cole, K. E., C. A. Strick, T. J. Paradis, K. T. Ogborne, M. Loetscher, R. P. Gladue, W. Lin, J. G. Boyd, B. Moser, D. E. Wood, et al 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.[Abstract/Free Full Text]
  10. Loetscher, M., P. Loetscher, N. Brass, E. Meese, B. Moser. 1998. Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine binding and gene localization. Eur. J. Immunol. 28:3696.[Medline]
  11. Sauty, A., M. Dziejman, R. A. Taha, A. S. Iarossi, K. Neote, E. A. Garcia-Zepeda, Q. Hamid, A. D. Luster. 1999. The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells. J. Immunol. 162:3549.[Abstract/Free Full Text]
  12. Gasperini, S., M. Marchi, F. Calzetti, C. Laudanna, L. Vicentini, H. Olsen, M. Murphy, F. Liao, J. Farber, M. A. Cassatella. 1999. Gene expression and production of the monokine induced by IFN-{gamma} (MIG), IFN-inducible T cell {alpha} chemoattractant (I-TAC), and IFN-{gamma}-inducible protein-10 (IP-10) chemokines by human neutrophils. J. Immunol. 162:4928.[Abstract/Free Full Text]
  13. Tensen, C. P., J. Flier, E. M. Van Der Raaij-Helmer, S. Sampat-Sardjoepersad, R. C. Van Der Schors, R. Leurs, R. J. Scheper, D. M. Boorsma, R. Willemze. 1999. Human IP-9: a keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3). J. Invest. Dermatol. 112:716.[Medline]
  14. Mazanet, M. M., K. Neote, C. C. Hughes. 2000. Expression of IFN-inducible T cell {alpha} chemoattractant by human endothelial cells is cyclosporin A-resistant and promotes T cell adhesion: implications for cyclosporin A-resistant immune inflammation. J. Immunol. 164:5383.[Abstract/Free Full Text]
  15. Mach, F., A. Sauty, A. S. Iarossi, G. K. Sukhova, K. Neote, P. Libby, A. D. Luster. 1999. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells. J. Clin. Invest. 104:1041.[Medline]
  16. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101:746.[Medline]
  17. Sorensen, T. L., M. Tani, J. Jensen, V. Pierce, C. Lucchinetti, V. A. Folcik, S. Qin, J. Rottman, F. Sellebjerg, R. M. Strieter, et al 1999. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J. Clin. Invest. 103:807.[Medline]
  18. Balashov, K. E., J. B. Rottman, H. L. Weiner, W. W. Hancock. 1999. CCR5+ and CXCR3+ T cells are increased in multiple sclerosis and their ligands MIP-1{alpha} and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873.[Abstract/Free Full Text]
  19. Flier, J., D. M. Boorsma, D. P. Bruynzeel, P. J. Van Beek, T. J. Stoof, R. J. Scheper, R. Willemze, C. P. Tensen. 1999. The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions. J. Invest. Dermatol. 113:574.[Medline]
  20. Hancock, W. W., W. Gao, V. Csizmadia, K. L. Faia, N. Shemmeri, A. D. Luster. 2001. Donor-derived IP-10 initiates development of acute allograft rejection. J. Exp. Med. 193:975.[Abstract/Free Full Text]
  21. Jaffe, E. A., R. L. Nachman, C. G. Becker, C. R. Minick. 1973. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745.
  22. Kim, C. H., H. E. Broxmeyer. 1999. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J. Leukocyte Biol. 65:6.[Abstract]
  23. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
  24. Ebert, L. M., S. R. McColl. 2001. Coregulation of CXC chemokine receptor and CD4 expression on T lymphocytes during allogeneic activation. J. Immunol. 166:4870.[Abstract/Free Full Text]
  25. Romagnani, P., F. Annunziato, E. Lazzeri, L. Cosmi, C. Beltrame, L. Lasagni, G. Galli, M. Francalanci, R. Manetti, F. Marra, et al 2001. Interferon-inducible protein 10, monokine induced by interferon {gamma}, and interferon-inducible T-cell {alpha} chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) {alpha}{beta}+ CD8+ single-positive T cells, TCR{gamma}{delta}+ T cells, and natural killer-type cells in human thymus. Blood 97:601.[Abstract/Free Full Text]
  26. Rabin, R. L., M. K. Park, F. Liao, R. Swofford, D. Stephany, J. M. Farber. 1999. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J. Immunol. 162:3840.[Abstract/Free Full Text]
  27. Roth, S. J., M. W. Carr, T. A. Springer. 1995. C-C chemokines, but not the C-X-C chemokines interleukin-8 and interferon-{gamma} inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol. 25:3482.[Medline]
  28. Ding, Z., K. Xiong, T. B. Issekutz. 2000. Regulation of chemokine-induced transendothelial migration of T lymphocytes by endothelial activation: differential effects on naive and memory T cells. J. Leukocyte Biol. 67:825.[Abstract]
  29. Romagnani, P., F. Annunziato, L. Lasagni, E. Lazzeri, C. Beltrame, M. Francalanci, M. Uguccioni, G. Galli, L. Cosmi, L. Maurenzig, et al 2001. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J. Clin. Invest. 107:53.[Medline]
  30. Salcedo, R., J. H. Resau, D. Halverson, E. A. Hudson, M. Dambach, D. Powell, K. Wasserman, J. J. Oppenheim. 2000. Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J. 14:2055.[Abstract/Free Full Text]
  31. Arenberg, D. A., P. J. Polverini, S. L. Kunkel, A. Shanafelt, J. Hesselgesser, R. Horuk, R. M. Strieter. 1997. The role of CXC chemokines in the regulation of angiogenesis in non-small cell lung cancer. J. Leukocyte Biol. 62:554.[Abstract]
  32. Carlos, T. M., J. M. Harlan. 1994. Leukocyte-endothelial adhesion molecules. Blood 84:2068.[Abstract/Free Full Text]
  33. Issekutz, T. B.. 1992. Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1. J. Immunol. 149:3394.[Abstract]
  34. Issekutz, T. B.. 1991. Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody: a likely role for VLA-4 in vivo. J. Immunol. 147:4178.[Abstract]
  35. Oppenheimer-Marks, N., L. S. Davis, D. T. Bogue, J. Ramberg, P. E. Lipsky. 1991. Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. J. Immunol. 147:2913.[Abstract]
  36. Chuluyan, H. E., T. J. Schall, T. Yoshimura, A. C. Issekutz. 1995. IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1{alpha}, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1. J. Leukocyte Biol. 58:71.[Abstract]
  37. Issekutz, A. C., H. E. Chuluyan, N. Lopes. 1995. CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms. J. Leukocyte Biol. 57:553.[Abstract]
  38. Mantovani, A., S. Sozzani, A. Vecchi, M. Introna, P. Allavena. 1997. Cytokine activation of endothelial cells: new molecules for an old paradigm. Thromb. Haemost. 78:406.[Medline]
  39. Santamaria Babi, L. F., B. Moser, M. T. Perez Soler, R. Moser, P. Loetscher, B. Villiger, K. Blaser, C. Hauser. 1996. The interleukin-8 receptor B and CXC chemokines can mediate transendothelial migration of human skin homing T cells. Eur. J. Immunol. 26:2056.[Medline]
  40. Oppenheimer-Marks, N., R. I. Brezinschek, M. Mohamadzadeh, R. Vita, P. E. Lipsky. 1998. Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells in vitro and in the SCID mouse-human rheumatoid arthritis model in vivo. J. Clin. Invest. 101:1261.[Medline]
  41. Weber, K. S., P. von Hundelshausen, I. Clark-Lewis, P. C. Weber, C. Weber. 1999. Differential immobilization and hierarchical involvement of chemokines in monocyte arrest and transmigration on inflamed endothelium in shear flow. Eur. J. Immunol. 29:700.[Medline]
  42. Piali, L., C. Weber, G. LaRosa, C. R. Mackay, T. A. Springer, I. Clark-Lewis, B. Moser. 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.[Medline]
  43. Shields, P. L., C. M. Morland, M. Salmon, S. Qin, S. G. Hubscher, D. H. Adams. 1999. Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J. Immunol. 163:6236.[Abstract/Free Full Text]
  44. Hoogewerf, A. J., G. S. Kuschert, A. E. Proudfoot, F. Borlat, I. Clark-Lewis, C. A. Power, T. N. Wells. 1997. Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570.[Medline]
  45. Luster, A. D., S. M. Greenberg, P. Leder. 1995. The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J. Exp. Med. 182:219.[Abstract/Free Full Text]
  46. Tanaka, Y., D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, S. Shaw. 1993. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1{beta}. Nature 361:79.[Medline]
  47. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279:381.[Abstract/Free Full Text]
  48. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M. Baggiolini, J. L. Virelizier, F. Arenzana-Seisdedos. 1997. HIV coreceptor downregulation as antiviral principle: SDF-1{alpha}-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:139.[Abstract/Free Full Text]
  49. Wang, J. M., A. Hishinuma, J. J. Oppenheim, K. Matsushima. 1993. Studies of binding and internalization of human recombinant monocyte chemotactic and activating factor (MCAF) by monocytic cells. Cytokine 5:264.[Medline]
  50. Sauty, A., R. A. Colvin, L. Wagner, S. Rochat, F. Spertini, A. D. Luster. 2001. CXCR3 internalization following T cell-endothelial cell conta