Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1

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1 Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1 Ziqiang Ding, Ke Xiong, and Thomas B. Issekutz Departments of Pediatrics, Microbiology/Immunology, and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada Abstract: Lymphocyte infiltration in inflammation is induced by the dual actions of chemokines and cell adhesion molecules. The role of LFA-1 and VLA-4 in chemokine-induced T cell transendothelial migration (TEM) across cytokine-activated endothelium has not been examined. LFA-1, but not VLA-4, mediated blood T cell TEM to RANTES, macrophage inflammatory protein-1 (MIP-1 ), and stromal cell-derived factor-1 (SDF- 1), and across tumor necrosis factor (TNF- ) or interferon- (IFN- ) -stimulated endothelial cells (EC). Chemokine stimulation in combination with TNF- activation of EC induced TEM, which was partially mediated by VLA-4. SDF-1 increased a 1 -integrin activation epitope on T cells and enhanced VLA-4-mediated adhesion. Thus, LFA-1 mediates TEM under most conditions, but VLA-4 can also mediate TEM, although, in contrast to LFA-1, this requires exogenous chemokines and EC activation. In addition, an LFA-1- and VLA-4- independent pathway of lymphocyte TEM can also be induced by SDF-1. J. Leukoc. Biol. 69: ; Key Words: adhesion molecules chemotaxis cytokine endothelial cells inflammation INTRODUCTION Chronic inflammation is characterized by the accumulation of lymphocytes in inflamed tissues. This lymphocyte recruitment is thought to be mediated by the combined actions of chemokines (CKs) and cell adhesion molecules (CAM) facilitating the interactions between lymphocytes and endothelial cells (EC) and also lymphocytes and extracellular matrix [1, 2]. CKs are a subfamily of the cytokines, which are chemotactic for leukocytes. There are about 50 human CKs identified to date [3 6]. Based on the number and arrangement of the first two cysteine residues in the structure, CKs are subdivided into four groups, CXC ( ), CC ( ),C( ), and CX 3 C( ). CXC chemokines that have been shown to act on T lymphocytes include interleukin-8 (IL-8), stromal cell-derived factor-1 (SDF-1), interferon- producing factor-10 (IP-10), Mig, and ITAC. CC chemokines are chemotactic for a wide range of cells, including T cells and monocytes. Some members of the CC chemokines are monocyte chemotactic protein-1 (MCP-1), RANTES, macrophage inflammatory protein (MIP)-1 /, MIP-3 /, and TARC. Most of these CKs are reported to be mainly chemotactic for memory or activated T cells and are thought to be important in memory T cell recruitment in inflammation. The CAMs on lymphocytes and EC also contribute to the selective migration of T cells to various tissues [2, 7 9]. Lymphocytes express several members of the integrin family including LFA-1 (CD11a/CD18, L 2 ), VLA-4 (CD49d/CD29, 4 1 ), and 4 7 ; and endothelial cells express ligands for these receptors, including ICAM-1 and -2, VCAM-1, and MAdCAM-1. Both VLA-4 and 4 7 have been shown to mediate T cell rolling and firm adhesion, and LFA-1 can mediate firm adhesion and transendothelial migration (TEM) [10 12]. Compared with our understanding of these integrins in lymphocyte adhesion, less is known about their role in migration across EC monolayers. Blockade of LFA-1 partially blocks spontaneous lymphocyte migration across unstimulated human umbilical vein endothelial cells (HUVEC) and across IL-1-treated HUVEC [10 12]. Blocking LFA-1 and ICAM-1 also inhibits MCP-1-induced T cell TEM across unactivated HUVEC [13]. However, the effect of blocking LFA-1 and other adhesion molecules on TEM stimulated by other CKs, such as SDF-1, which is substantially more chemotactic than MCP-1 for T cells, and can induce TEM of both naive and memory T cells, has not been studied [14 16]. There are also no reports that have examined the CAMs mediating T cell TEM in response to CKs across cytokine-activated EC, which is the situation that occurs in inflammation. The role of VLA-4 in TEM is less clear and more controversial. VLA-4 was shown to play a minor role, especially compared with LFA-1, in TEM across unstimulated EC in response to MCP-1 [13]. VLA-4 on normal and phorbol esteractivated T cells mediated lymphocyte adhesion to IL-1-activated HUVEC, but did not mediate T cell TEM [11]. In contrast, cutaneous lymphocyte antigen (CLA)-positive, but not CLA, T cells were shown to utilize VLA-4 for TEM, suggesting that engagement of CLA by E-selectin triggers signals in the T cell for the active participation of VLA-4 in transmigra- Correspondence: Dr. Thomas B. Issekutz, Department of Pediatrics, Division of Immunology, Rheumatology and Infectious Diseases, IWK Grace Health Center, 5850 University Avenue, Halifax, Nova Scotia, B3J 3G9, Canada. tissekutz@iwkgrace.ns.ca Received June 13, 2000; revised October 18, 2000; accepted October 19, Journal of Leukocyte Biology Volume 69, March

2 tion [17 19]. Thus, we hypothesized that VLA-4, in addition to mediating rolling and firm adhesion, may have a role in T cell migration across an endothelial cell monolayer in a situation where both T cells and EC are activated. Chemokines can enhance the affinity of 2 and 1 integrins and facilitate lymphocyte binding to EC and to immobilized ligands [20 23]. Combined stimulation of lymphocytes by CKs and EC by cytokines, as occurs in inflammation, is thus likely to have a major effect on the adhesive interactions between T cells and EC that mediate TEM. In vivo T cell infiltration into inflamed tissues is in part mediated by VLA-4, in addition to LFA-1 [24 27]. However, the contribution of VLA-4 to T cell rolling and firm adhesion versus transmigration of the endothelium in vivo in inflammation is unknown. Our previous studies showed that T cell TEM was significantly increased by several CKs and by EC activation with IFN- or TNF- ; and T cell TEM to the CKs was greatly enhanced across cytokine-activated endothelium [16]. This report demonstrates that LFA-1 is essential for T cell TEM induced by RANTES, MIP-1, and SDF-1, and LFA-1 mediates TEM across IFN- - and TNF- -activated endothelium. VLA-4 is not required under these conditions, however, a major contribution by VLA-4 to T cell TEM is demonstrated when lymphocyte migration is stimulated by CKs across TNF- -activated EC. SDF-1 stimulates T cells to express a 1 integrin activation-associated epitope and to enhance the avidity of VLA-4. These studies also demonstrate that SDF-1- induced TEM appears to involve an LFA-1- and VLA-4- independent component. MATERIALS AND METHODS Reagents Recombinant human TNF- (specific activity U/mg) and IFN- (10 7 U/mg) were provided by Genentech (South San Francisco, CA). Recombinant human IL-1 (specific activity U/mg) was a generous gift from Dr. D. Urdal (Immunex, Seattle, WA). Recombinant human RANTES, MIP-1, and SDF-1 were obtained from R&DSystems (Minneapolis, MN). Anti-CD45 mab (4B2) was obtained from the 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 immunoglobulin was purchased from DAKO (Glostrup, Denmark). Anti-LFA-1 mab (60.3) was kindly provided by Dr. J. M. Harlan (University of Washington, Seattle, WA). Anti- VLA-4 (HP2/1 and HP1/2) and a mab to an activation epitope of 1 (HUTS-21) were generous gifts from Dr. F. Sanchez-Madrid (Universidad Autonoma de Madrid, Spain) [28]. Anti- 1 integrin (3S3) was kindly provided by Dr. J. Wilkins (University of Manitoba, Winnipeg). Isolation and culture of endothelial cells HUVEC were isolated by collagenase digestion as described by Jaffe et al. [29]. Briefly, human umbilical veins were flushed with Ringer lactate, then incubated with 0.5 mg/ml collagenase type II (Sigma Chemical, St. Louis, MO) at 37 C for 30 min. Detached EC were collected, washed, then cultured in gelatin-coated flasks (Nunc, Naperville, IL) with RPMI 1640 medium (Sigma) containing 20% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT), 50 g/ml endothelial cell growth supplement (Sigma), 90 g/ml heparin, 2 mm L-glutamine, 50 M 2-mercaptoethanol (2-ME), 100 U/mL penicillin, and 100 g/ml streptomycin. Confluent HUVEC in the flasks were gently trypsinized and seeded onto polycarbonate Transwell filters of 6.5-mm diameter and 5- m pore size (Costar Corning, Cambridge, MA). The Transwell filters were prepared by coating with 0.01% gelatin at 37 C overnight followed by 3 g of human fibronectin (GIBCO, Grand Island, NY) at 37 C for 3 h. Then 0.1 ml of EC ( cells) was 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 EC monolayers was assessed by microscopic observation and by measuring the permeability of the monolayer by using 125 I-labeled albumin diffusion as previously reported [30, 31]. Preparation of lymphocytes Lymphocytes were isolated from human blood by dextran sedimentation, gradient centrifugation, and passage over a nylon wool column. In brief, acid citrate dextrose heparin anticoagulated blood was gently mixed with a half volume of 3% dextran (Pharmacia, Uppsala, Sweden) in saline, and erythrocytes were allowed to sediment for 30 min. The supernatant containing leukocyte-rich plasma was harvested and layered onto Ficoll-Paque (Pharmacia) and centrifuged at 900 g for 20 min. The blood mononuclear cells on top of the Ficoll-Paque were collected and washed three times with Tyrode s solution. The cells were resuspended in RPMI medium with 10% human platelet-poor plasma and were applied to a nylon wool column. After 60 min of incubation, the unbound leukocytes were eluted, washed, resuspended in fresh RPMI medium plus 10% plasma, and cultured overnight in tissue culture flasks. In preliminary experiments overnight culture of the T cells was not found to affect T cell TEM to the stimuli used in these studies. The nonadherent cells contained 96% T cells, 3% B cells, and 1% monocytes by immunofluorescence staining, and were 98% viable by trypan blue dye exclusion. In some experiments, memory (CD45RA )-enriched T cells were purified by negative selection through panning or magnetic-activated cell sorting (MACS). For panning, T cells were incubated with a mab to CD45RA (G1 15) at 200 g/10 8 cells/ml in RPMI medium plus 10% FCS at 4 C for 45 min. The cells were then washed twice, resuspended in HEPES-buffered HBSS containing 10% FCS and incubated at 4 C for 45 min in culture dishes coated with goat anti-mouse Ig. The nonadherent cells were harvested as CD45RA (memory T cell-enriched) subtype of cells. The purity was 97% by immunofluorescence staining with anti- CD45RA. For MACS isolation, T cells were incubated with biotinylated G1 15 (anti-cd45ra) and then incubated with streptavidin-conjugated magnetic beads. Finally, cells were passed through a column in a magnetic field and the flow-through cells were collected as memory cells. Unfractionated and memory T cells were labeled by incubating cells/ml in HBSS 15 mm HEPES 10% FCS with 50 Ci/mL Na 2 51 CrO 4 (Amersham, Oakville, Ontario, Canada) at 37 C for 45 min. Cells were washed three times with RPMI medium and resuspended in RPMI medium 5 mg/ml HSA for the TEM assay. The cells were left untreated or pretreated with 20 g/ml of anti-cd45, anti-lfa-1, anti-vla-4, or anti-lfa-1 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. Based on preliminary titrations this concentration of the mabs was found to be optimal. Measurement of lymphocyte TEM HUVEC were left untreated or were stimulated by adding TNF- (100 U/mL), TNF- (100 U/mL) plus IL-1 (20 U/mL), or IFN- (100 U/mL) for 4 or 18 h to the lower chamber of the Transwells. The endothelial monolayers in the Transwell inserts were rinsed once with RPMI medium, then 100 L of labeled T cells ( cells/ml) was placed in the upper chamber, and the inserts were transferred to new wells (lower chambers) of a 24-well plate containing 0.6 ml of fresh RPMI medium with 5 mg/ml HSA. In some experiments, various CKs were also added to the lower chambers. All CKs were used at a concentration of 50 ng/ml, since previous studies showed that this concentration strongly stimulated T cell TEM [16]. The Transwell chambers were then incubated at 37 C in 5% CO 2. After 4 h, the T cells that had migrated through the EC monolayer into the lower chambers were recovered. The radioactivity in these samples was determined by gamma counting. The percentage of migrated cells was calculated by dividing the radioactivity of the migrated cells by the radioactivity of the total cells added to the upper chamber. Spontaneous release of 51 Cr from the labeled cells during the 4-h migration assay was 2%. Ding et al. Chemokines in lymphocyte migration 459

3 Analysis of lymphocyte 1 integrin expression by flow cytometry T cells were left untreated or treated with SDF-1 at 37 C for 30 min. Then cells were washed once and stained with 3S3 (anti- 1 mab), HUTS-21 (antiactivated 1 mab), and HP2/1 (anti- 4 mab) at 4 C for 30 min. Cells were washed and stained with fluorescein isothiocyanate-conjugated sheep antimouse IgG (Sigma Chemical). Finally, cells were washed, resuspended in 1% paraformaldehyde in phosphate-buffered saline (PBS), and analyzed by flow cytometry. Measurement of lymphocyte adhesion HUVEC were grown to confluence in gelatin-coated 96-well tissue culture plates. 51 Cr-labeled T lymphocytes ( cells in 100 L) were added to wells with or without SDF-1 (50 ng/ml) in triplicate. Some of the cells were pretreated with anti-lfa-1 (20 g/ml), anti-vla-4 (20 g/ml), or both mabs. The cells were allowed to adhere at 37 C for 60 min. Nonadherent cells were removed by four washes with RPMI. The bound T cells were lysed with 1 N NaOH, collected into tubes, and the radioactivity measured by gamma counting. Percent cell adhesion was calculated by dividing the radioactivity of bound cells by the radioactivity of total input cells. In some experiments, fibronectin (1 g/well), collagen (2 g/well), and HSA (10 g/well) were immobilized in 96-well culture plates by incubating at 37 C for 2 h. After coating, the wells were gently washed three times with PBS, then 51 Cr-labeled T cells were added to each well and the adhesion assay was carried out as described above for lymphocyte adhesion to HUVEC. Statistical analysis Data were expressed as means SEM of multiple separate assays. Analysis of variance (ANOVA) and Student s unpaired t test were used to compare the differences between means. RESULTS Effect of LFA-1 and VLA-4 integrins on chemokine-induced T cell TEM Previous studies showed that RANTES, MIP-1, and SDF-1 could stimulate T cell TEM; however, the role of LFA-1 and VLA-4 in T cell TEM to these CKs has not been examined. As shown in Figure 1A, 7.9% of T cells spontaneously migrated across resting EC. RANTES, MIP-1, and SDF-1 each increased TEM to 13.7, 15.7, and 45.5%, respectively. Anti- LFA-1 significantly inhibited the increased T cell migration induced by RANTES, MIP-1, and SDF-1 by 57, 71, and 37%, respectively. Anti-VLA-4 alone had no effect on the migration to these CKs, and the blockade of both LFA-1 and VLA-4 was not significantly more effective than blocking LFA-1 alone. In contrast to RANTES and MIP-1, a large portion of SDF-1- induced T cell TEM (51%) was independent of LFA-1 and VLA-4. Because RANTES and MIP-1 act primarily on memory (CD45RO ) T lymphocytes, while SDF-1 induces migration of both naive and memory T cells [32 34], the role of LFA-1 and VLA-4 in memory T cell TEM was examined. As shown in Figure 1B, RANTES, MIP-1, and SDF-1 induced marked increases in memory T cell TEM, which were greater than the migration by unfractionated T cells (Fig. 1A). Anti-LFA-1 mab strongly inhibited the increased lymphocyte migration induced by RANTES, MIP-1, and SDF-1 by 100, 89, and 66%, respectively. Anti-VLA-4 alone had no effect, and the blockade of both LFA-1 and VLA-4 was not significantly more Fig. 1. Effect of anti-lfa-1 and anti-vla-4 on chemokine-induced T cell transendothelial migration across unstimulated HUVEC. Unfractionated (A) and memory (CD45RA ) (B) T cells were labeled with 51 Cr and their TEM in response to RANTES, MIP-1, and SDF-1 was determined. The lymphocytes were either left untreated or treated with anti-cd45, anti-lfa-1, anti-vla- 4, or anti-lfa-1 plus anti-vla-4 mabs and then added on top of the EC monolayers in Transwell chambers. The T cells were allowed to migrate for 4 h, and the percentage of cells that had migrated was calculated by dividing the radioactivity of the migrated cells by the radioactivity of the total cells added to the upper chamber. Each column represents the mean SEM of 3 10 assays. *P 0.05, **P 0.01 compared with no Ab treatment. effective than blocking LFA-1 alone. Compared to the migration induced by RANTES and MIP-1, which were nearly abolished by anti-lfa-1, there was a small but consistent portion (17%) of the SDF-1-induced migration independent of LFA-1 and VLA-4. Effect of LFA-1 and VLA-4 on chemokineinduced TEM across IFN- -activated endothelium IFN- activation of the endothelium significantly enhanced T cell TEM [35 37]. The role of LFA-1 and VLA-4 in chemokine-induced T cell migration across IFN- -activated endothelium was examined. Treatment of HUVEC monolayers with IFN- significantly (P 0.01) increased T cell migration to 460 Journal of Leukocyte Biology Volume 69, March

4 TEM across TNF- -activated EC was again only partially (81%) inhibited by dual blockade of LFA-1 and VLA-4 as observed with resting and IFN- -activated EC. Memory T cell migration to CKs is selectively enhanced by endothelial cytokine activation [16]. Treatment of the HUVEC with TNF- induced a greater increase in memory than unfractionated T cell TEM and enhanced RANTES, MIP-1, and SDF-1-induced memory T cell migration (Fig. 3B). The effect of blocking LFA-1 and VLA-4 on memory T cell TEM to these CKs was similar to that on unfractionated T cell TEM. Blocking VLA-4 did not inhibit memory T cell TEM. Blocking LFA-1 inhibited 47 85% of the increased memory T cell TEM, and Fig. 2. Effect of anti-lfa-1 and anti-vla-4 on chemokine-induced T cell transendothelial migration across IFN- -treated endothelium. HUVEC monolayers in Transwell chambers were either stimulated with 100 U/mL of IFN- for 18 h or left untreated. Unfractionated T lymphocytes were labeled with 51 Cr and their TEM in response to RANTES, MIP-1, and SDF-1 across the activated HUVEC was determined in the presence or absence of the indicated mabs as outlined in Figure 1. The percentage of cells that had migrated was calculated by dividing the radioactivity of the migrated cells by the radioactivity of the total cells added to the upper chamber. Each column represents the mean SEM of three to seven assays. *P 0.05, **P 0.01 compared with no Ab treatment. 16.4% (Fig. 2), and enhanced T cell TEM to RANTES, MIP-1, and SDF-1. Anti-LFA-1 mab completely blocked lymphocyte TEM induced by IFN- activation of EC and also TEM to RANTES and MIP-1 across IFN- -activated EC. In contrast, anti-lfa-1 mab only partially inhibited TEM to SDF-1 across IFN- -activated EC (56% inhibition). Anti- VLA-4 mab, either alone or with anti-lfa-1, had no effect on TEM to these stimuli. Effect of LFA-1 and VLA-4 on chemokineinduced TEM across TNF- -activated endothelium TNF- activation of EC significantly increases endothelial surface ICAM-1, VCAM-1, and E-selectin expression [16, 38, 39]. As shown in Figure 3A, stimulation of HUVEC monolayers with TNF- for 18 h increased TEM of unfractionated T cells to 16.9%. RANTES, MIP-1, and SDF-1 further increased TEM across TNF- activated EC to 29 57%, which is also greater than the migration to these CKs across resting EC. Anti-LFA-1 completely blocked T cell TEM induced by TNF- treatment of the HUVEC. In contrast, anti-lfa-1 only partially inhibited T cell TEM to RANTES, MIP-1, and SDF-1 (66.4, 72.1, and 56.2% inhibition, respectively) across TNF- -activated EC. Anti-VLA-4 alone did not inhibit TEM, however, blockade of both LFA-1 and VLA-4 more strongly inhibited T cell TEM to RANTES (P 0.05), MIP-1 (P 0.001), and SDF-1 (P 0.05) across TNF- -activated EC, than did LFA-1 blockade alone. These results suggest that VLA-4 is important in T cell TEM in response to stimulation by both CKs and TNF- activation of the endothelium. SDF-1-induced T cell Fig. 3. Effect of anti-lfa-1 and anti-vla-4 on chemokine-induced T cell TEM across TNF- -activated endothelium. HUVEC monolayers in Transwell chambers were either stimulated with 100 U/mL of TNF- for 18 h or left untreated. Unfractionated (A) and memory (CD45RA ) (B) T cells were labeled with 51 Cr and their TEM in response to RANTES, MIP-1, and SDF-1 across the activated HUVEC was determined in the presence or absence of the indicated mabs as outlined in Figure 1. The percentage of cells that had migrated was calculated by dividing the radioactivity of the migrated cells by the radioactivity of the total cells added to the upper chamber. Each column represents the mean SEM of three to eight assays. **P 0.01 compared to no Ab treatment. P 0.05, P 0.01 compared to anti-lfa-1 treatment. Ding et al. Chemokines in lymphocyte migration 461

5 be increased in SDF-1-induced T cell TEM across TNF- IL-1-activated EC. Fig. 4. Effect of anti-lfa-1 and anti-vla-4 on SDF-1-induced memory T cell TEM across TNF- or TNF- plus IL-1-stimulated endothelium. HUVEC monolayers in Transwell chambers were stimulated with TNF- 100 U/mL (A) or TNF- 100 U/mL IL-1 20 U/mL (B) for 4horleft untreated. Memory (CD45RA ) T cells were labeled with 51 Cr and their TEM in response to SDF-1 across the activated HUVEC was determined in the prescence or absence of the indicated mabs as outlined in Figure 1. Each bar represents the mean SEM of three to five assays. *P 0.05, **P 0.01 compared with no Ab treatment. P 0.01 compared with anti-lfa-1 treatment. Effect of SDF-1 on lymphocyte surface VLA-4 expression and function These results suggested that VLA-4 could mediate a substantial component (20 30%) of T cell TEM across TNF- -activated EC in the presence of CK stimulation. This VLA-4- dependent migration was greatest in response to SDF-1. Therefore, the effect of SDF-1 on VLA-4 expression, activation, and function was examined. Incubation of T cells with SDF-1 did not alter the total expression of 1 and 4 integrins (data not shown) on the T cells, but increased the expression of an activation-associated epitope of 4 1, which was recognized by the HUTS-21 mab (Fig. 5). SDF-1 stimulation of T cells increased the percent of T cells expressing this active 1 epitope from 14.9 to 26.4% and increased the mean fluorescence intensity of HUTS-21 staining from 3.6 to 5.0. The effect of SDF-1 on T cell adhesion to immobilized fibronectin was also examined (Fig. 6). About 8 10% of unstimulated lymphocytes bound to immobilized fibronectin and collagen. SDF-1 stimulation doubled T cell fibronectin adhesion to 20%, but did not alter T cell adhesion to collagen. SDF-1-induced T cell binding to fibronectin was completely inhibited by anti-vla-4 mab (data not shown). Effect of LFA-1 and VLA-4 on lymphocyte adhesion to HUVEC The above results suggested that SDF-1 could enhance the activity of VLA-4 and induce VLA-4 to mediate T cell TEM. Therefore the effect of SDF-1 on VLA-4 and LFA-1 mediated lymphocyte adhesion to resting and cytokine-activated HUVEC was examined (Fig. 7). The adhesion of lymphocytes to resting EC was 7.5%. TNF- treatment of the endothelium blocking both LFA-1 and VLA-4 inhibited % of the increased migration. Effect of LFA-1 and VLA-4 on SDF-1-induced lymphocyte TEM across endothelium activated with TNF- or TNF- plus IL-1 for 4 h Adhesion molecules, such as E-selectin, reach peak expression on HUVEC after 4 h of cytokine treatment and decline thereafter [40]. Therefore, the role of LFA-1 and VLA-4 in SDF-1- induced lymphocyte TEM across EC stimulated with TNF- or TNF- plus IL-1 for 4 h was examined (Fig. 4). Treatment of the HUVEC for 4 h with these cytokines did not significantly increase T cell TEM and did not enhance migration to SDF-1. Anti-LFA-1 strongly inhibited (51%) the increased lymphocyte TEM to SDF-1 across TNF- -activated EC (P 0.05). Anti- VLA-4 inhibited the migration by 24%, and the blockade of both LFA-1 and VLA-4 inhibited TEM by 84% (P 0.01). In contrast to the TEM across TNF- -activated EC, SDF-1- induced T cell TEM across TNF- IL-1-activated EC was inhibited only by 27% with anti-lfa-1 (P 0.05). Anti- VLA-4 mab alone did not affect the migration, whereas blocking both LFA-1 and VLA-4 inhibited 76% of the migration (P 0.01). Therefore, the contribution of VLA-4 appeared to Fig. 5. Effect of SDF-1 on the expression of a 1 integrin activation-associated epitope on T lymphocytes. Human T cells were treated with SDF ng/ml at 37 C for 30 min or left untreated. The cells were then stained with a mab to an activation epitope of 1 (HUTS-21) or an isotype-matched control mab followed by FITC-conjugated sheep anti-mouse IgG and analyzed by flow cytometry. The dotted line represents the negative control antibody. The dashed line indicates the untreated T cells stained with HUTS-21, and the solid line the SDF-1-treated cells stained with HUTS-21. Results from one of three similar experiments is presented. 462 Journal of Leukocyte Biology Volume 69, March

6 Fig. 6. Effect of SDF-1 on T lymphocyte adhesion to fibronectin. T cells were labeled with 51 Cr and added to 96-well plates coated with HSA (10 g/well), fibronectin (1 g/well), and collagen (2 g/well), respectively. SDF-1 at 50 ng/ml was added to the indicated wells, and cells were allowed to adhere for 1 h at 37 C. The nonadherent cells were washed off, and the percent cell adhesion was calculated by dividing the radioactivity of bound cells by the radioactivity of total input cells. Each column indicates the mean SEM of quadruplicate measurements. A representative result from one of four experiments is shown. increased this adhesion to 43.2%, and anti-lfa-1 significantly (P 0.01) inhibited this increased T cell adhesion by 27%. Anti-VLA-4 alone had no effect, whereas the combination of anti-lfa-1 and anti-vla-4 inhibited 90% of the increased adhesion. IFN- treatment of the HUVEC also enhanced lymphocyte adhesion to 18.2%, but blockade of LFA-1 completely inhibited this adhesion and VLA-4 did not contribute to this adhesion. SDF-1 significantly enhanced lymphocyte adhesion to unstimulated EC (25.6%) and this was completely blocked by anti-lfa-1 (Fig. 7). Anti-VLA-4 had no effect on this adhesion, and blocking both LFA-1 and VLA-4 was not more effective than blocking LFA-1 alone. Treatment of lymphocytes with SDF-1 and EC with TNF- increased T cell adhesion to 52.9%, greater than that induced by either stimulus alone. Blocking LFA-1 or VLA-4 alone did not reduce this adhesion, but it was markedly inhibited by the combined blockade of LFA-1 and VLA-4 (86% inhibition), suggesting that both LFA-1 and VLA-4 can mediate this binding. The lack of effect of LFA-1 blockade alone suggests an enhanced role for VLA-4 in SDF-1-stimulated T cell adhesion to TNF- -activated HUVEC. In contrast to combined stimulation with SDF-1 and TNF-, T cell adhesion stimulated by SDF-1 and IFN- was comparable to that of SDF-1 stimulation alone (Fig. 7), and anti- LFA-1 completely inhibited this adhesion. This coincides with the fact that IFN- -treated HUVEC does not express the VLA-4 ligand, VCAM-1. DISCUSSION At inflammatory sites the up-regulation of adhesion molecules on EC and the local production of CKs are key factors determining the selective recruitment of leukocytes [1, 2, 41]. The effect of CKs on the utilization of LFA-1 ( L 2 ) and VLA-4 ( 4 1 ) in T cell TEM across cytokine-activated EC, as would occur at an inflammatory site in vivo, has not been previously reported. Earlier studies have shown that specific CKs (MCP-1, RANTES, IP-10, MIP-1, and ) can enhance integrin-mediated adhesion to immobilized ligands or to unstimulated endothelium [20, 21, 42 45]. This report demonstrates that CKs (RANTES, MIP-1, and SDF-1) acting on the T cells modify the adhesion pathways used by T cells to migrate through cytokine-activated endothelium. Previously, we showed that the CKs, RANTES, MIP-1, and SDF-1, and cytokine (TNF- or IFN- ) treatment of EC each alone induced significant T cell TEM; and the combination of these CKs with endothelial activation was additive, demonstrating that endothelial activation of HUVEC can enhance CK-stimulated T cell transmigration [16]. The studies presented here demonstrate that blockade of LFA-1 abolished T cell TEM induced by IFN- and TNF- treatment of the HUVEC, and migration to RANTES and MIP-1 across unstimulated and IFN- activated HUVEC was strongly or completely inhibited (Figs. 1 and 2). Blockade of VLA-4 had no effect on this migration. These results agree with previous reports that LFA-1 plays a critical role in lymphocyte TEM across cytokine-stimulated EC [11, 12] and in response to MCP-1 and RANTES [13, 14, 46, 47], and extend these findings to RANTES and MIP-1 across IFN- -stimulated HUVEC. They also show that the response to SDF-1 was also partly LFA-1-dependent and VLA-4-independent across unstimulated and IFN- -stimulated HUVEC (Figs. 1 and 2), but suggest that an additional unrecognized pathway is also involved in TEM to SDF-1. Our results on the mechanism of human lymphocyte TEM across TNF- -activated HUVEC are unexpected based on Fig. 7. Effect of anti-lfa-1 and anti-vla-4 on TNF- -, IFN- -, and SDF- 1-stimulated T cell adhesion to HUVEC. EC cultured in 96-well plates were either left untreated or stimulated with TNF- 100 U/mL or IFN- 100 U/mL for 18 h. SDF-1 (50 ng/ml) was added to the indicated wells immediately before the T cells. 51 Cr-labeled T cells were either left untreated or treated with anti-lfa-1, anti-vla-4, and anti-lfa-1 plus anti-vla-4 mabs and added to the EC monolayers; and the T cells were allowed to adhere for 1hat 37 C. The nonadherent cells were washed off, and the percent cell adhesion was calculated as in Figure 6. Each column represents mean SEM of three assays. **P 0.01 compared to no Ab treatment. P 0.01 compared to anti-lfa-1 treatment. Ding et al. Chemokines in lymphocyte migration 463

7 previous observations of T cell adhesion and TEM [10, 11]. Treatment of the endothelium for 18 h with TNF- increased TEM through an LFA-1-dependent and VLA-4-independent pathway, but the addition of RANTES, MIP-1, or SDF-1 to stimulate T cell migration across this endothelium made this migration partially VLA-4 dependent (Fig. 3). This was observed with all three CKs but was greatest with memory T cells responding to SDF-1, in which 40% of the enhanced migration was mediated by VLA-4 when LFA-1 was blocked (Fig. 3). SDF-1 was found to induce the expression of an activationdependent epitope on 1 and enhance VLA-4-dependent adhesion to fibronectin and to TNF- -activated HUVEC (Figs. 5 7). SDF-1 also increased T cell adhesion to unstimulated and IFN- -stimulated EC in an LFA-1-dependent manner, suggesting that it activated both VLA-4 and LFA-1 (Fig. 7). These studies show that the mechanism of human T cell TEM can be driven to involve VLA-4, as well as LFA-1, by at least three CKs, and that in the presence of VCAM-1 expressed on TNF-, but not on IFN- -treated EC [48], VLA-4 can mediate TEM by human lymphocytes. Studies in rats had previously shown that VLA-4 mediated part of the infiltration of T cells into inflamed tissues [24, 25]. However, human T cells were not found to use VLA-4 for TEM [10, 11]. This report suggests that human T cells require a trigger from exogenous CKs in order to utilize VLA-4 for TEM even in the presence of VCAM-1 on the endothelium. Another interesting observation is the fact that VLA-4 could mediate a large component of the adhesion of T cells to TNF- -activated HUVEC (Fig. 7) without the need for the lymphocytes to be stimulated by exogenously added CK as appeared to be required for TEM (Fig. 3). One explanation may be that TNF- -activated EC produce enough CKs such as RANTES [49, 50] to induce VLA-4-dependent adhesion, but in the absence of an exogenous chemotactic gradient, VLA-4- dependent TEM is not observed, although LFA-1-dependent TEM can still occur. Another possibility may be that the exogenous CKs induce a higher avidity of VLA-4 and this is required to mediate TEM, but is not required for adhesion to HUVEC. VLA-4 has been shown to have multiple activation states that alter its bind to various ligands [51, 52]. An increased avidity of VLA-4 may be needed to permit the T cell to bind to the endothelium during its migration through EC monolayers. A higher avidity of VLA-4 may also be needed to allow the T cell to crawl through the subendothelial matrix of fibronectin as has been suggested [20]. Because VLA-4 has been shown to mediate leukocyte rolling in vitro and in vivo [53 56], one can consider a model in which low-avidity VLA-4 mediates rolling on cytokine-activated EC in vivo, and in the presence of appropriate CK activation of the T cell, VLA-4 avidity is enhanced to permit initially firm adhesion to VCAM-1, and if sufficiently activated to promote migration out of the blood vessel. Our results with human T cells in vitro demonstrate that VLA-4 can not only mediate lymphocyte adhesion, but also TEM if VLA-4 avidity is enhanced by CK activation of the T cell. In contrast to the above findings that the effect of VLA-4 in T cell TEM could only be demonstrated when LFA-1 was blocked, in vivo studies have shown that anti-vla-4 mab alone was inhibitory in lymphocyte migration to inflammatory sites in the skin, arthritic joints, and the central nervous system in rats [24, 25, 27, 57]. This might be because blockade of VLA-4 in vivo could inhibit T cell rolling and subsequent firm adhesion and migration. This would not be observed in our TEM assays. However, combined blockade of VLA-4 and LFA-1 in vivo demonstrated a more marked inhibition of T cell migration to tissues than blockade of either adhesion pathway alone [26]. The results presented here show that this also applies to human T cell TEM when tested in the presence of dual stimulation by cytokine activation of the endothelium and an exogenous CK signal. Cytokine activation of EC can enhance CAM expression and facilitate lymphocyte adhesion and TEM [36 38]. Treatment of HUVEC for 18 h with TNF- and IFN- each increased T cell TEM and enhanced TEM stimulated by CKs, even though only TNF- but not IFN- markedly increased endothelial CAM expression [data not shown and ref. 16]. Treatment of HUVEC for 4 h with TNF- or TNF- plus IL-1, which dramatically increases ICAM-1 and VCAM-1 expression, did not increase T cell TEM and did not enhance T cell TEM to SDF-1, in contrast to 18-h treatment of the EC (Fig. 4 vs. Fig. 3). This suggests that increased CAM expression is not sufficient to enhance T cell TEM, but increased migration induced by SDF-1 across these EC is mediated by LFA-1 and VLA-4. Endothelial cells express CXCR4, the receptor for SDF-1, but not the receptors for RANTES and MIP-1. Incubation of EC with SDF-1 for 4 h, as occurs in the TEM assay, did not affect EC CAM expression (data not shown), and TEM to SDF-1 is completely prevented if the SDF-1 is added on top of the EC [16]. Thus it seems unlikely that the effects of SDF-1 on T cell TEM are mediated by a direct action on the EC rather than on the T lymphocyte. Lymphocyte TEM induced by SDF-1, but not by RANTES and MIP-1 was only partially inhibited by blocking LFA-1 and VLA-4 (Figs. 1 3). This seems unlikely to be the result of incomplete blockade of LFA-1 or VLA-4, since several blocking mabs were used to inhibit these integrins, and TEM to RANTES and MIP-1, even across activated HUVEC, was reduced to baseline by these mabs. However, it is possible that SDF-1 induces a state of integrin activation that is incompletely inhibited by these mabs. Alternatively, SDF-1-stimulated T cell TEM may involve an unrecognized pathway. Masuyama et al. [58] described a new mab, 4C8, which blocks T cell migration through resting and IFN- -stimulated HUVEC. It is unlikely that the LFA-1- and VLA-4-independent migration is mediated by the 4C8 antigen, since this TEM appears to require SDF-1. E- and P-selectin are also unlikely to be contributing to this TEM because these are not expressed on the unstimulated HUVEC, although one cannot exclude that they are induced in response to contact with SDF-1-activated lymphocytes. SDF-1 itself might also directly mediate T cell adhesion and migration, since ECs can immobilize CKs on surface proteoglycans and these can mediate lymphocyte activation, adhesion, and migration [42, 59]. The CX3C chemokine, fractalkine, has also been shown to mediate cell rolling and adhesion, and is expressed after TNF- treatment of the endothelium [60]. Further studies to define the contribution of each of these receptor ligand interactions to the SDF-1-induced TEM will be needed. 464 Journal of Leukocyte Biology Volume 69, March

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