Published online before print August 1, 2003
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,1
* Departments of Microbiology & Immunology and
Pediatrics, Dalhousie Inflammation Group, Dalhousie University, Halifax, Nova Scotia, Canada
1 Correspondence: Department of Pediatrics, Division of Immunology, Rheumatology and Infectious Diseases, IWK Health Centre, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9. E-mail: thomas.issekutz{at}iwk.nshealth.ca
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, tumor necrosis factor 
, and interleukin-1 to up-regulate endothelial cell adhesion molecule expression with CXCL10 or CCL5 induced an additive increase in lymphoblast migration. Thus, CXCR3 ligands are more chemotactic than CCR5 ligands in vitro; however, in vivo, CXCL10 and CCL5 have comparable T cell-recruiting activities to cutaneous sites and are more potent than the other CXCR3 and CCR5 chemokines. Therefore, in vitro chemotaxis induced by these chemokines is not necessarily predictive of their in vivo lymphocyte-recruiting activity.
Key Words: inflammation • chemokine • chemotaxis • skin
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines are grouped into subfamilies based on the position of conserved cysteine residues in the N-terminal portion of the molecule and exert their action through seven-transmembrane G-protein-coupled receptors that are grouped in the same manner as the chemokine that binds to them. Several inflammatory conditions, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), are characterized by the infiltration of activated/memory (CD45RO+) T cells. These infiltrating T cells have an increased expression of the chemokine receptors CXCR3 and CCR5 as compared with T cells present in the blood [5
6
7
8
9
]. CXCR3 is expressed on human T cells and natural killer cells, and its ligands CXCL9 [monokine induced by interferon- (IFN-; MIG)], CXCL10 [IFN-inducible protein 10 (IP-10)], and CXCL11 [IFN-inducible T cell- chemoattractant (I-TAC)] are effective T cell chemoattractants [10
11
12
13
]. These chemokines are preferentially expressed in inflammatory lesions. Acute cardiac rejection is accompanied by intragraft production of CXCL9, CXCL10, and CXCL11 [14
]. CXCL9 and CXCL10 are expressed by macrophages and reactive astrocytes present in the plaques of actively demyelinating lesions of the central nervous system of patients with MS [5
, 15
]. CXCL10 has also been found to be abundantly expressed in various inflammatory lesions characterized by T cell infiltration, including delayed-type hypersensitivity in the skin [16
], experimental autoimmune encephalomyelitis [17
], and transplant rejection [18
].
CCR5 is expressed on human monocytes and CD45RO+ T cells [19
, 20
]. CCL3 [macrophage-inflammatory protein-1 (MIP-1)], CCL4 (MIP-1ß), and CCL5 [regulated on activation, normal T expressed and secreted (RANTES)], which bind to CCR5, can induce the chemotaxis of activated/memory T cells but not naïve, human T cells [21
, 22
]. These chemokines have also been found to be expressed in many inflammatory lesions [23
]. There is mRNA for all three chemokines expressed in the joints of patients with RA [24
], and the quantity of CCL3 and CCL4 protein is increased two- and 25-fold, respectively, in RA synovial fluid [25
] as compared with control synovial fluid. In MS, CCL3 is strongly associated with microglia/macrophages in central nervous system lesions but not in normal brain tissue [5
].
In spite of this evidence suggesting an important function for CXCR3 and CCR5 chemokines in inflammatory sites, there are few studies that have examined the relative activities of these chemokines on T cell recruitment in vivo. Studies in severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood lymphocytes injected intraperitoneally showed that subcutaneous (s.c.) CXCL10 induces the accumulation of human T cells at the injection site [26 ]. Further studies that have grafted human skin onto these SCID mice indicate that CXCL10 and CCL5 were able to recruit human T cells to the skin [27 ]. However, the effects of these and other CXCR3 and CCR5 chemokines on the migration of normal and activated T cells from the blood in a normal animal have not been studied.
In vitro studies have used transfected cell lines overexpressing CXCR3 to examine the relative potency of CXCL9, CXCL10, and CXCL11 in receptor binding, calcium flux, and chemotaxis [7 , 28 29 30 ]. Human CCR5 has also been overexpressed in transfected cell lines [31 32 33 ] to determine the relative potency of CCL3, CCL4, and CCL5. However, studies comparing the activities of these chemokines at inducing chemotaxis in vitro and T cell recruitment in vivo are lacking.
Therefore, the objective of this study was to examine the ability of CXCR3 and CCR5 chemokine ligands to induce T cell recruitment in vivo and to compare this migration with the ability of these chemokines to stimulate T cell chemotaxis in vitro. CXCR3 chemokines, particularly CXCL10, were potent chemoattractants in vitro, and the CCR5 chemokines were only weakly active at stimulating T cell chemotaxis. In contrast, T cells migrated in significant numbers to skin sites containing CXCR3 and CCR5 ligands in vivo, especially CXCL10 and CCL5, and interleukin (IL)-1 and tumor necrosis factor (TNF-) enhanced this.
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(CCL3), MIP-1ß (CCL4), IP-10 (CXCL10), I-TAC (CXCL11), MIG (CXCL9), stromal cell-derived factor-1 (CXCL12), and recombinant mouse (rm)TNF- (specific activity=5x107 U/mg), were purchased from PeproTech Inc. (Rocky Hill, NJ). The purity of these products is >98% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure liquid chromatography analysis, and the endotoxin level is <0.01 ng/mg (1 EU/mg) of cytokine or chemokine. The murine cytokine IL-1 was provided by Hoffmann-LaRoche (Nutley, NJ), and rat rIFN- was a gift from Dr. Peter Van der Meide (TNO Primate Centre, Rijswijk, The Netherlands).
Lymphocyte isolation
Spleen T lymphocytes were isolated from normal donors. In brief, the splenocytes were isolated by finely mincing the spleen to produce a single-cell suspension. The stroma was allowed to settle for 5 min at 1 g. The resultant cell suspension was resuspended in RPMI medium and washed, and the red blood cells were lysed with ammonium chloride. The cells were then resuspended in RPMI plus 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) and were applied onto a nylon wool column. After 60 min of incubation, the unbound lymphocytes were eluted, washed, and resuspended in fresh RPMI.
The isolation of lymph node lymphocytes and lymphoblast-enriched fractions was performed as described previously [34 ]. Briefly, rats were injected s.c. in each footpad with 106 pfu vaccinia virus. Four days later, the draining lymph nodes (axillary and popliteal) were removed, and lymphocytes were obtained by gently mincing the nodes to produce a cell suspension. The stroma was allowed to settle for 5 min at 1 g. The lymphocyte-rich supernatant was washed in RPMI and applied to a continuous linear-density gradient of Percoll (Amersham Inc., Oakville, Ontario, Canada). This allowed for fractionation into lymphoblast-enriched and lymphoblast-depleted cell populations. The low-density lymphocytes, confirmed by cell sizing on a Coulter counter (Coulter Electronics, Hialeah, FL) were pooled, washed in RPMI, and panned using a tissue-culture plate coated with 100 µg/ml goat anti-rat immunoglobulin G (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) to remove B cells. The nonadherent cells were collected, washed, and radiolabeled as described below. The resultant population was >95% T cells, as determined by immunofluorescence staining.
Measurement of lymphocyte chemotaxis in vitro
The chemotaxis of T cells in response to chemokines was measured across 5 µm pore-size polycarbonate filters (6.5 mm diameter) in 24-well Transwell chambers (Costar Corning Co., Cambridge, MA) precoated with 0.01% gelatin and 60 g/ml human fibronectin. Lymphoblasts and normal spleen T cells were incubated with 50 µCi Na251CrO4 for 45 min at 37°C per 5 x 107 cells/ml RPMI (Amersham Inc.). The labeled cells were then washed in RPMI and resuspended at 1–2 x 106 cells/ml in RPMI plus 5 mg/ml human serum albumin (HSA), and 100 µl of this cell suspension was added to the upper chamber of each Transwell. Chemokines were added to the lower chamber in a total volume of 600 µl RPMI plus HSA 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 amount of T cell migration was measured by counting. Percent migration was calculated by dividing the radioactivity on the cells in the lower chamber by the total input radioactivity of the labeled cells added to the upper chamber.
Measurement of in vivo lymphocyte migration
The migration of radiolabeled lymphoblasts and spleen T cells to cutaneous sites injected with lymphocyte-recruiting agents in rats was measured as described previously [34
]. Briefly, 107 lymphoblasts in 100 µl RPMI were incubated with 1 µCi 111In for 10 min at 20°C, washed twice, and resuspended at a concentration of 2.5 x 107 cells/ml in RPMI for injections. Spleen T cells (5x107 cells/ml) were incubated with 50 µCi Na251CrO4 for 45 min at 37°C in RPMI + 10% FBS, washed twice, and resuspended at a concentration of 5 x 107 cells/ml RPMI for injection. Normal rats were anesthetized with halothane and injected intravenously (i.v.) with 5–10 x 106 T lymphoblasts and 2 x 107 spleen T cells. Immediately afterward, the skin on the backs of the rats was shaved, and 50 µl chemokines and cytokines were injected intradermally (i.d.) into two or three sites. Two to four additional sites were also injected with diluent alone (RPMI+1 mg/ml HSA). In most experiments, the rats were killed 20 h later, as this has been shown to be optimal for the measurement of maximal lymphocyte accumulation [35
]. The skin on the backs of the animals was removed, excess blood in the superficial veins was squeezed out, and the injected areas were punched out with a leather punch, resulting in a circular piece of tissue 12 mm in diameter. Samples of the spleen, cervical lymph nodes, liver, lung, and blood were removed, and the radioactivity in these tissues and the injected skin sites was determined by counting. In some experiments, animals were injected with i.d. stimuli 3.5 h before and at the time of the i.v. injection of radiolabeled cells. These animals were killed 3 h after the labeled cells were given to evaluate the kinetics of lymphocyte recruitment into skin sites of various ages.
Histology
In some experiments, skin sites were collected in buffered formalin for histological analysis. The skin sites were embedded in paraffin, and 5 µm sections were cut, stained with hematoxylin and eosin, and examined for the presence of infiltrating leukocytes in the skin.
Statistics
One-way ANOVA with Dunnett or Tukey-Kramer post-test for multiple comparisons was performed using InStat (GraphPad Software, San Diego, CA).
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Figure 1. Effect of CXCR3 and CCR5 chemokines on lymph node T lymphoblast and spleen T cell chemotaxis. Rat lymph node T lymphoblasts (A and C) and spleen T cells (B and D) were labeled with 51Cr and added to the upper chamber of a 24-well Transwell chamber. The indicated concentrations of CXCR3 (A and B) and CCR5 (C and D) chemokines were added to the lower chamber. After incubation for 1 h, chemotaxis to the lower chamber was measured. Values are mean ± SEM of triplicate wells. +, P< 0.05; x, P< 0.01; *, P< 0.001, as compared with spontaneous migration to media. Results are mean ± SEM of two to six experiments.
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As the chemokines that bind to CCR5 were quite weak at inducing T lymphoblast chemotaxis, the ability of CCL5 to stimulate T cell adhesion to fibronectin in 96-well plates was also examined and compared with adhesion induced by CXCL10. CCL5 induced a significant increase in T lymphoblast adhesion from a background of 8.2 ± 1.5% of input T cells adhered in control wells to 32 ± 7% in wells containing CCL5 (data not shown). CXCL10 also increased T lymphoblast adhesion fivefold over medium controls from 8.2 ± 1.5% to 41.3 ± 9.1%, and CCL3 also induced significant T cell adhesion (data not shown). Thus, although CCL5 and CCL3 were relatively weak stimulators of T lymphoblast chemotaxis in vitro, they were effective at inducing increased cell adhesion similar to the potent chemoattractant CXCL10.
Effect of CXCR3 and CCR5 ligands on T cell migration to the skin
The migration of T cells from the blood to cutaneous sites injected with chemokines that bind to CXCR3 and CCR5 was examined. Lymphoblasts from antigen-stimulated lymph nodes and normal spleen T cells were radiolabeled and injected i.v. into normal recipients who were given i.d. injections of chemokines. The accumulation of radiolabeled cells in the skin was assessed 20 h later, which was shown in previous studies to be optimal for measurement of T cell recruitment to the skin [35
]. There was a progressive increase in T lymphoblast (Fig. 2
) and spleen T cell migration to the CXCR3 chemokines. CXCL9, CXCL10, and CXCL11 each significantly increased T lymphoblast migration to the skin at 100 and 300 ng/site (P<0.01), and spleen T cells were also recruited by all three chemokines. CXCL10 was the most potent chemokine in attracting both cell populations and induced up to a three- to fourfold increase in migration to the skin as compared with control injection sites.
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Figure 2. Effect of CXCR3 and CCR5 chemokines on T lymphoblast and spleen T cell migration to the skin. Rats were injected i.v. with 111In-labeled T lymphoblasts (A and C) and 51Cr-labeled spleen T cells (B and D) and i.d. injections on the back in duplicate sites with 50 µl containing the indicated amounts of CXCL9, CXCL10, CXCL11 (A and B) or CCL3, CCL4, CCL5 (C and D) or control diluent. Animals were killed 20 h later, and the radioactivity in each lesion was determined. Each point represents the mean increase over control injection sites, which were 75 ± 7 cpm/site for T lymphoblasts and 30 ± 4 cpm/site for spleen T cells, and shows the mean ± SEM of four to 16 animals. Significance compared with control sites is shown as +, P < 0.05; x, P< 0.01; *, P< 0.001.
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Kinetics of lymphocyte migration in response to CXCR3 and CCR5 chemokines
The kinetics of migration of T lymphoblasts and spleen T cells in response to i.d. chemokines was examined. Chemokines and IFN- were injected i.d. 3.5 h before and immediately after the i.v. injection of radiolabeled T cells. The animals were killed 3 h later, and the accumulation of the labeled cells in skin sites (which were 3 h and 6.5 h of age) was determined (Fig. 3
). CXCL10 and CXCL11 induced a rapid and significant increase in migration of T lymphoblasts and spleen T cells in the first 3 h. This was followed by a decline in the migration of T cells to the chemokine-injected sites, which were 3.5–6.5 h old. T cell migration to IFN- was also rapid with significant migration in the first 3 h, but in contrast to the chemokines, this migration markedly increased at the later time point (3.5–6.5 h).
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Figure 3. Kinetics of T lymphoblast and spleen T cell migration in response to CXCR3 and CCR5 ligands. Rats were injected i.d. in duplicate sites with 300 ng CXCL9, CXCL10, and CXCL11 (A and B); CCL3, CCL4, and CCL5 (C and D); and 300 U rIFN- and control diluent. Recipients were given i.d. injections 3.5 h before and simultaneously with the i.v. injection of 111In-labeled T lymphoblasts (A and C) and 51Cr-labeled spleen T cells (B and D). Animals were killed 3 h later, and the radioactivity of each lesion was determined. Each point represents the mean increase over diluent control-injected sites in four animals and is shown on the abscissa at the average age of the lesion during the 3 h the labeled cells were circulating in the animal. Control sites were 67 ± 4 and 55 ± 5 for the lymphoblasts, and 20.4 ± 2.5 and 18.6 ± 2.3 for spleen T cells in the 1.5 and 5 h lesions, respectively. Significance compared with control sites is shown as +, P< 0.05; x, P< 0.01; *, P< 0.001.
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Effect of combinations of chemokines on T lymphocyte migration
Chemokines that bind to different chemokine receptors may have different roles in mediating transendothelial migration; therefore, combining chemokines might significantly enhance T cell migration to the skin. Figure 4
shows the effect of injecting a combination of chemokines on T cell recruitment. Coinjection of CCL5 and CXCL10 resulted in a significant (P<0.01), additive increase in the accumulation of lymphoblasts as compared with injection of either chemokine alone. However, coinjection of CCL3 with CXCL10 or CCL5 was equivalent to the injection of CXCL10 or CCL5 alone. In lesions coinjected with other combinations of CXCR3 and CCR5 chemokines, the accumulation to the combination of chemokines was marginally greater than to the more-potent chemokine alone. Similar results were found using normal spleen T cells (data not shown).
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Figure 4. Effect of coinjecting two chemokines on T lymphoblast migration to the skin. Rats were injected i.v. with 111In-labeled T lymphoblasts and given i.d. injections on the back in duplicate sites with 300 ng CXCL10 (A–C), CXCL11 (B, D), CXCL9 (E), CCL3 (C, F), CCL5 (A, D–F), or control diluent. Twenty hours later, the radioactivity in each lesion was determined. Each point represents the mean increase over control-injection sites, which were 77 ± 3 cpm/site. The bars represent mean ± SEM of four animals. Significances of coinjected sites compared with the injection of each chemokine alone are shown as x, P < 0.01.
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Figure 5. Histological appearance of 20-h-old lesions injected with chemokines. Rats were injected i.v. with 111In-labeled T lymphoblasts and given i.d. injections on the back in duplicate sites with 300 ng CXCL9 and CCL5 (B) or control diluent (A). Twenty hours later, the sites were excised, and some skin sites were collected in buffered formalin for histological analysis. The skin sites were embedded in paraffin, and 5-µm sections were cut, stained with hematoxylin and eosin, and examined for the presence of infiltrating leukocytes in the skin. This figure represents a representative field of view at 400x original magnification. The radioactivity in each lesion was also determined, which was 71 and 297 cpm/site for A and B, respectively.
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, TNF-, or IL-1 on T lymphoblast accumulation to dermal sites was determined. As shown in Figure 6
, IL-1, IFN-, and TNF- each stimulated T lymphoblast recruitment to the skin, and IFN- was substantially more active than the other two cytokines. Coinjection of CXCL10 with these cytokines resulted in a significant (P<0.05 or P<0.01), additive increase in T lymphoblast migration in comparison with the injection of either stimulus alone. Coinjection of CCL5 also resulted in a significant increase in T cell migration to IL-1 and TNF- but not to IFN-, the most potent stimulus for T cell recruitment. The results were similar when the migration of spleen T cells was examined (data not shown).
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Figure 6. Effect of coinjecting cytokines and chemokines on T lymphoblast migration to the skin. Rats were injected i.v. with 111In-labeled T lymphoblasts and given i.d. injections containing 300 ng CXCL10 (A–C) or CCL5 (D–F) in combination with 10 ng mIL-1 (A, D), 10 ng TNF- (C, F), 300 U IFN- (B, E), or diluent control. Twenty hours later, the radioactivity in each lesion was determined. Each point represents the mean increase over control injection sites, which were 80 ± 3 cpm/site. The bars represent the mean ± SEM of four animals. Significances of coinjected sites compared with the injection of each stimulus alone are shown as +, P < 0.05; x, P< 0.01.
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The chemokines CXCL9, CXCL10, and CXCL11, which bind to CXCR3, were chemotactic for in vivo antigen-activated T lymphoblasts and for unstimulated spleen T cells. CXCL10 was particularly potent, inducing significant chemotaxis of both cell populations. The relative chemotactic potency of the three chemokines was CXCL10 > CXCL11 > CXCL9. These relative activities of the three chemokines are similar to the relative binding affinities, CXCL10 > CXCL11 
CXCL9, reported by Wang et al. [30
], using human embryo kidney (HEK)-293 cells transfected with rat CXCR3.
The three chemokines that bind to CCR5 were much less effective chemoattractants in vitro. The largest response to CCL3, CCL4, and CCL5 was less than a twofold increase at low concentrations of CCL5, at intermediate concentrations of CCL3, and at high concentrations for CCL4. This relative potency is similar to that of human T cells, in which CCL5 was the most effective at inducing chemotaxis and transendothelial migration, followed by CCL3 [44 , 45 ]. Studies examining CCR5 transfected into Chinese hamster ovary-K1, HEK-293, and L1.2 cells have also found that CCL5 and CCL3 are potent stimulators of CCR5 [31 , 33 , 46 ]. It should be noted however that CCL3 and CCL5 can also bind to CCR1, and CCL5 is a ligand for CCR3. However, CCR1 and CCR3 are present on only a small (<3%) fraction of human blood T cells, and the proportion of T cells expressing CCR5 is much higher in inflammatory sites than CCR1 or CCR3 [47 48 49 50 51 52 53 ].
To evaluate the efficacies of these chemokines in vivo, the ability of the chemokines to recruit T cells from the blood to dermal sites was determined. The novelty of this approach is that the activity of cells that have been activated in vivo can be evaluated by in vitro chemotaxis and in vivo migration. Normal and in vivo antigen-stimulated T cells migrated into the skin in response to all six of the chemokines tested (Fig. 2) . The migration was assessed at 20 h, as our previous studies have shown that this is the time of maximum T cell accumulation into dermal sites injected with inflammatory stimuli, as there is minimal migration out of the site within this time [35 ]. The response to CXCL10 and CCL5 was generally greater than to the other chemokines. The relative potency of CXCR3 chemokines in vivo was similar to their chemotactic activities in vitro. However, the results for the CCR5 chemokines were quite different from that observed in the in vitro chemotaxis assay. CCL5 was a much more potent stimulus in vivo than in vitro, using the same T cell population. CCL5 induced nearly as much T cell recruitment to the skin as CXCL10, although it was much less active at stimulating T cell chemotaxis. Similarly, CCL3 was quite a weak stimulator of chemotaxis, yet in vivo, it was certainly effective at recruiting T cells.
The basis for this difference in in vivo lymphocyte-recruiting activity and in vitro chemotaxis by CCR5 ligands is not readily apparent. One possibility may be that CCL5 and CCL3, by binding to proteoglycans on the surface of endothelial cells in the presence of the shear stress produced by blood flow, are much more active stimulators of in vivo lymphocyte adhesion and transendothelial migration than suggested by their in vitro chemotactic activities. It has recently been shown that CXCL12 presented apically on the surface of endothelial cells in flow chambers can stimulate rapid lymphocyte transendothelial migration [54 ]. It is possible that CCL5 and CCL3 could act in a similar manner. Our finding that CCL5 and CCL3 increased T lymphoblast adhesion, and CCL5 was nearly as active as CXCL10 at enhancing adhesion to fibronectin (data not shown) is consistent with CCL5 being a potent activator of T cell integrins and with its strong in vivo-recruiting activity. Another possible explanation for the in vivo activity of CCL5 and CCL3 may be an indirect effect of these chemokines on resident cells, such as tissue macrophages, mast cells, or endothelial cells, to stimulate T cell recruitment. The ability of CCL5 to stimulate the production of a T cell chemotactic factor from rat peritoneal macrophages, mast cells, and whole skin explants was examined, but no chemotactic activity was detected (data not shown).
The kinetics of T cell migration to i.d. chemokines was also examined and compared with the kinetics of IFN-, a potent T cell-recruiting cytokine in vivo [35
].As shown in Figure 3
, the chemokines induced a rapid accumulation of T cells within 3 h, with less accumulation in the later time points, and IFN- continued to stimulate the migration of T cells into the skin. The rapid and transient effect of the injected chemokines is also in keeping with these agents acting directly on the T cells to promote lymphocyte recruitment.
Our studies also examined the effect of combining two chemokines together on T cell recruitment. The combination of the two most potent stimuli, CXCL10 and CCL5, resulted in a significant enhancement in accumulation, as compared with either stimulus alone (Fig. 4A) . There may be several explanations for this increased T cell recruitment. T cells expressing the receptors for these ligands may represent different subsets of T cells, causing the two chemokines to recruit different subsets of lymphocytes. However, in humans it has been shown that CCR5 is expressed on a subset of CXCR3+ T cells in inflammatory lesions, and in rodents, receptor expression is likely to be similar to that in humans [5 , 6 , 55 , 56 ]. CCL5 and CXCL10 may be acting optimally at different steps to promote T cell infiltration into the skin. In keeping with the chemotaxis results, CXCL10 may be acting in a soluble state to induce recruitment, and CCL5, which is relatively weak at inducing chemotaxis, may act through binding to the endothelium to enhance transendothelial migration. A marked increase in T cell recruitment was also not observed when CCL5 was combined with the other CXCR3 ligands, CXCL9 and CXCL11, which were less potent, and when CXCL10 was combined with CCL3, suggesting that CXCL10 and CCL5 may have special potentiating roles in promoting T cell recruitment.
During inflammation, endothelium is activated by cytokines, such as IFN-, IL-1, and TNF-. It is generally accepted that these cytokines increase transendothelial migration by increasing the level of CAMs on the surface of the endothelium [36
, 38
, 39
, 57
] as well as stimulating the production of chemokines [40
, 58
, 59
]. Our results demonstrate that combining IL-1 or TNF- with CXCL10 or CCL5 significantly increased T cell migration to the skin as compared with migration in response to either stimulus alone (Fig. 6)
. There was only a small increase in lymphocyte migration when the chemokines CXCL10 and CCL5 were injected together with IFN-, which is a potent stimulator of T cell recruitment, as well as a potent inducer of CXCL10 production by many cell types. Our findings suggest that cytokines, which are less potent stimulators of T cell recruitment, markedly potentiate T cell migration to exogenous, T cell-recruiting chemokines.
These studies, which compare the in vitro and in vivo potency of multiple chemokines that bind to the same receptor, highlight the complexity of the chemokine–chemokine receptor network. They demonstrate that chemotatic activity in vitro may not directly correlate with the capacity to stimulate lymphocyte recruitment to an inflammatory site in vivo. This may be a result of the ability of these chemokines to act sequentially in the transmigration process to enhance recruitment when produced together within an inflammatory site or to secondary effects of the chemokines on other cell types. However, it underscores the importance of assessing the activity of chemokines in vivo as well as in vitro.
Received November 12, 2002; revised June 25, 2003; accepted July 1, 2003.
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