| dc.description | In chronic inflammatory reactions such as rheumatoid arthritis and multiple sclerosis, T cells that migrate into lesions preferentially express the chemokine receptors CXCR3, CCR5 and CCR2. Chemokines that bind these receptors are also found in the inflammatory lesions, and in particular, CCL2 is increased within lesions where there is an influx of monocytes and T cells. However, the contribution of these chemokines in recruiting T cells from the blood to the site of inflammation is unclear. In addition, the relative role of each of the chemokines that bind CXCR3 (CXCL9, CXCL10, CXCL11) and CCR5 (CCL3, CCL4, CCL5) in this process is unknown. The in vitro chemotaxis and in vivo migration of antigen-activated T lymphoblasts and unactivated spleen T cells to chemokines was examined. T lymphoblasts exhibit chemotaxis to CXCR3 ligands with a relative potency of CXCL10 > CXCL11 > CXCL9, with much less chemotaxis to the CCR5 ligands. In vivo, rat T lymphoblasts were recruited in large numbers to skin sites injected with CXCL10 and CCL5, and less to CCL3, CCL4, CXCL9, and CXCL11. The cytokines IFN-gamma, TNF-alpha, and IL-1alpha were co-injected with chemokines to upregulate endothelial cell adhesion molecule expression. Combining CXCL10 or CCL5 with these cytokines resulted in an additive increase in migration. Experiments using unactivated T cells demonstrated a similar pattern of migration although there was less overall migration of these cells. CXCR3 ligands are more chemotactic than CCR5 ligands for activated rat T cells in vitro while in vivo, CXCL10 and CCL5 are the most active at recruiting T cells out of the blood to an intradermal skin site. Thus, neither a chemokine's affinity for its receptor, nor its in vitro chemotactic activity is predictive of its capacity to recruit lymphocytes from the blood during inflammation. To study the role of CCR2 in the infiltration of leukocytes, cDNA for rat CCR2 was cloned by RT-PCR, and a FLAG tag added. Stably transfected CHO cells, expressing high levels of CCR2, were used to immunize hamsters and generate hybridomas. Five hybridomas, CR2.1--CR2.5, producing mAbs specific for rat CCR2 were identified. CCR2 was expressed at a high level on 80--90% of rat blood monocytes. Treatment of monocytes with two CCR2 mAbs induced CD11b/CD18 upregulation, L-selectin shedding, increased adhesion, and inhibited monocyte chemotaxis, similar to CCL2. CCR2 was also expressed on ∼10% of neutrophils, 20% of B cells but only on 3--5% of T cells. Immunization in vivo with vaccinia virus and in vitro anti-TCR activation increased CCR2 expression on T cells 5- and 12-fold, respectively. CCL2 stimulated the chemotaxis of both these CCR2+ T cells in vitro, and their migration to dermal inflammatory sites in vivo. These T cells also preferentially migrated to CCL2 in the presence of blockade of CXCR3, another chemokine receptor upregulated on activated T cells. These results demonstrate that some CCR2 mAbs may be potent activators of monocytes; and although CCR2 is expressed on few resting T cells, it is markedly up regulated after T cell activation and can mediate T cell infiltration in inflammation in vivo. | en_US |
