The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2–15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3–10% of CD4 and 10–40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5+ and CCR2+ T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1α and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC50 of 0.09 and 0.6–1.0 μg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.

The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases ∼5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.

Collagen-producing bone marrow-derived cells (fibrocytes) have been detected in animal models and patients with fibrotic diseases. In vitro data suggest that they develop from monocytes with the help of accessory cells and profibrotic soluble factors. Using a mouse model of renal fibrosis, unilateral ureteral obstruction, we found the number of circulating fibrocytes was not reduced when monocytes were depleted with a monoclonal antibody against CCR2 or when CCR2-/- mice with very low numbers of circulating or splenic monocytes were analyzed. The absence of CCR2, however, interfered with migration of fibrocytes into the kidney. The phenotype of splenic and renal fibrocytes was very similar and distinct from classical monocytes as fibrocytes expressed no CD115, medium levels of CCR2, and high levels of CD11b and Ly-6G. Using a depleting monoclonal antibody against Ly-6G or bone marrow chimeric mice expressing the diphtheria toxin receptor under the control of CD11b, we could efficiently deplete fibrocytes from the kidney. Depletion of fibrocytes or reduced migration of fibrocytes into the kidney resulted in lower renal expression of collagen-I. Thus, fibrocytes develop outside the kidney independent of infiltrating monocytes and rely on CCR2 for migration into target organs.

The predominance of CCR5-positive mononuclear cells in the synovial effusions of patients with arthritis suggests an important role for CCR5 in the process of joint inflammation, and identifies CCR5 as a possible new target for therapeutic intervention.