Hilke Brühl

Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.

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.

Expression and Characterization of the Chemokine Receptors CCR2 and CCR5 in Mice

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.

Dual Role of CCR2 during Initiation and Progression of Collagen-Induced Arthritis: Evidence for Regulatory Activity of CCR2 + T Cells

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.

Identification of Antigen-Capturing Cells as Basophils

Binding of intact Ag is a hallmark of Ag-specific B cells. Apart from B cells, a small number of non-B cells can bind Ag with comparable efficacy as B cells and are found in the peripheral blood, spleen, and bone marrow of mice. This population has been observed for a long time and recently named “Ag-capturing cells.” Their identity remained enigmatic. In this study, we show that these cells are basophilic granulocytes. Their ability to capture Ags is dependent on surface IgE receptors and on Ag-specific plasma IgE molecules appearing after immunization. Several surface markers including surface bound IgE, IL-3R, CD45, CD16/32, and the chemokine receptor CCR2 were used to clearly identify these cells. Cross-linkage of surface Igs results in the release of large amounts of IL-4 and IL-6. The data identify basophils as Ag-capturing cells and support the concept of basophils as important regulators of humoral immune responses.

Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model

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.

Depletion of CCR5-expressing cells with bispecific antibodies and chemokine toxins: a new strategy in the treatment of chronic inflammatory diseases and HIV.

The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5+ cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5+ leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3+ T cells against CCR5+ target cells. The Ab specifically depletes CCR5+ T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5+ monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of Pseudomonas exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5+ Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5− Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells.

Differences in CCR5 expression on peripheral blood CD4+CD28− T-cells and in granulomatous lesions between localized and generalized Wegener’s granulomatosis

Wegeners granulomatosis (WG) is an autoimmune disease characterized by granulomatous lesions and a necrotizing vasculitis. Th1-type-cells lacking CD28 are expanded independent of age and immunosuppressive therapy in WG. To address their migratory properties of CD4(+)CD28(-) T-cells we studied the expression of the inducible inflammatory Th1-type chemokine receptor CCR5 in localized WG and generalized WG. Expansion of CD4(+)CD28(-) T-cells was more prominent in generalized WG compared to localized WG. In localized WG a larger fraction of CD4(+)CD28(-) T-cells displayed CCR5 expression compared to generalized WG. CCR5 expression was also higher in granulomatous lesions in localized WG. Higher levels of CCR5 expression on CD4(+)CD28(-) T-cells in localized WG may favor stronger CCR5-mediated recruitment of this T-cell subset into granulomatous lesions in localized WG. Expansion of Th-1-type CD4(+)CD28(-)CCR5(+) effector memory T-cells might contribute to disease progression and autoreactivity, either directly, by maintaining the inflammatory response, or as a result of bystander activation.

Intrapulmonary targeting of RANTES/CCL5-responsive cells prevents chronic fungal asthma

Regulated upon activation in normal T cells, expressed, and secreted (RANTES)/CCL5 is abundantly expressed during atopic asthma, suggesting that it is an important mediator of this disease. The contribution of intrapulmonary RANTES/CCL5‐sensitive cells during Aspergillus fumigatus‐induced airway disease in mice was assessed in this study. The intranasal delivery of a chimeric protein comprised of RANTES/CCL5 and a truncated version of Pseudomonas exotoxin A (RANTES‐PE38) significantly attenuated serum IgE, peribronchial eosinophilia, and airway hyperreactivity when it was administered from day 0 to 15 after intratracheal conidia challenge in A. fumigatus‐sensitized mice but had little effect when delivered from day 15 to 30 after conidia challenge. Intranasal RANTES‐PE38 treatment enhanced macrophage recruitment and accelerated fungal clearance in the lungs of RANTES‐PE38‐treated mice. These data reveal a major role for RANTES/CCL5 and its receptors in the development of fungal asthma yet reveal only a modest role in the chronic remodeling of the allergic airway in this disease.

Important role of interleukin-3 in the early phase of collagen-induced arthritis.

OBJECTIVE Activation of basophils contributes to memory immune responses and results in exacerbation of collagen-induced arthritis (CIA). We undertook the present study to analyze the production and biologic effects of interleukin-3 (IL-3), a strong activator of basophils, in CIA. METHODS Arthritis was induced by immunization with type II collagen. Mice were treated with blocking monoclonal antibodies against IL-3 or with recombinant IL-3. Clinical scoring, histologic analysis, fluorescence-activated cell sorter analysis, enzyme-linked immunosorbent assay, and cell culturing were performed to assess disease activity and IL-3 production. RESULTS IL-3 was produced in large quantities by collagen-specific CD4+ T cells in the spleen and was present in the synovial tissue during onset of arthritis, but was down-regulated in paws with severe inflammation. Blockade of IL-3 during the time of arthritis onset resulted in profound improvement of the disease, with reductions in synovial leukocyte and cytokine levels, peripheral blood basophil levels, and anticollagen antibody titers. Blockade of IL-3 during the late phase of arthritis had no beneficial effect. Administration of recombinant IL-3 during onset of arthritis induced a marked exacerbation of the disease, with increased peripheral blood basophil and plasma IL-6 levels and increased titers of anticollagen antibody. In studies of the regulation of IL-3 expression in CD4+ T cells, IL-6 and IL-4 suppressed the release of IL-3 by activated CD4+ T cells, whereas lipopolysaccharide and CpG DNA up-regulated IL-3 secretion in activated CD4+ T cells by acting on costimulatory cells. CONCLUSION Taken together, the present results demonstrate for the first time that IL-3 has an important role in the early phase of CIA.

Chondroitin sulfate A released from platelets blocks RANTES presentation on cell surfaces and RANTES-dependent firm adhesion of leukocytes

The sequestration of chemokines on the surface of microvascular endothelium is an early event in the selective recruitment of leukocytes. The sequestration and presentation of chemokines must be tightly controlled to confine the extravasation of leukocytes and to prevent uncontrolled inflammation. We investigated whether soluble molecules released under physiological conditions could control chemokine immobilization on cell surfaces and function as regulatory chemokine binding molecules. We determined that human serum contains a molecule that suppresses RANTES (CCL5) binding to endothelial cells, PBMC and CHO cells. Using platelet‐rich and platelet‐free plasma, serum from patients with thrombocytopenia, and purified platelets, we identified platelets as the source of the chemokine‐binding molecule and further identified it as chondroitin sulfate A. In contrast to platelet‐derived fully‐sulfated chondroitin sulfate A, low‐sulfated chondroitin sulfate A present in plasma was almost inactive. Under physiological flow conditions chondroitin sulfate A was found to block RANTES‐mediated firm adhesion of monocytes to endothelial cells. It also prevented RANTES‐mediated influx of calcium in CCR5‐transfected CHO cells while internalization of CCR5 was only marginally reduced. Taken together, chondroitin sulfate A released from platelets appears to act as an important regulatory molecule for cellular responses to chemokines.