Differential contribution of immune effector mechanisms to cortical demyelination in multiple sclerosis.

Cortical demyelination is a widely recognized hallmark of multiple sclerosis (MS) and correlate of disease progression and cognitive decline. The pathomechanisms initiating and driving gray matter damage are only incompletely understood. Here, we determined the infiltrating leukocyte subpopulations in 26 cortical demyelinated lesions of biopsied MS patients and assessed their contribution to cortical lesion formation in a newly developed mouse model. We find that conformation-specific anti-myelin antibodies contribute to cortical demyelination even in the absence of the classical complement pathway. T cells and natural killer cells are relevant for intracortical type 2 but dispensable for subpial type 3 lesions, whereas CCR2+ monocytes are required for both. Depleting CCR2+ monocytes in marmoset monkeys with experimental autoimmune encephalomyelitis using a novel humanized CCR2 targeting antibody translates into significantly less cortical demyelination and disease severity. We conclude that biologics depleting CCR2+ monocytes might be attractive candidates for preventing cortical lesion formation and ameliorating disease progression in MS.

IL-3 promotes the development of experimental autoimmune encephalitis.

Little is known about the role of IL-3 in multiple sclerosis (MS) in humans and in experimental autoimmune encephalomyelitis (EAE). Using myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE, we show that CD4+ T cells are the main source of IL-3 and that cerebral IL-3 expression correlates with the influx of T cells into the brain. Blockade of IL-3 with monoclonal antibodies, analysis of IL-3 deficient mice, and adoptive transfer of leukocytes demonstrate that IL-3 plays an important role for development of clinical symptoms of EAE, for migration of leukocytes into the brain, and for cerebral expression of adhesion molecules and chemokines. In contrast, injection of recombinant IL-3 exacerbates EAE symptoms and cerebral inflammation. In patients with relapsing-remitting MS (RRMS), IL-3 expression by T cells is markedly upregulated during episodes of relapse. Our data indicate that IL-3 plays an important role in EAE and may represent a new target for treatment of MS.

Flow Cytometry Detection of Chemokine Receptors for the Identification of Murine Monocyte and Neutrophil Subsets.

Chemokine receptors are differentially expressed on leukocyte subpopulations dictating their ability to migrate both in physiological and pathological conditions. Their expression is modulated during leukocyte differentiation and maturation and they can be used as markers to identify and characterize the frequency and the activation state of leukocytes present in a tissue. Here, we will describe flow cytometry approaches to detect chemokine receptors identifying subpopulations of circulating monocytes and neutrophils.

IL-3 contributes to development of lupus nephritis in MRL/lpr mice.

MRL/lpr mice develop a spontaneous autoimmune disease that closely resembles human systemic lupus erythematosus (SLE) with DNA autoantibodies, hypergammaglobulinemia, immune complex glomerulonephritis, and systemic vasculitis. Little is known about the role of IL-3 in SLE. In order to study this we analyzed the expression of IL-3 in murine lupus and determined whether blockade of IL-3 with a monoclonal antibody or injection of recombinant IL-3 affects lupus nephritis in MRL/lpr mice. During disease progression IL-3 levels were increased in the plasma and in the supernatant of cultured splenocytes from MRL/lpr mice. Administration of IL-3 aggravated the disease with significantly higher renal activity scores, more renal fibrosis, and more glomerular leukocyte infiltration and IgG deposition. Blockade of IL-3 significantly improved acute and chronic kidney damage, reduced the glomerular infiltration of leukocytes and the glomerular deposition of IgG, and decreased the development of renal fibrosis. Furthermore, DNA autoantibody production, proteinuria, and serum creatinine levels were significantly lower in the anti-IL-3 group. Thus, IL-3 plays an important role in the pathogenesis of SLE and the progression of lupus nephritis. Hence, blockade of IL-3 may represent a new strategy for treatment of lupus nephritis.

B-cell inhibition by cross-linking CD79b is superior to B-cell depletion with anti-CD20 antibodies in treating murine collagen-induced arthritis.

Depletion of B cells with the anti-CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B-cell proliferation induced via the B-cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti-CD79b also induces death in resting and activated B cells. B-cell inhibition is mediated by cross-linkage of CD79b, but independent of Fc-receptor engagement. In the model of collagen-induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti-collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B-cell depletion with anti-CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B-cell-mediated autoimmune diseases.

Basophils inhibit proliferation of CD4⁺ T cells in autologous and allogeneic mixed lymphocyte reactions and limit disease activity in a murine model of graft versus host disease.

Basophils are known to modulate the phenotype of CD4(+) T cells and to enhance T helper type 2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4(+) T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin-4 (IL-4) and IL-6. The inhibitory effect of basophils on T-cell proliferation can be blocked with antibodies against IL-4 and IL-6 and is absent in IL-4/IL-6 double-deficient mice. In addition, we show that basophils and IL-4 have beneficial effects on disease activity in a murine model of acute graft-versus-host disease (GvHD). When basophils were depleted with the antibody MAR-1 before induction of GvHD, weight loss, GvHD score, mortality and plasma tumour necrosis factor levels were increased while injection of IL-4 improved GvHD. Basophil-depleted mice with GvHD also have increased numbers of CD4(+) T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4(+) T-cell proliferation in an IL-4-dependent manner.

Chondroitin sulfate activates B cells in vitro, expands CD138+ cells in vivo, and interferes with established humoral immune responses.

Glycosaminoglycans have anti-inflammatory properties and interact with a variety of soluble and membrane-bound molecules. Little is known about their effects on B cells and humoral immune responses. We show that CS but not dextran or other glycosaminoglycans induces a pronounced proliferation of B cells in vitro compared with TLR4 or TLR9 ligands. With the use of inhibitors and KO mice, we demonstrate that this proliferation is mediated by the tyrosine kinases BTK and Syk but independent of CD44. Antibodies against Ig-α or Ig-ß completely block CS-induced B cell proliferation. Injection of CS in mice for 4-5 days expands B cells in the spleen and results in a marked increase of CD138(+) cells in the spleen that is dependent on BTK but independent of CD4(+) T cells. Long-term treatment with CS for 14 days also increases CD138(+) cells in the bone marrow. When mice were immunized with APC or collagen and treated with CS for up to 14 days during primary or after secondary immune responses, antigen-specific humoral immune responses and antigen-specific CD138(+) plasma cells in the bone marrow were reduced significantly. These data show that CD138(+) cells, induced by treatment with CS, migrate into the bone marrow and may displace other antigen-specific plasma cells. Overall, CS is able to interfere markedly with primary and fully established humoral immune responses in mice.

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.

Adiponectin stimulates release of CCL2, -3, -4 and -5 while the surface abundance of CCR2 and -5 is simultaneously reduced in primary human monocytes.

The adipokine adiponectin is well known to affect the function of immune cells and upregulation of CCL2 by adiponectin in monocytes/macrophages has already been reported. In the current study the effect of adiponectin on CCL2, -3, -4, and -5 and their corresponding receptors CCR1, CCR2, and CCR5 has been analyzed. Adiponectin elevates mRNA and protein of the CC chemokines in primary human monocytes. Simultaneously the surface abundance of CCR2 and CCR5 is reduced while CCR1 is not affected. Downregulation of CCR2 by adiponectin is blocked by a CCR2 antagonist although expression of the CCL2 regulated genes CCR2 and TGF-beta 1 is not altered in the adiponectin-incubated monocytes. CCL2, -3, and -5 concentrations measured in supernatants of monocytes of normal-weight (NW), overweight (OW), and type 2 diabetic (T2D) patients positively correlate with BMI and are increased in obesity and T2D. In contrast CCL4 is similarly abundant in the supernatants of all of these monocytes. The degree of adiponectin-mediated induction of the chemokines CCL3, -4, and -5 negatively correlates with their basal levels and upregulation of CCL3 and CCL5 is significantly impaired in OW and T2D cells. Serum concentrations of these chemokines are almost equal in the three groups and do not correlate with the levels in monocyte supernatants. In conclusion these data demonstrate that adiponectin stimulates release of CCL2 to CCL5 in primary human monocytes, and induction in cells of overweight probands is partly impaired. Adiponectin also lowers surface abundance of CCR2 and CCR5 and downregulation of CCR2 seems to depend on autocrine/paracrine effects of CCL2.

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.

Targeting of Gr-1+,CCR2+ monocytes in collagen-induced arthritis.

OBJECTIVE: The chemokine receptor CCR2 is highly expressed on monocytes and considered a promising target for treatment of rheumatoid arthritis. However, blockade of CCR2 with a monoclonal antibody (mAb) during progression of collagen-induced arthritis results in a massive aggravation of the disease. In this study we investigated why CCR2 antibodies have proinflammatory effects, how these effects can be avoided, and whether CCR2+ monocytes are useful targets in the treatment of arthritis. METHODS: Arthritis was induced in DBA/1 mice by immunization with type II collagen. Mice were treated with mAb against CCR2 (MC-21), IgE, or isotype control antibodies at various time points. Activation of basophils and depletion of monocyte subsets were determined by fluorescence-activated cell sorter analysis and enzyme-linked immunosorbent assay. RESULTS: Crosslinkage of CCR2 activated basophils to release interleukin-6 (IL-6) and IL-4. In vivo, IL-6 release occurred only after exposure to high doses of MC-21, whereas application of low doses of the mAb circumvented the release of IL-6. Regardless of the dose level used, the antibody MC-21 efficiently depleted Gr-1+,CCR2+ monocytes from the synovial tissue, peripheral blood, and spleen of DBA/1 mice. Activation of basophils with high doses of MC-21 or with antibodies against IgE resulted in a marked aggravation of collagen-induced arthritis and an increased release of IL-6. In contrast, low-dose treatment with MC-21 in this therapeutic setting had no effect on IL-6 and led to marked improvement of arthritis. CONCLUSION: These results show that depletion of CCR2+ monocytes may prove to be a therapeutic option in inflammatory arthritis, as long as the dose-dependent proinflammatory effects of CCR2 mAb are taken into account.

In vitro and in vivo properties of a dimeric bispecific single-chain antibody IgG-fusion protein for depletion of CCR2+ target cells in mice.

The chemokine receptor CCR2 is highly expressed on leukocytes in several inflammatory diseases of both mice and men. Apart from blockade of CCR2 to prevent chemokine-dependent cell migration, depletion of CCR2(+) cells might be a promising strategy for treatment of inflammatory diseases. We therefore designed a bispecific antibody construct with the ability to deplete CCR2(+) target cells in vitro and in vivo. The bispecific antibody construct consists of two single-chain antibody variable fragments (scFv) - one recognizing murine CD3epsilon and the other recognizing murine CCR2 - joined by a short linker and fused to a modified hinge region and the C(H)2 and C(H)3 domains of murine IgG1 for dimerization. The protein was expressed in mammalian cells and purified via its C-terminal histidine tail. In vitro this construct leads to efficient antigen-specific and costimulation-independent activation of T cells and strong lysis of CCR2(+) target cells. In vivo the construct induces an almost complete depletion of CCR2(+)CD11b(+) monocytes from the peripheral blood and spleens of BALB/c mice within 24 h. This recombinant protein construct is a dimeric, bispecific antibody with markedly improved serum levels compared to conventional bispecific single-chain antibodies and the ability to deplete CCR2(+)CD11b(+) monocytes in vivo.

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.

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 approximately 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.

Post-translational and cell type-specific regulation of CXCR4 expression by cytokines.

We have investigated the regulation and function of the chemokine receptor CXCR4 on neutrophils. CXCR4 is hardly detectable on neutrophils in the peripheral blood. However, overnight culture strongly up-regulates CXCR4 expression on the cell surface. The functional activity of CXCR4 on cultured neutrophils was confirmed by stromal cell-derived factor (SDF)-induced migration and up-regulation of the integrins CD11b and CD11c. CXCR4 surface expression on neutrophils but not on lymphocytes and monocytes is rapidly down-regulated after stimulation with TNF-alpha and IFN-gamma, resulting in significantly decreased SDF-induced functional responses of neutrophils. In contrast to surface expression, CXCR4 mRNA expression was several-fold increased in cytokine-stimulated neutrophils, suggesting a post-translational regulation. By confocal microscopy we demonstrate that CXCR4 is internalized after stimulation with TNF-alpha and IFN-gamma. Tauhe down-modulation of CXCR4 surface expression in response to TNF-alpha and IFN-gamma was fully reversible after cytokine removal. Further, CXCR4 down-modulation could be completely blocked by hypertonic sucrose and significantly reduced by chlorpromazine indicating the involvement of clathrin-coated pits. Internalization of CXCR4 by cytokines in a cell type-specific manner is a novel and functionally important mechanism of chemokine receptor regulation.

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.

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

Wegener's 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.

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.