C-C chemokine receptor type 2 mediates glomerular injury and interstitial fibrosis in focal segmental glomerulosclerosis.

BACKGROUND: Glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of chronic kidney injury leading to end-stage renal disease. Inflammatory mechanisms contribute to glomerular and interstitial scarring, including chemokine-mediated recruitment of leucocytes. In particular, accumulation of C-C chemokine receptor type 2 (CCR2)-expressing macrophages promotes renal injury and fibrotic remodelling in diseases like glomerulonephritis and diabetic nephropathy. The functional role of CCR2 in the initiation and progression of primary glomerulosclerosis induced by podocyte injury remains to be characterized. METHODS: We analysed glomerular expression of CCR2 and its chemokine ligand C-C motif chemokine ligand 2 (CCL2) in human focal segmental glomerulosclerosis (FSGS). Additionally, CCL2 expression was determined in stimulated murine glomeruli and glomerular cells in vitro. To explore pro-inflammatory and profibrotic functions of CCR2 we induced adriamycin nephropathy, a murine model of FSGS, in BALB/c wild-type and Ccr2-deficient mice. RESULTS: Glomerular expression of CCR2 and CCL2 significantly increased in human FSGS. In adriamycin-induced FSGS, progressive glomerular scarring and reduced glomerular nephrin expression was paralleled by induced glomerular expression of CCL2. Adriamycin exposure stimulated secretion of CCL2 and tumour necrosis factor-α (TNF) in isolated glomeruli and mesangial cells and CCL2 in parietal epithelial cells. In addition, TNF induced CCL2 expression in all glomerular cell populations, most prominently in podocytes. In vivo, Ccr2-deficient mice with adriamycin nephropathy showed reduced injury, macrophage and fibrocyte infiltration and inflammation in glomeruli and the tubulointerstitium. Importantly, glomerulosclerosis and tubulointerstitial fibrosis were significantly ameliorated. CONCLUSIONS: Our data indicate that CCR2 is an important mediator of glomerular injury and progression of FSGS. CCR2- targeting therapies may represent a novel approach for its treatment.

Ly6Chigh Monocytes Oscillate in the Heart During Homeostasis and After Myocardial Infarction-Brief Report.

OBJECTIVE: Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. APPROACH AND RESULTS: Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6Chigh monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6Chigh monocytes were CCR2high, whereas only a minority was CCR1high and CCR5high. We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6Chigh monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6Chigh monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6Chigh monocyte infiltration after ZT13 MI but not ZT5 MI. CONCLUSIONS: CCR2 surface expression on Ly6Chigh monocytes changes in a time-of-day-dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.

Chemokine (C-C Motif) receptor 1 is required for efficient recruitment of neutrophils during respiratory infection with modified vaccinia virus Ankara.

UNLABELLED: Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1(-/-) mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE: Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

C-C motif chemokine CCL3 and canonical neutrophil attractants promote neutrophil extravasation through common and distinct mechanisms.

Initial observations suggested that C-C motif chemokines exclusively mediate chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremaster muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1α) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1(-/-)) or 5 (Ccr5(-/-)). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3Kγ-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of ß(2) integrins, of α(4) integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils.

Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells.

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.

Rhoh deficiency reduces peripheral T-cell function and attenuates allogenic transplant rejection.

Rhoh is a hematopoietic system-specific GTPase. Rhoh-deficient T cells have been shown to have a defect in TCR signaling manifested during their thymic development. Our aims were to investigate the phenotype of peripheral Rhoh-deficient T cells and to explore in vivo the potential benefit of Rhoh deficiency in a clinically relevant situation, in which T-cell inhibition is desirable. In murine allogenic kidney transplantation, Rhoh deficiency caused a significant 75% reduction of acute and chronic transplant rejection accompanied by 75% lower alloantigen-specific antibody levels and significantly better graft function. This effect was independent of the lower T-cell numbers in Rhoh-deficient recipients, because injection of equal numbers of Rhoh-deficient or control T cells into kidney transplanted mice with SCID led again to a significant 60% reduction of rejection. Mixed lymphocyte reaction revealed that the weaker alloreactivity was associated with a 85% lower cytotoxicity and a 50-80% lower cytokine release in Rhoh-deficient T cells without an influence on the secretion itself. Antigen uptake and presentation in DC were unaffected by Rhoh deficiency. These findings stress the importance of Rhoh for the function of peripheral T cells.

Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long-term renal allograft outcome.

The chemokine (C-C motif) receptor 5 (CCR5) has been implicated in experimental and clinical allograft rejection. To dissect the function of CCR5 in acute and chronic renal allograft rejection, bilaterally nephrectomized WT and Ccr5-/- C57BL/6 mice were used as recipients of WT BALB/c renal allografts and analyzed 7 and 42 days after transplantation. Lesion scores (glomerular damage, vascular rejection, tubulointerstitial inflammation) and numbers of CD4+, CD8+, CD11c+ and alpha smooth muscle actin (alphaSMA)+ cells were reduced in allografts from Ccr5-/- recipients during the chronic phase. Increasing creatinine levels indicated deterioration of allograft function over time. While mRNA expression of Th1-associated markers decreased between 7 and 42 days, Th2-associated markers increased. Markers for alternatively activated macrophages (arginase 1, chitinase 3-like 3, resistin-like alpha, mannose receptor, C type 1), were strongly upregulated (mRNA and/or protein level) only in allografts from Ccr5-/- recipients at 42 days. Ccr5 deficiency shifted intragraft immune responses during the chronic phase towards the Th2 type and led to accumulation of alternatively activated macrophages. Additionally, splenocytes from unchallenged Ccr5-/- mice showed significantly increased arginase 1 and mannose receptor 1 mRNA levels, suggesting constitutive alternative activation of splenic macrophages. We conclude that Ccr5 deficiency favors alternative macrophage activation. This finding may be relevant for other inflammatory diseases that involve macrophage activation and may also influence future therapeutic strategies targeting CCR5.

A simplified method for bronchoalveolar lavage in mice by orotracheal intubation avoiding tracheotomy.

Bronchoalveolar lavage (BAL) represents an important method to sample immune cells and soluble substances from the lungs of humans and animals suffering from respiratory disease. The mouse is the most commonly used model organism to study lung disease. Performing BAL in mice is difficult due to their small size and the currently used method requires tracheotomy, a complex and time-consuming procedure. Here, we describe a simple alternative procedure that avoids this step. To perform the BAL, a rigid, olive tip cannula is inserted from the mouth into the trachea under visual inspection. This novel method requires minimal training, is simple, fast, inexpensive and should be useful for researchers studying mouse models of human lung disease.

Evolutionary genetics: Ambiguous role of CCR5 in Y. pestis infection.

Mecsas and colleagues suggest that a deficiency in the chemokine receptor CCR5 in humans is unlikely to confer protection against plague, based on their study of Yersinia pestis infection in Ccr5-deficient mice. They were testing the hypothesis that a mutation in the CCR5 gene, frequently found in Caucasians, may have been selected for in the past because it provided protection against (bubonic) plague; the mutation, called CCR5Delta32, is characterized by a 32-base-pair deletion. We have also tested this hypothesis by using Y. pestis infection in mice and, in addition, we have done phagocytosis experiments with macrophages from wild-type and Ccr5-deficient mice. Although, like Mecsas et al., we did not see any difference in the survival of the two groups of mice, we did find that there was a significantly reduced uptake of Y. pestis by Ccr5-deficient macrophages in vitro. Our results indicate that the role of Ccr5 in Y. pestis infection may therefore be more complex than previously thought.

Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice.

The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.

CCL2 Is a Vascular Permeability Factor Inducing CCR2-Dependent Endothelial Retraction during Lung Metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.

Ccr5 but not Ccr1 deficiency reduces development of diet-induced atherosclerosis in mice.

OBJECTIVE: Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined. METHODS AND RESULTS: We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E-deficient (ApoE-/-) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE-/- Ccr5-/- mice, ApoE-/- Ccr1-/- mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE-/- Ccr5-/- mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-gamma content were increased in ApoE-/- Ccr1-/- mice. CONCLUSIONS: Genetic deletion of Ccr5 but not Ccr1 in ApoE-/- mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.

Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue.

Leukocyte infiltration of reperfused tissue is a key event in the pathogenesis of ischemia-reperfusion. However, the role of chemokine receptors Ccr1, Ccr2, and Ccr5 for each single step of the postischemic recruitment process of leukocytes has not yet been characterized. Leukocyte rolling, firm adherence, transendothelial, and extravascular migration were analyzed in the cremaster muscle of anaesthetized C57BL/6 mice using near-infrared reflected light oblique transillumination microscopy. Prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min after onset of reperfusion, migration parameters were determined in wild-type, Ccr1-/-, Ccr2-/-, and Ccr5-/- mice. Sham-operated wild-type mice without ischemia were used as controls. No differences were detected in numbers of rolling leukocytes among groups. In contrast, the number of firmly adherent leukocytes was increased significantly in wild-type mice as compared with sham-operated mice throughout the entire reperfusion phase. Already after 5 min of reperfusion, this increase was reduced significantly in Ccr1-/- and Ccr5-/- mice, whereas only in Ccr2-/- mice, was adherence attenuated significantly at 120 min after onset of reperfusion. Furthermore, after 120 min of reperfusion, the number of transmigrated leukocytes (>80% Ly-6G+ neutrophils) was elevated in wild-type mice as compared with sham-operated animals. This elevation was significantly lower in Ccr1-/-, Ccr2-/-, and Ccr5-/- mice. Leukocyte extravascular migration distances were comparable among groups. In conclusion, these in vivo data demonstrate that Ccr1, Ccr2, and Ccr5 mediate the postischemic recruitment of neutrophils through effects on intravascular adherence and subsequent transmigration.

Phenotyping renal leukocyte subsets by four-color flow cytometry: characterization of chemokine receptor expression.

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune (obstructive nephropathy) and immune-mediated (lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8+ cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets.

Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.

Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit(+) erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45(+) c-kit(lo) CX(3)CR1(-) immature (A1) cells and matured into CD45(+) c-kit(-) CX(3)CR1(+) (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.

Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency.

CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Valpha14-Jalpha18 gene segments in mice and Valpha24-Jalpha18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4(+)/CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vbeta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

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.

Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.

The inflammatory response is characterized by the induction (or repression) of hundreds of genes. The activity of many of these genes is controlled by MAPKs and the IkappaB kinase-NFkappaB pathway. To reveal the effects of blocking these pathways simultaneously, fibroblasts were infected with retroviruses encoding TAK1K63W, an inactive mutant of the protein kinase TAK1. Expression of this protein inhibited tumor necrosis factor (TNF)-induced activation of NFkappaB, JNK, and p38 MAPK and sensitized the cells to TNF-induced apoptosis. 23 different microarray experiments were used to analyze the expression of >7000 genes in these cells. We identified 518 genes that were regulated by TNF in both TAK1K63W-expressing cells and control cells, 37 genes induced by TNF only when TAK1K63W was present, and 48 TNF-induced genes that were suppressed by TAK1K63W. The TNF-inducible genes that were most strongly suppressed by TAK1K63W, ccl2, ccl7, ccl5, cxcl1, cxcl5, cxcl10, saa3, and slpi also had much lower basal levels of expression, indicating that TAK1 also played a role in their normal expression. Chromatin immunoprecipitation studies on four of these genes suggested that inactivation of TAK1 activity led to direct suppression of expression at the transcriptional level because of impaired recruitment of RNA polymerase II to their promoters. ccl2 induction by TNF or interleukin-1 was also suppressed in cells that expressed TAK1 antisense RNA or that were genetically deficient in JNK1/2 or p65 NFkappaB. These data suggest that regulation of the expression of a selected group of inflammation-related genes is funneled through TAK1, making it a potentially useful target for more specific anti-inflammatory drug development.

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice.

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.