Control mice received the same amount of isotype

Control mice received the same amount of isotype. CSF2 receptor subunit beta (CSF2rb)-deficient mice were used. Urinary monocytes and CSF2 were quantified and kidney expression was analysed. CSF2-blocking antibody was used in the nephrotoxic nephritis (NTN) model. Results Compared with WT mice, CCR2?/? chimeric mice showed reduced circulating CM and were guarded from NCGN. C/EBP?/? chimeric mice lacked NCM but developed NCGN much like WT chimeric mice. Kidney and urinary Ibrutinib Racemate CSF2 were upregulated in AAV mice. CSF2 increased the ability of ANCA-stimulated monocytes to generate interleukin-1 and to promote TH17 effector cell polarisation. CSF2rb?/? chimeric mice harboured reduced numbers of kidney TH17 cells and were guarded from NCGN. CSF2 neutralisation reduced renal damage in the NTN model. Finally, patients with active AAV displayed increased urinary CM figures, CSF2 levels and expression of GM-CSF in infiltrating renal cells. Conclusions CMs but not NCMs are important for inducing kidney damage in AAV. CSF2 is usually a crucial pathological factor by modulating monocyte proinflammatory functions and thereby TH17 cell polarisation. mRNA expression was strongly upregulated, confirming that CSF2 is usually locally expressed (physique 3D). Finally, we found a positive correlation between CSF2 protein level and the percentage of renal crescents (physique 3E). These results indicate that CSF2 is usually strongly upregulated in kidneys of AAV mice. We next explored the hypothesis that CSF2 is usually a critical mediator of AAV. Open in a separate window Physique 3 CSF2 expression is usually increased in kidneys from mice with anti-MPO-induced NCGN. (A) Immunofluorescence images show strong CSF2 expression in kidney sections from mice with AAV compared with Ctrl mice. A representative image at magnification 40 is usually shown for each group. 4′,6-Diamidin-2-phenylindol (DAPI) was used to stain nuclei (blue). (B) CSF2 level in urine by ELISA is usually increased in AAV mice compared with Ctrl mice. (C) CSF2 level by ELISA in renal lysates is usually increased in AAV mice compared with Ctrl mice. (D) mRNA by RT-PCR expression in kidney lysates is usually increased in AAV mice compared with Ctrl mice. (E) Correlation between the percentage of crescents and the amount of renal CSF2. **P 0.01, ***P 0.001. AAV, antineutrophil cytoplasmic antibody-associated vasculitis; AU, arbitrary unit; CSF2, colony-stimulating factor-2; Ctrl, Ibrutinib Racemate control; MPO, myeloperoxidase; NCGN, necrotising crescentic glomerulonephritis. To study the mechanistic role of CSF2 in AAV, MPO-deficient mice were immunised with murine MPO, irradiated and transplanted with BM cells from either C57BL/6J WT (WT) or CSF2rb-deficient (CSF2rb?/?) animals to generate MPO?/? CSF2rb+/+ mice (WT) and MPO?/? CSF2rb?/? chimeric (CSF2rb?/?) mice, respectively (physique 4A). Rabbit polyclonal to AADACL3 Anti-MPO titres and blood cell counts were comparable in both animal groups (online supplemental physique 4). Renal histology revealed strongly reduced crescent formation in CSF2rb?/? chimeric mice compared with the WT group (physique 4B). Urine analysis did not show group differences by dipstick analysis, albumin ELISA or NGAL ELISA (physique 4CCE). Amazingly, quantification of kidney-infiltrating leucocytes revealed no difference in either neutrophil, CM or in NCM influx (physique 4F and online supplemental physique 3). Ibrutinib Racemate These data demonstrate that CSF2 and its interaction with its CSF2rb receptor on myeloid cells are essential for crescent formation and that the effect is not mediated by reduced myeloid cell influx. Thus, we next explored CSF2 effects on monocyte functions in the presence of MPOCANCA. Ibrutinib Racemate Open in a separate window Physique 4 Csf2rb?/? chimeric mice are guarded from anti-MPO-induced NCGN. (A) Experimental settings describing the induction of NCGN. (B) Csf2rb?/? chimeric mice developed less renal damage compared with WT chimeric mice with a reduction of crescentic and necrotic glomeruli. A representative image of a kidney section stained with PAS at high magnification (40) is usually shown for each group. (C) Leu, Prot and Ery by urine dipstick are comparable in both groups. (D) Albuminuria and (E) NGAL urine levels by ELISA were comparable in both groups. (F) Renal infiltration of.