A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3 ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV.

Poor myeloid engraftment remains a barrier to experimental use of humanized mice. Focusing primarily on peripheral blood cells, we compared the engraftment profile of NOD-scid-IL2Rγc(-/-) (NSG) mice with that of NSG mice transgenic for human membrane stem cell factor (hu-mSCF mice), NSG mice transgenic for human interleukin (IL)-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and stem cell factor (SGM3 mice). hu-mSCF and SGM3 mice showed enhanced engraftment of human leukocytes compared to NSG mice, and this was reflected in the number of human neutrophils and monocytes present in these strains. Importantly, discrete classical, intermediate, and nonclassical monocyte populations were identifiable in the blood of NSG and hu-mSCF mice, while the nonclassical population was absent in the blood of SGM3 mice. Granulocyte-colony stimulating factor (GCSF) treatment increased the number of blood monocytes in NSG and hu-mSCF mice, and neutrophils in NSG and SGM3 mice; however, this effect appeared to be at least partially dependent on the stem cell donor used to engraft the mice. Furthermore, GCSF treatment resulted in a preferential expansion of nonclassical monocytes in both NSG and hu-mSCF mice. Human tubulointerstitial CD11c(+) cells were present in the kidneys of hu-mSCF mice, while monocytes and neutrophils were identified in the liver of all strains. Bone marrow-derived macrophages prepared from NSG mice were most effective at phagocytosing polystyrene beads. In conclusion, hu-mSCF mice provide the best environment for the generation of human myeloid cells, with GCSF treatment further enhancing peripheral blood human monocyte cell numbers in this strain.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

These data suggest that GCSF, which is raised in patient serum, may play an important role in exacerbating disease in ANCA vasculitis. In addition, GCSF therapy for neutropenia should be used with caution in these patients.

SummaryAntibodies against neutrophil proteins myeloperoxidase (MPO) and proteinase 3 are thought to cause disease in anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. There have been a number of recent developments in the animal models of ANCA vasculitis in both mice and rats. These include models based on an immune response to MPO generated in MPO-deficient mice, with other models using MPO-sufficient mice and rats. In addition, there is a report of the use of humanized mice where immunodeficient mice have been engrafted with human haematopoietic stem cells and injected with patient ANCA. Antibodies to another protein lysosomal-associated protein-2 have been found in patients with ANCA vasculitis, and evidence from a rat model suggests that they are also pathogenic. These models all have advantages and disadvantages, which are discussed. We also consider what these models have taught us about the pathogenesis of ANCA vasculitis. Experiments using genetically modified mice and pharmacological inhibition have given insights into disease mechanisms and have identified potential therapeutic targets. Toll-like receptor stimulation modifies disease by acting both at the level of tissue injury and in the generation of the autoimmune response. Complement is also potentially important with data to support the role of the alternative pathway and C5a in particular. Intracellular pathways have been examined, with a role showing p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase g. Serine proteases are now known to contribute to disease by release of interleukin-1b in ANCA-activated neutrophils and monocytes. Other potential therapies studied in these models include the use of bortezemib and strategies to modify antibody glycosylation.

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To explore the role of antigen-specific CD4؉ T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1 ؊/؊ mice with CD4 ؉ T cells from OT2 ؋ RAG1 ؊/؊ mice. These OT2 ؋ RAG1 ؊/؊ mice have a transgenic T-cell receptor specific for this peptide. When CD4؉ T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T H 1 and T H 17 cells, using Fab 2 fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 ؋ RAG1 ؊/؊ mice were polarized in vitro, and T H 1 or T H 17 cell lines were injected into mice that were also given peptideconjugated Fab 2 or unconjugated Fab 2 control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T H 17 cells and peptide-conjugated Fab 2 but in none of the other three groups. These results suggest that T H 17 but not T H 1 cells can induce crescentic glomerulonephritis.