Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model
Authors: Stephen P. McAdoo, Maria Prendecki, Anisha Tanna, Tejal Bhatt, Gurjeet Bhangal, John McDaid, Esteban S. Masuda, H. Terence Cook, Frederick W.K. Tam, Charles D. Pusey
Journal: Kidney International (2020)

The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV. The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV. Translational StatementThe anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides are a group of rare diseases characterized by the inflammation of blood vessels, crescentic glomerulonephritis, and lung hemorrhage. Current treatments are not completely effective, as many patients develop refractory or relapsing disease, or treatment-related toxicities. This article investigates the role of spleen tyrosine kinase (SYK), an immunoreceptor-associated signaling protein, in the pathogenesis of ANCA vasculitis and its potential as a therapeutic target. In a preclinical model, SYK upregulation was identified at sites of renal and pulmonary inflammation, and treatment with an SYK inhibitor was completely effective in treating established lung and kidney disease, without hematological toxicity. SYK expression is also confirmed in glomerular inflammatory lesions in human ANCA-associated vasculitides. Clinical studies of SYK inhibition in IgA nephropathy are ongoing, and these novel data suggest that this approach should also be considered in ANCA vasculitis. The anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides are a group of rare diseases characterized by the inflammation of blood vessels, crescentic glomerulonephritis, and lung hemorrhage. Current treatments are not completely effective, as many patients develop refractory or relapsing disease, or treatment-related toxicities. This article investigates the role of spleen tyrosine kinase (SYK), an immunoreceptor-associated signaling protein, in the pathogenesis of ANCA vasculitis and its potential as a therapeutic target. In a preclinical model, SYK upregulation was identified at sites of renal and pulmonary inflammation, and treatment with an SYK inhibitor was completely effective in treating established lung and kidney disease, without hematological toxicity. SYK expression is also confirmed in glomerular inflammatory lesions in human ANCA-associated vasculitides. Clinical studies of SYK inhibition in IgA nephropathy are ongoing, and these novel data suggest that this approach should also be considered in ANCA vasculitis. Spleen tyrosine kinase (SYK) is a cytosolic protein tyrosine kinase that is expressed in most leucocyte populations, where it has diverse immune functions. It has a well characterized role in mediating signaling from classical immunoreceptors such as the B-cell receptor and the Fc receptor, and also from some integrins and C-type lectins.1Mocsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: a crucial player in diverse biological functions.Nat Rev Immunol. 2010; 10: 387-402Crossref PubMed Scopus (745) Google Scholar Targeting SYK has thus emerged as a potential treatment approach for a variety of immune and inflammatory diseases,2Geahlen R.L. Getting Syk: spleen tyrosine kinase as a therapeutic target.Trends Pharmacol Sci. 2014; 35: 414-422Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar and a number of specific SYK inhibitors are in development.3Thorarensen A. Kaila N. New spleen tyrosine kinase inhibitors: patent applications published during 2011-2013.Pharm Pat Anal. 2014; 3: 523-541Crossref PubMed Scopus (6) Google Scholar We have previously shown that fostamatinib, a small molecule kinase inhibitor with selectivity for SYK, is an effective treatment in experimental models of immune-complex glomerulonephritis.4Smith J. McDaid J.P. Bhangal G. et al.A spleen tyrosine kinase inhibitor reduces the severity of established glomerulonephritis.J Am Soc Nephrol. 2010; 21: 231-236Crossref PubMed Scopus (64) Google Scholar,5McAdoo S.P. Reynolds J. Bhangal G. et al.Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.J Am Soc Nephrol. 2014; 25: 2291-2302Crossref PubMed Scopus (37) Google Scholar SYK also mediates proinflammatory responses induced by IgA1 derived from patients with IgA nephropathy,6Kim M.J. McDaid J.P. McAdoo S.P. et al.Spleen tyrosine kinase is important in the production of proinflammatory cytokines and cell proliferation in human mesangial cells following stimulation with IgA1 isolated from IgA nephropathy patients.J Immunol. 2012; 189: 3751-3758Crossref PubMed Scopus (51) Google Scholar and full results from a Phase II clinical study assessing SYK inhibition with fostamatinib in proliferative IgA nephropathy are awaited (NCT02112838). The anti-neutrophil cytoplasm antibody (ANCA)–associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotizing inflammation of small blood vessels and pauci-immune crescentic glomerulonephritis, in which ANCA are thought to play a directly pathogenic role.7Jennette J.C. Falk R.J. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease.Nat Rev Rheumatol. 2014; 10: 463-473Crossref PubMed Scopus (242) Google Scholar It has previously been shown that SYK activation occurs following ANCA-induced neutrophil activation,8Hewins P. Williams J.M. Wakelam M.J. Savage C.O. Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18.J Am Soc Nephrol. 2004; 15: 796-808Crossref PubMed Scopus (57) Google Scholar suggesting that SYK inhibition may be a potential therapeutic approach in AAV, though in vivo data are lacking. Here, we have investigated the effect of SYK inhibition in an experimental model of myeloperoxidase (MPO)-ANCA–induced systemic vasculitis (experimental autoimmune vasculitis [EAV]) that was developed in our laboratory.9Little M.A. Smyth C.L. Yadav R. et al.Antineutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.Blood. 2005; 106: 2050-2058Crossref PubMed Scopus (225) Google Scholar,10Little M.A. Smyth L. Salama A.D. et al.Experimental autoimmune vasculitis: an animal model of anti-neutrophil cytoplasmic autoantibody-associated systemic vasculitis.AmJ Pathol. 2009; 174: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar It is characterized by ANCA-induced enhancement of leucocyte–endothelial cell interactions and the development of both alveolar hemorrhage and necrotizing glomerulonephritis by 4 weeks after disease induction. In contrast to our previous studies in immune-complex glomerulonephritis, this model has a distinct pauci-immune mechanism of tissue injury, similar to that in AAV. We performed immunohistochemical staining for total (T)- and activated (i.e., phosphorylated [P]-) SYK. In healthy rat lung tissue, this analysis demonstrated that T-SYK was expressed in large airway cuboidal epithelial cells and associated lymphoid tissue (Figure 1a), consistent with previously described patterns of SYK expression in hematopoetic and some epithelial cell types.11Ulanova M. Puttagunta L. Marcet-Palacios M. et al.Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells.Am J Physiol Lung Cell Mol Physiol. 2005; 288: L497-L507Crossref PubMed Scopus (86) Google Scholar There was minimal T-SYK detection in alveolar squamous epithelium (Figure 1b). In lung tissue taken from animals 6 weeks after induction of EAV (Figure 1c), alveolar lumens were consolidated with erythrocytes, consistent with the development of lung hemorrhage. In addition, large mononuclear cells with cytoplasmic T-SYK expression were seen. Staining of serial sections identified a population of mononuclear cells positive for ED-1 (the rat homologue of CD68), T-SYK, and P-SYK (Figure 1d–f, respectively) in diseased lung, and dual staining confirmed T-SYK expression in ED-1+ve cells (Figure 1g), suggesting an infiltrating population of monocytes/macrophages expressing activated SYK at sites of alveolar hemorrhage. A small number of T-SYK+ve ED-1-ve cells were also observed, suggesting additional cell populations that express SYK in this model, potentially lymphocytes or neutrophils. As previously described, in normal rat kidney tissue, T-SYK was detected in distal tubular epithelial cells but not in normal glomeruli. In kidney tissue taken from animals with established EAV, T-SYK was detected within inflamed glomeruli, particularly within areas of endocapillary proliferation and crescent formation, whereas there was no SYK detection in unaffected glomeruli (Figure 1h). Upregulation of SYK expression was confirmed by the finding of increased SYK mRNA in diseased renal tissue, by both in situ hybridization (Figure 1i and j) and by real-time quantitative polymerase chain reaction (RT-qPCR; Figure 1k). Dual staining showed co-localization of T-SYK and ED-1+ve cells within inflammatory glomerular lesions (Figure 1l). As observed in lung tissue, a small population of T-SYK+ve ED-1–ve cells was seen in some glomeruli. Staining of serial sections suggested that P-SYK localizes to infiltrating ED-1+ve monocytes/macrophages in and around glomeruli (Figure 1m and n). P-SYK staining in kidney sections was both cytoplasmic and nuclear; SYK is known to have a nuclear localization signal in B lymphocytes,12Zhou F. Hu J. Ma H. et al.Nucleocytoplasmic trafficking of the Syk protein tyrosine kinase.Mol Cell Biol. 2006; 26: 3478-3491Crossref PubMed Scopus (56) Google Scholar and we have previously described nuclear staining for P-SYK in human kidney disease.13McAdoo S.P. Bhangal G. Page T. et al.Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.Kidney Int. 2015; 88: 52-60Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In order to confirm SYK phosphorylation in EAV kidney tissue, we performed immunoblotting for P-SYK in kidney cortex, and showed upregulation compared with control kidney tissue (Figure 1o). These immunohistochemical data suggest that SYK is expressed and activated at sites of renal and pulmonary inflammation in EAV. We therefore sought to examine the effect of SYK inhibition using fostamatinib in vivo. EAV was induced in Wistar Kyoto rats (n = 8 per group) by immunization with recombinant human MPO in complete Freunds adjuvant (CFA). After onset of vasculitis was confirmed by the development of proteinuria and hematuria 4 weeks after disease induction, animals were treated with fostamatinib of 30 mg/kg, 20 mg/kg, or vehicle preparation by twice daily oral gavage, and assessed for disease severity at 6 weeks, after a total period of 14 days of treatment with drug or vehicle. Control animals were immunized with CFA alone and followed until 6 weeks (n = 4). To obtain comparative lung and renal histology at treatment initiation (week 4), an additional group of animals was used after induction of EAV (n = 6). Four weeks after induction of EAV, the majority of animals had macroscopic evidence of lung hemorrhage, and in vehicle-treated animals, this progressed to severe lung hemorrhage in all animals by week 6 (Figure 2a). Fostamatinib treatment resulted in a dose-dependent reduction in the severity of pulmonary hemorrhage, as determined by macroscopic assessment of lung tissue at the time of cull (Figure 2a and b; median lung hemorrhage score 3, 1, and O for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P < 0.0001), and by histologic assessment for hemosiderin-laden cells in lung parenchyma (Figure 2c and d; median Perls' stain detection 3.1, 0.5, and 0.2 au for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.009). In addition, there was a dose-dependent reduction in ED-1 positive macrophage infiltration (Figure 2e and f; median 2.0, 0.4, and 0.1 ED-1 cells/field for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P < 0.0001). Notably, treatment with fostamatinib 30 mg/kg led to complete reversal of lung hemorrhage in all rats. Introduction of fostamatinib treatment 4 weeks after disease induction was associated with rapid resolution of urinary abnormalities during the 2-week treatment period. Compared to vehicle-treated animals, there were significant reductions in both hematuria (Figure 3a and b; median dipstick hematuria 3, 0, and 0 for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.002) and proteinuria (Figure 3c and d; median proteinuria 2.8, 0.7, and 0.2 mg/day for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.0004). These improvements were associated with a dose-dependent preservation of excretory renal function (Figure 3e; median serum creatinine 51.5, 45.8, and 45 μmol/l for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.027). Histologic examination of renal tissue at 4 weeks, prior to treatment initiation, confirmed features of early proliferative glomerulonephritis and macrophage infiltration, that by 6 weeks progressed to focal necrotizing and crescentic glomerulonephritis in vehicle-treated animals (Figure 4). There was a dose-dependent reduction in proliferative glomerular lesions (Figure 4a and c; median 12%, 5%, and 0% glomerular abnormalities for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.026) and ED-1+ve monocyte/macrophage glomerular infiltration (Figure 4b and c; median 1.8, 0.3, and 0.2 ED-1+ cells per glomerular cross-section for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.0004). In keeping with reduced glomerular cell number, analysis of intra-renal gene expression (Figure 4d) confirmed a significant reduction in monocyte/macrophage-associated proinflammatory cytokine and enzyme production (monocyte chemoattractant protein–1 [MCP-1], C–C motif chemokine ligand 3 [CCL3], Il-1β, tumor necrosis factor– α [TNFα], matrix metallopeptidase 9 [MMP9]) with fostamatinib treatment, without a significant effect on the expression of T cell–associated cytokines (IL-2, IL-6, IL-4). There was a corresponding decrease in intra-renal SYK expression following fostamatinib treatment.Figure 4Spleen tyrosine kinase (SYK) inhibition improves renal histopathology in experimental autoimmune vasculitis (EAV). (a) Quantification of glomerular abnormalities at treatment initiation (week 4 [W4]) and at 6 weeks after disease induction, with (c) representative photomicrographs demonstrating focal necrotizing glomerulonephritis and crescent formation in vehicle-treated animals and preserved glomerular histology after fostamatinib (Fosta) treatment (periodic acid–Schiff [PAS]–stained sections, original magnification ×400). (b) Quantification of ED-1 (rat homologue of CD68)–positive cells infiltrating glomeruli at treatment initiation (W4) and at 6 weeks after disease induction, showing a dose-dependent reduction with fostamatinib treatment, with representative photomicrographs demonstrating immunoperoxidase staining for ED-1 (b; with hematoxylin counterstain; original magnification ×400). (d) Heat-map indicating changes in intra-renal gene expression following treatment with fostamatinib, as determined by real-time quantitative polymerase chain reaction and expressed as fold-change compared to normal kidney tissue. All data are reported as median ± interquartile range; statistical comparison by Mann-Whitney or Kruskal-Wallis test, with Dunn's post-test comparison to vehicle group (lower indicator); ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.0001. CCL3, C–C motif chemokine ligand 3; CFA, complete Freunds adjuvant; MCP-1, monocyte chemoattractant protein 1; MMP9, matrix metallopeptidase 9; ns, not significant; TNF, tumor necrosis factor. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Short-term fostamatinib treatment did not significantly affect circulating MPO-ANCA levels in this study (Figure 5a and b; median MPO-ANCA titre 6 weeks after disease induction—111, 87, and 99 au for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.19). Nor was there a significant effect on IgG subclass following treatment (Supplementary Figure S1). Direct immunofluorescence for deposited IgG in renal tissue demonstrated a pauci-immune pattern of renal injury and confirmed no difference in glomerular immune deposits between control animals and any treatment group (Figure 5c and d). Fostamatinib treatment did not significantly depress hemoglobin concentrations compared to vehicle-treated animals (Figure 5e). However, there was a mild reduction in peripheral blood white cell count following fostamatinib treatment compared to vehicle-treated animals, consistent with previous reports that have attributed this phenomenon to a margination effect (Figure 5f; median white blood cell count 6 weeks after disease induction—9.2, 6.2, and 6.5 x 109/l for vehicle, 20 mg/kg, and 30 mg/kg, respectively, P = 0.047). However, white blood cell counts were not reduced beyond those of control animals immunized with CFA alone (Figure 5f). Although there was no difference in circulating MPO-ANCA levels after treatment, we observed that SYK inhibition could inhibit MPO-ANCA–induced cellular responses. Myeloid progenitors express high levels of the ANCA antigen MPO,14van der Veen B.S. de Winther M.P. Heeringa P. Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease.Antioxid Redox Signal. 2009; 11: 2899-2937Crossref PubMed Scopus (338) Google Scholar and it has been shown that primed rat bone marrow–derived neutrophils and monocytes produce proinflammatory cytokines following ANCA stimulation.15Kanzaki G. Nagasaka S. Higo S. et al.Impact of anti-glomerular basement membrane antibodies and glomerular neutrophil activation on glomerulonephritis in experimental myeloperoxidase-antineutrophil cytoplasmic antibody vasculitis.Nephrol Dial Transplant. 2016; 31: 574-585Crossref PubMed Scopus (16) Google Scholar We confirmed that EAV serum is reactive to bone marrow mononuclear and polymorphonuclear cells by indirect immunofluorescence (Figure 5g), and that SYK is phosphorylated following stimulation of bone-marrow cells with rat MPO-ANCA (Figure 5h). We then sought to examine effects of SYK inhibition using R406, the active metabolite of fostamatinib, on MPO-ANCA–induced responses in vitro. After TNFα-priming, undifferentiated bone marrow cells produced high levels of MCP-1 following stimulation with rat-derived ANCA-IgG (whereas control IgG had no additional effect). Pretreatment with R406 was associated with a dose-dependent reduction in MCP-1 production (Figure 5i; 62%, 77%, and 75% reduction with 0.2, 1.0, and 2.0 μM R406, respectively, P = 0.0004). There was a similar reduction in production of reactive oxygen species, induced by MPO-ANCA following pretreatment with R406 (Figure 5j). We and others have demonstrated that SYK expression is upregulated, and that SYK is activated, at sites of glomerular inflammation in renal AAV.13McAdoo S.P. Bhangal G. Page T. et al.Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.Kidney Int. 2015; 88: 52-60Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,16Ryan J. Ma F.Y. Han Y. et al.Myeloid cell-mediated renal injury in rapidly progressive glomerulonephritis depends upon spleen tyrosine kinase.J Pathol. 2016; 238: 10-20Crossref PubMed Scopus (13) Google Scholar Our previous studies, however, were conducted using a commercially available rabbit polyclonal antibody that is no longer available. We therefore have validated our previous description of SYK expression in AAGN using an alternative mouse mouse monoclonal antibody directed against human SYK (Figure 6). SYK was detected within inflammatory lesions in AAGN, including areas of glomerular crescent formation (Figure 6a and f), fibrinoid necrosis (Figure 6d), and periglomerular inflammation (H), but not within normal or sclerotic glomeruli (Figure 6b and c), suggesting that it is a feature of active disease. Staining of serial sections indicates significant co-localization of SYK to CD68+ve cells in these inflammatory lesions (Figure 6d–i), and double staining confirms co-expression of SYK and CD68 in cells present within glomerular crescents (Figure 6j). Occasional SYK+ve CD68–ve cells were also observed within the glomerular tuft, similar to the pattern of staining observed in rat tissue, which may represent other cell types, such as neutrophils, that may be present during the course of AAGN. The findings reported here are consistent with our previous work showing that SYK inhibition with fostamatinib is effective in both preventing and treating established crescentic glomerulonephritis in anti–glomerular basement membrane disease models.4Smith J. McDaid J.P. Bhangal G. et al.A spleen tyrosine kinase inhibitor reduces the severity of established glomerulonephritis.J Am Soc Nephrol. 2010; 21: 231-236Crossref PubMed Scopus (64) Google Scholar,5McAdoo S.P. Reynolds J. Bhangal G. et al.Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.J Am Soc Nephrol. 2014; 25: 2291-2302Crossref PubMed Scopus (37) Google Scholar These data additionally suggest that fostamatinib may inhibit ANCA-induced inflammatory responses, and that it may therefore have therapeutic potential in both pauci-immune and immune-complex–mediated glomerular inflammation. In addition, these new data show that the striking treatment effect of fostamatinib can be achieved with significantly reduced doses compared to our previous work in experimental autoimmune glomerulonephritis, and without significant bone marrow toxicity. It has been shown that ANCA-induced SYK activation in neutrophils is mediated by signaling through Fc receptors for IgG (FcγR) and the integrin CD18,8Hewins P. Williams J.M. Wakelam M.J. Savage C.O. Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18.J Am Soc Nephrol. 2004; 15: 796-808Crossref PubMed Scopus (57) Google Scholar and it is likely that disruption of signaling via both these receptors on effector myeloid cells is responsible for the therapeutic effect observed in this study. In particular, both pulmonary and renal disease in this model are characterized by macrophage infiltration, and the unique susceptibility of the WKY rat strain to other forms of experimental glomerulonephritis has been attributed, at least in part, to genetic polymorphisms that control monocyte/macrophage activation, including those in FcγRIII genes.17Aitman T.J. Dong R. Vyse T.J. et al.Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans.Nature. 2006; 439: 851-855Crossref PubMed Scopus (554) Google Scholar, 18Behmoaras J. Bhangal G. Smith J. et al.Jund is a determinant of macrophage activation and is associated with glomerulonephritis susceptibility.Nat Genet. 2008; 40: 553-559Crossref PubMed Scopus (70) Google Scholar, 19Behmoaras J. Smith J. D'Souza Z. et al.Genetic loci modulate macrophage activity and glomerular damage in experimental glomerulonephritis.J Am Soc Nephrol. 2010; 21: 1136-1144Crossref PubMed Scopus (20) Google Scholar In humans, these cells are known to express ANCA autoantigens, including MPO, and they produce proinflammatory mediators following ANCA-stimulation,20Hattar K. Bickenbach A. Csernok E. et al.Wegener's granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release-role of autocrine cell activation.J Leukocyte Biol. 2002; 71: 996-1004PubMed Google Scholar,21Weidner S. Neupert W. Goppelt-Struebe M. Rupprecht H.D. Antineutrophil cytoplasmic antibodies induce human monocytes to produce oxygen radicals in vitro.Arthritis Rheum. 2001; 44: 1698-1706Crossref PubMed Scopus (69) Google Scholar which is dependent upon FcγR cross-linking22Ralston D.R. Marsh C.B. Lowe M.P. Wewers M.D. Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release. Role of surface proteinase-3, alpha1-antitrypsin, and Fcgamma receptors.J Clin Invest. 1997; 100: 1416-1424Crossref PubMed Scopus (170) Google Scholar; thus, it is possible that inhibition of SYK-mediated functions in both neutrophils and monocytes is responsible for the reduction in disease severity observed with fostamatinib treatment. In keeping with a role for SYK activity in monocytes/macrophages in mediating disease, our current and previous immunohistochemical analyses suggest that these cell types are the predominant glomerular leucocyte expressing SYK in both EAV and human AAV, although other cell types, including neutrophils, may be involved. We also observed a significant reduction in macrophage-associated gene transcripts in renal tissue following fostamatinib treatment, including IL-1β, MMP9 and MCP-1. MCP-1 is known to contribute to the pathogenesis of experimental glomerulonephritis and it is present in the glomeruli of patients with AAGN.23Lloyd C.M. Minto A.W. Dorf M.E. et al.RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis.J ExpMed. 1997; 185: 1371-1380Crossref Scopus (425) Google Scholar, 24Wada T. Yokoyama H. Furuichi K. et al.Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).FASEB J. 1996; 10: 1418-1425Crossref PubMed Scopus (178) Google Scholar, 25Rovin B.H. Rumancik M. Tan L. Dickerson J. Glomerular expression of monocyte chemoattractant protein-1 in experimental and human glomerulonephritis.Lab invest. 1994; 71: 536-542PubMed Google Scholar Indeed, urinary levels of this cytokine correlate with disease activity in AAV,26Tam F.W. Sanders J.S. George A. et al.Urinary monocyte chemoattractant protein-1 (MCP-1) is a marker of active renal vasculitis.Nephrol Dial Transplant. 2004; 19: 2761-2768Crossref PubMed Scopus (79) Google Scholar and urinary MCP-1 has been proposed as a novel predictive biomarker in patients,27Moran S.M. Monach P.A. Zgaga L. et al.Urinary soluble CD163 and monocyte chemoattractant protein-1 in the identification of subtle renal flare in anti-neutrophil cytoplasmic antibody-associated vasculitis.Nephrol Dial Transplant. 2020; 35: 283-291PubMed Google Scholar perhaps supporting potential clinical application of SYK inhibition in AAV. It is notable that we did not observe a significant effect on circulating MPO-ANCA levels following fostamatinib treatment in this study, in contrast to our previous findings in experimental autoimmune glomerulonephritis,