Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis

Abstract

Antineutrophil cytoplasm antibody (ANCA)–associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14+ monocytes or CD16+ neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis. Antineutrophil cytoplasm antibody (ANCA)–associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14+ monocytes or CD16+ neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis. Antineutrophil cytoplasm antibody (ANCA)–associated vasculitis is characterized by small-vessel inflammation and immune reactivity against the neutrophil and monocyte components proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA activate neutrophils and monocytes in vitro,1.Falk R.J. Terrell R.S. Charles L.A. et al.Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro.Proc Nat Acad Sci USA. 1990; 87: 4115-4119Crossref PubMed Scopus (1122) Google Scholar and are capable of inducing disease in certain animal models.2.Xiao H. Heeringa P. Hu P. et al.Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (997) Google Scholar,3.Little 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 (239) Google Scholar ANCA titers do not closely follow disease activity, as patients in clinical remission frequently have persistent ANCA positivity, and serial measurements have been demonstrated to be of limited use during disease remission in guiding individualized patient treatment.4.Tomasson G. Grayson P.C. Mahr A.D. et al.Value of ANCA measurements during remission to predict a relapse of ANCA-associated vasculitis—a meta-analysis.Rheumatology. 2012; 51: 100-109Crossref PubMed Scopus (234) Google Scholar A recently demonstrated transcriptional profiling of purified CD8+ T cells has identified a subset of genes associated with a poor prognosis, and is therefore a potential biomarker in ANCA–associated vasculitis (AAV).5.McKinney E.F. Lyons P.A. Carr E.J. et al.A CD8+ T cell transcription signature predicts prognosis in autoimmune disease.Nat Med. 2010; 16: 586-591Crossref PubMed Scopus (283) Google Scholar Glomerulonephritis, a common feature of AAV, is pauci-immune and characterized by infiltration of glomerular monocytes/macrophages and T cells.6.Ruth A.J. Kitching A.R. Kwan R.Y. et al.Anti-neutrophil cytoplasmic antibodies and effector CD4+ cells play nonredundant roles in anti-myeloperoxidase crescentic glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1940-1949Crossref PubMed Scopus (128) Google Scholar Importantly, the extent of glomerular inflammation on renal biopsy predicts renal outcome.7.Berden A.E. Ferrario F. Hagen E.C. et al.Histopathologic classification of ANCA-associated glomerulonephritis.J Am Soc Nephrol. 2010; 21: 1628-1636Crossref PubMed Scopus (535) Google Scholar The more inflamed the glomeruli, with predominance of crescents or focal necrosis, the better the renal outcome compared with mixed and sclerotic lesions, suggesting that these lesions respond the most to therapy. However, there are currently no noninvasive means of monitoring the activity of those proinflammatory glomerular-infiltrating leukocytes. Calprotectin (S100A8/S100A9, MRP8/14) is a heterodimer complex of 2 intracellular calcium-binding proteins S100A8 (MRP 8) and S100A9 (MRP 14). These proteins are expressed by neutrophils, monocytes, and early differentiated macrophages.8.Edgeworth J. Freemont P. Hogg N. Ionomycin-regulated phosphorylation of the myeloid calcium-binding protein p14.Nature. 1989; 342: 189-192Crossref PubMed Scopus (86) Google Scholar,9.Nacken W. Roth J. Sorg C. et al.S100A9/S100A8: Myeloid representatives of the S100 protein family as prominent players in innate immunity.Microsc Res Tech. 2003; 60: 569-580Crossref PubMed Scopus (288) Google Scholar Phagocytes have been demonstrated to release this complex after their interaction with activated, inflamed endothelium.10.Frosch M. Strey A. Vogl T. et al.Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis.Arthritis Rheum. 2000; 43: 628-637Crossref PubMed Scopus (321) Google Scholar Once secreted, the complex binds to activated endothelial cells via heparan sulfate proteogylcans,11.Robinson M.J. Tessier P. Poulsom R. et al.The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate glycosaminoglycans on endothelial cells.J Biol Chem. 2002; 277: 3658-3665Crossref PubMed Scopus (190) Google Scholar,12.Srikrishna G. Panneerselvam K. Westphal V. et al.Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.J Immunol. 2001; 166: 4678-4688Crossref PubMed Scopus (131) Google Scholar and induces proinflammatory effects, such as increased secretion of IL8, upregulation of ICAM-1, and further recruitment of leukocytes. In addition, the interaction results in impairment of the endothelial monolayer integrity and induction of both caspase-dependent and caspase-independent cell death mechanisms resulting in both apoptosis and necrosis.13.Viemann D. Strey A. Janning A. et al.Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells.Blood. 2005; 105: 2955-2962Crossref PubMed Scopus (247) Google Scholar,14.Viemann D. Barczyk K. Vogl T. et al.MRP8/MRP14 impairs endothelial integrity and induces a caspase-dependent and -independent cell death program.Blood. 2007; 109: 2453-2460Crossref PubMed Scopus (114) Google Scholar These characteristics make calprotectin a potentially important mediator of tissue damage in AAV, in which endothelial activation and vascular damage are prominent. A previous study demonstrated the formation of MRP8/MRP14 (calprotectin) complexes on glomerular infiltrating macrophages, which correlated with the severity of different forms of glomerulonephritis,15.Frosch M. Vogl T. Waldherr R. et al.Expression of MRP8 and MRP14 by macrophages is a marker for severe forms of glomerulonephritis.J Leukoc Biol. 2004; 75: 198-206Crossref PubMed Scopus (65) Google Scholar whereas in renal vasculitis most activated macrophages express this complex and produce TNFα and IL 1.16.Rastaldi M.P. Ferrario F. Crippa A. et al.Glomerular monocyte-macrophage features in ANCA-positive renal vasculitis and cryoglobulinemic nephritis.J Am Soc Nephrol. 2000; 11: 2036-2043PubMed Google Scholar In addition, high serum calprotectin levels have been described in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, renal allograft rejection, juvenile idiopathic arthritis, and Kawasaki disease,17.Haga H.J. Brun J.G. Berntzen H.B. et al.Calprotectin in patients with systemic lupus erythematosus: relation to clinical and laboratory parameters of disease activity.Lupus. 1993; 2: 47-50PubMed Google Scholar, 18.Soyfoo M.S. Roth J. Vogl T. et al.Phagocyte-specific S100A8/A9 protein levels during disease exacerbations and infections in systemic lupus erythematosus.J Rheumatol. 2009; 36: 2190-2194Crossref PubMed Scopus (80) Google Scholar, 19.De Rycke L. Baeten D. Foell D. et al.Differential expression and response to anti-TNFalpha treatment of infiltrating versus resident tissue macrophage subsets in autoimmune arthritis.J Pathol. 2005; 206: 17-27Crossref PubMed Scopus (94) Google Scholar, 20.Burkhardt K. Radespiel-Troger M. Rupprecht H.D. et al.An increase in myeloid-related protein serum levels precedes acute renal allograft rejection.J Am Soc Nephrol. 2001; 12: 1947-1957PubMed Google Scholar, 21.Frosch M. Ahlmann M. Vogl T. et al.The myeloid-related proteins 8 and 14 complex, a novel ligand of toll-like receptor 4, and interleukin-1beta form a positive feedback mechanism in systemic-onset juvenile idiopathic arthritis.Arthritis Rheum. 2009; 60: 883-891Crossref PubMed Scopus (140) Google Scholar, 22.Hirono K. Foell D. Xing Y. et al.Expression of myeloid-related protein-8 and -14 in patients with acute Kawasaki disease.J Am Coll Cardiol. 2006; 48: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar whereas increasing levels have been associated with relapse in juvenile-onset arthritis23.Foell D. Wulffraat N. Wedderburn L.R. et al.Methotrexate withdrawal at 6 vs. 12 months in juvenile idiopathic arthritis in remission: a randomized clinical trial.JAMA. 2010; 303: 1266-1273Crossref PubMed Scopus (210) Google Scholar and decreasing levels with improvements in joint swelling in rheumatoid arthritis.24.Andres Cerezo L. Mann H. Pecha O. et al.Decreases in serum levels of S100A8/9 (calprotectin) correlate with improvements in total swollen joint count in patients with recent-onset rheumatoid arthritis.Arthritis Res Ther. 2011; 13: R122Crossref PubMed Scopus (57) Google Scholar In this study, we report on serum calprotectin levels, calprotectin surface expression on circulating leukocytes and in renal biopsies, and calprotectin (S100A8 and S100A9) gene expression in purified leukocyte subsets in patients with active AAV, AAV in remission, and healthy controls. We demonstrate that calprotectin is expressed on the surface of leukocytes and in the serum at higher basal levels in patients than in healthy controls, whereas disease activity is associated with increasing serum levels. Glomerular inflammation is characterized by infiltration with calprotectin-positive leukocytes, and the extent of glomerular inflammation is closely related to the degree of calprotectin expression. Overall, we find that calprotectin is a potential novel biomarker of disease activity in AAV, and the altered basal levels in patients suggest a potential role in disease etiology. Immunohistochemistry (IHC) was on renal of acute AAV The patients were to the classification as focal crescentic mixed and sclerotic In addition, there were patients with no active Calprotectin was to active glomerular lesions, found crescents or in of and lesions were for calprotectin expression was a the levels of expression in the crescentic lesions compared with the sclerotic or lesions of whereas there was a to levels of expression with decreasing of inflammation. with renal lesions did not demonstrate calprotectin the of cells on the renal biopsy in of of the patients and were of on the extent of active whereas there was prominent calprotectin The AAV classification has been to predict renal with renal in focal lesions by crescentic lesions, and that calprotectin expression the with the in the crescentic lesions by focal calprotectin but may macrophages, and are associated with poor renal The calprotectin was and in a glomerular to that of macrophages and of the glomerular calprotectin-positive cells were positive for the neutrophil marker more of the cells were positive for macrophage was which demonstrated cells the which were positive for with of glomerular of which were calprotectin positive and By macrophages were for Serum was AAV patients with acute disease patients patients disease remission and healthy controls. patients had and with patients measured the the patient of the patients with serum calprotectin and at the of and and and and and and and active cytoplasm proteinase in a active cytoplasm proteinase The and of calprotectin levels were as healthy controls, acute AAV and remission AAV were healthy and both acute and remission AAV as as acute and remission In addition, there was a serum calprotectin levels and total neutrophil count was no with monocyte count or serum We measured serum calprotectin in patients on who demonstrated significantly levels of calprotectin than those patients with both active and AAV patients had serum calprotectin measured at more than the after patient had increase in serum calprotectin at which correlated with a clinical relapse and for In the patients who remission, there was a in serum calprotectin levels to months during remission The ANCA decreased in the patients was no in calprotectin levels in those acute patients in had been in the previous compared with those who were to were no in this to calprotectin may be a useful of disease We therefore serum patients in the with patients with limited systemic disease renal who were for 12 months with and or and and in high relapse were had at 12 relapse had was for a total of were the patients treatment However, patients were for of patients which were at and 6 months of treatment was with of to as as 12 and 14 had been or was in those patients who had of this of 14 were and were The patients who did not relapse had treatment at 12 of the patients relapsed 12 months on and patients relapsed after 12 months and of the had calprotectin measured the relapse and of In the calprotectin levels were significantly than in those with disease during remission levels limited disease at compared with disease remission However, there was a increase in calprotectin levels after 12 months at 14 vs. The 14 patients who on to relapse during the study a of months had significantly higher levels of calprotectin at the on than the patients who were on treatment at these the there was a to higher in the but this did not These that to calprotectin levels, on predict with demonstrated that at both and 6 months there was a that was associated with a of at a was associated with a of and of and a of whereas at 6 months a was associated with a of and of the at was whereas at 6 months was of those with renal of patients and ANCA and the early were at a patients were on expression was in a subset of healthy active AAV and remission AAV patients had renal whereas had disease with no renal were and were whereas were ANCA were on the flow by with were by a and neutrophil were identified by and characteristics was a of of neutrophils and monocytes with expression of calprotectin in AAV patients during active disease and compared with healthy of neutrophils calprotectin healthy controls, acute AAV, AAV, active AAV compared with controls, and active AAV compared with AAV, of monocytes calprotectin healthy controls, acute AAV, and AAV, We were to surface calprotectin expression on the in the of monocytes cell surface calprotectin the acute and was but did not was a in calprotectin acute patients and healthy for both neutrophil and monocyte cell surface expression. However, we compared the levels of S100A8 and S100A9 mRNA purified neutrophils and monocytes, the we found no serum calprotectin and mRNA expression. there were no in mRNA expression AAV patients and healthy not Glomerular infiltration and neutrophil are features of pauci-immune glomerulonephritis to AAV, whereas circulating monocyte and neutrophil activation is found during active A previous study has demonstrated glomerular macrophage expression of the heterodimer in patients with a of M.P. Ferrario F. Crippa A. et al.Glomerular monocyte-macrophage features in ANCA-positive renal vasculitis and cryoglobulinemic nephritis.J Am Soc Nephrol. 2000; 11: 2036-2043PubMed Google Scholar patients with active AAV who demonstrated prominent expression in of However, this heterodimer is more inflammatory R.S. C. G. et al.The calcium-binding proteins MRP8 and MRP14 form a heterodimer in a subset of monocytes/macrophages in acute but in inflammatory Immunol. PubMed Scopus Google Scholar We found glomerular infiltration of calprotectin-positive cells in the renal of patients with calprotectin expression was active crescents and in of focal necrosis, but was in sclerotic or Calprotectin expression the lesions that predict better renal outcome to the with in the focal lesions and the in crescentic lesions, whereas outcome is in focal glomerulonephritis by crescentic In acute AAV, circulating monocytes and neutrophils are We found that patients with AAV have elevated levels of cell surface calprotectin on both neutrophils and monocytes compared with healthy controls, with levels increasing further during active In addition, serum levels of calprotectin these with significantly higher levels in active disease compared with remission and those found in healthy controls. those patients in clinical remission had elevated serum calprotectin levels and leukocyte suggesting that there is a degree of subclinical innate immune These are of in AAV remission, in which there is persistent monocyte and H. et in ANCA-associated levels of in remission are associated with a higher relapse in the 2009; Full Text Full Text PDF PubMed Scopus Google Scholar with augmented D. T. et of T cell during disease remission in cytoplasmic the role of cells and Rheum. 2010; PubMed Scopus Google Scholar and elevated monocyte H. et expression of Toll-like by monocytes and cells in ANCA-associated 2011; PubMed Scopus Google Scholar Importantly, levels of calprotectin after but of in those patients who on to of that to calprotectin at or 6 months may be a useful of with a of at is significantly better than a ANCA which has recently been in to a of for disease G. Grayson P.C. Mahr A.D. et al.Value of ANCA measurements during remission to predict a relapse of ANCA-associated vasculitis—a meta-analysis.Rheumatology. 2012; 51: 100-109Crossref PubMed Scopus (234) Google Scholar However, as we did not have levels, there a that higher levels of The levels at and 6 months were measured patients were which may for the levels compared with those patients with renal These results are to those found in H.J. Brun J.G. Berntzen H.B. et al.Calprotectin in patients with systemic lupus erythematosus: relation to clinical and laboratory parameters of disease activity.Lupus. 1993; 2: 47-50PubMed Google M.S. Roth J. Vogl T. et al.Phagocyte-specific S100A8/A9 protein levels during disease exacerbations and infections in systemic lupus erythematosus.J Rheumatol. 2009; 36: 2190-2194Crossref PubMed Scopus (80) Google Scholar The most elevated levels in have been with arthritis and renal with a the serum and and no cell surface levels and serum C. M. H. et of the S100A8/A9 complex in cells and cell surface S100A8/A9 on leukocyte in systemic lupus Res Ther. 2011; 13: PubMed Scopus Google Scholar In addition, a study (calprotectin) levels in patients with early demonstrated significantly higher levels of in patients disease compared with the of P. A. R. et of S100A8 and S100A9 in of activation and joint during and human Rheum. 2012; PubMed Scopus Google Scholar with severe demonstrate elevated levels of the levels are than those in vasculitis with no serum levels and severity on the M.A. Vogl T. Foell D. et al.Expression and role of myeloid-related in clinical and Med. 2009; PubMed Scopus Google Scholar to the of serum we were not to demonstrate intracellular or surface expression of the heterodimer in human endothelial cells at or after with not suggesting that most serum calprotectin is circulating neutrophils and We were not to demonstrate a the serum levels of calprotectin and the mRNA expression levels in and did we find patients and controls, suggesting that expression and secretion of calprotectin are not to the of the mRNA by the However, both serum levels and cell surface calprotectin expression are augmented during disease activity, and in early systemic disease higher levels are associated with disease These suggest that serum calprotectin levels may treatment and may be of more clinical than which is the in patients during remission and not A previous study in patients with juvenile idiopathic arthritis with demonstrated that higher levels of were of and subclinical D. Wulffraat N. Wedderburn L.R. et al.Methotrexate withdrawal at 6 vs. 12 months in juvenile idiopathic arthritis in remission: a randomized clinical trial.JAMA. 2010; 303: 1266-1273Crossref PubMed Scopus (210) Google Scholar Calprotectin of a and a marker of activation of innate has recently been demonstrated to have a role in and in induction of autoimmune T K. Vogl T. M. et al.The Toll-like receptor and are in the of CD8+ T Med. 2010; 16: PubMed Scopus Google Scholar expression has been demonstrated in the and has been to to renal in animal H.J. et receptor on renal cells to the induction of glomerulonephritis via and Am Soc Nephrol. 2007; PubMed Scopus (94) Google Scholar expression on both cells and glomerular endothelial cells has been to have a role in neutrophil recruitment and renal in the myeloperoxidase glomerulonephritis et renal cell and 2010; Full Text Full Text PDF PubMed Scopus Google Scholar In animal S100A8 was to in macrophages R. et a expression of on macrophages via toll-like receptor and receptor expression in during arthritis.Arthritis Rheum. 2010; PubMed Scopus Google Scholar has been demonstrated that neutrophil D. M. et a protein complex in against 2009; PubMed Scopus Google Scholar and that are by neutrophils. The formation of may have a role in vasculitis and the autoimmune response in patients with small-vessel K. M. et neutrophils in autoimmune small-vessel Med. 2009; PubMed Scopus Google Scholar Overall, we demonstrate that calprotectin is in patients with AAV and a marker of immune activation and disease of patients with disease and to further in disease with further to demonstrate calprotectin be to have a role in vasculitis as a potential The were patients after and and 3 was on for macrophages and calprotectin the was a in with for at antibodies were or antibody was as a at the as the The antibodies were at in a for activity was with The were in and with the were and with and were The were to the recently described classification for A.E. Ferrario F. Hagen E.C. et al.Histopathologic classification of ANCA-associated glomerulonephritis.J Am Soc Nephrol. 2010; 21: 1628-1636Crossref PubMed Scopus (535) Google Scholar and the of calprotectin in both the and the was to 3 by was and was the for and and for for The antibodies were for at at the following and and The were for and and for the The was to the AAV patients were identified the vasculitis at in and and E.F. Lyons P.A. Carr E.J. et al.A CD8+ T cell transcription signature predicts prognosis in autoimmune disease.Nat Med. 2010; 16: 586-591Crossref PubMed Scopus (283) Google Scholar the for of with or activity was assessed and disease at was by with elevated vasculitis activity and acute inflammatory of patients were with positive or in the of of a pauci-immune glomerulonephritis, or were patients disease relapse which was as in in to disease were induction or patients were assessed by and had no related to active and had a vasculitis activity of were the In addition, serum a of patients with limited systemic AAV, the K. N. P.A. et of versus for induction of remission in early systemic cytoplasmic Rheum. 2005; PubMed Scopus Google Scholar by the were Serum was patients with active as as patients in disease remission and healthy controls. The serum the patients in the was the serum and of patients at months the study and of treatment with active at months in remission on and at months a of patients had In these was at 12 with of in those patients who had Serum was at Calprotectin levels were assessed by to the was and at the following was patients in and after The was at for to was for at by was with and calprotectin as as The cells were with the antibodies on for by were in flow The is described in in Lyons et P.A. M. A. et of human the of positive and 2007; PubMed Scopus Google Scholar monocytes were cells by positive CD14+ were the by positive CD16+ after a was to the was with or and to by the for of of a were the and and expression for S100A8 and S100A9 was the in P.A. M. A. et of human the of positive and 2007; PubMed Scopus Google Scholar The are on was of were A was or more a with was a was were for and patients in the were is by a clinical was by the the the the of the the and is a The for is in of a The Serum was as of the and and the and by the We are to who the