ANCA: Fuel for the fire or the spark that ignites the flame?
Authors: David J. Salant
Journal: Kidney International (1999)

Anti-neutrophil cytoplasmic autoantibodies (ANCA) have proved to be good serological markers of active pauci-immune crescentic glomerulonephritis (GN) and necrotizing vasculitides, especially Wegener's granulomatosis and microscopic polyarteritis nodosa[1FALK R.J. JENNETTE J.C. Renal vasculitis,.Immunologic Renal Diseases. edited by NEILSON EG, COUSER WG. Lippincott-Raven, Philadelphia1997: 1099-1118Google Scholar]. It is less clear what role, if any, they play in the pathogenesis. Although titers of ANCA, against either myeloperoxidase (MPO) or proteinase 3 (PR3), tend to correlate with disease activity, there is presently no direct evidence that they are either necessary or sufficient to cause vasculitis and GN. This is not to say that they are unimportant. Indeed, it is hard to imagine that autoantibodies that are so closely associated with a relatively limited group of clinicopathological syndromes could represent a nonspecific epiphenomenon. It is just that none of Koch's postulates has been fulfilled. That is, the pathogen (ANCA) has not been identified in the lesions, it has yet to be shown that removing or neutralizing ANCA cures the disease, and giving ANCA to experimental animals has not been found to cause the disease. It is argued that glomerular and/or vascular immune deposits containing ANCA IgG may have been present at the onset of disease in pauci-immune GN only to be shed into the circulation or phagocytosed by the time a biopsy is taken. For example, a model was developed in which rats were immunized with MPO and then, when circulating anti-MPO antibodies were present, MPO and hydrogen peroxide were perfused into the renal artery to form MPO-anti-MPO complexes[2Brouwer E. Huitema M.G. Klok P.A. De Weerd H. Cohen Tervaert J.W. Weening J.J. Kallenberg C.G.M. Antimyeloperoxidase-associated proliferative glomerulonephritis: An animal model.J Exp Med. 1993; 177: 905-914Google Scholar]. This led to the development of crescentic GN even though glomerular-bound immune complexes subsequently disappeared. Unfortunately, this model still begs the question because the antigen was purposefully planted in the glomerulus to serve as a target, which is clearly different than in spontaneous ANCA-associated GN. Additional evidence against a primary role for ANCA derives from a mouse model that spontaneously develops necrotizing vasculitis and pauci-immune crescentic GN with circulating anti-MPO autoantibodies[3Kinjoh K. Kyogoku M. Good R.A. Genetic selection for crescent formation yields mouse strain with rapidly progressive glomerulonephritis and small vessel vasculitis.Proc Natl Acad Sci USA. 1993; 90: 3413-3417Google Scholar]. Most notably, anti-MPO-producing hybridomas from these mice do not cause necrotizing vasculitis or crescentic GN when transferred to naive recipients. Thus, there is no evidence that ANCA are sufficient to cause vasculitis and it has yet to be shown that they are necessary for the onset of disease. Alternative possible agents that might initiate vascular injury include bacterial products, viral infections, or a cell-mediated immune reaction. Again, there is no direct evidence for any of these, although patients with pauci-immune GN often have an infectious prodrome, and T cell reactivity to the ANCA antigens, MPO and PR3, has been demonstrated in patients with necrotizing vasculitis. Most of the evidence supporting a biological effect and possible pathological role for ANCA is derived from in vitro studies. These have shown that the major target antigens, MPO and PR3, which are normally contained within neutrophil and monocyte granules, are translocated to the outer cell surface when the cells are activated with cytokines like interleukin (IL)-1 or tumor necrosis factor-α or as they undergo apoptosis[1FALK R.J. JENNETTE J.C. Renal vasculitis,.Immunologic Renal Diseases. edited by NEILSON EG, COUSER WG. Lippincott-Raven, Philadelphia1997: 1099-1118Google Scholar],[4Gilligan H.M. Bredy B. Brady H.R. Hébert M.J. Slayter H.S. Xu Y.H. Rauch J. Shia M.A. Koh J.S. Levine J.S. Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming.J Exp Med. 1996; 184: 2231-2241Google Scholar]. If cytokine-primed leukocytes are then exposed to anti-MPO or anti-PR3, the antibodies bind the antigen, cross-link FcγII receptors, and cause the cells to degranulate, undergo a respiratory burst and damage co-cultured endothelial cells[5Porges A.J. Redecha P.B. Kimberly W.T. Csernok E. Gross W.L. Kimberly R.P. Anti-neutrophil cytoplasmic antibodies engage and activate human neurophils via Fc gamma RIIa.J Immunol. 1994; 153: 1271-1280Google Scholar],[6Savage C.O.S. Pottinger B.E. Gaskin G. Pusey C.D. Pearson J.D. Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells.Am J Pathol. 1992; 141: 335-342Google Scholar]. Furthermore, as shown recently with monocytes[7Ralston 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 Fc gamma receptors.J Clin Invest. 1997; 100: 1416-1424Google Scholar] and with neutrophils by Cockwell and associates in this issue of Kidney International[8Cockwell P. Brooks C.J. Adu D. Savage C.O.S. Interleukin-8: A pathogenetic role in antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis.Kidney Int. 1999; 55: 852-863Google Scholar], ANCA also stimulate the release of the potent neutrophil chemoattractant, IL-8. Interleukin-8 is one of an expanding family of small peptides, called chemokines, that are powerful leukocyte chemotaxins and activators. The chemokines are subdivided into four subfamilies—CC, CXC, CX3C and C—based on the number of conserved cysteines and the number of amino acids separating the first two cysteines[9Luster A.D. Chemokines—Chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Google Scholar]. IL-8 belongs to the CXC subfamily and has high specificity for neutrophils[10Baggiolini M. Dewald B. Moser B. Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines.Adv Immunol. 1994; 55: 97-179Google Scholar]. Each of the chemokines is remarkably selective for a limited number of leukocytes and the presence of particular chemokines within an inflammatory focus dictates what types of leukocytes will be present[9Luster A.D. Chemokines—Chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Google Scholar]. This high degree of specificity is conferred by the chemokine receptors (CR) expressed on the leukocyte surface. The receptors for IL-8, CXCR1 and CXCR2, are expressed exclusively on neutrophils[9Luster A.D. Chemokines—Chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Google Scholar]. All known chemokine receptors belong to the superfamily of G-protein-coupled receptors with seven transmembrane spanning domains. Both CXCR1 and CXCR2 have high affinity for IL-8 and activate a pertussis-sensitive signaling cascade that involves phospholipid hydrolysis, intracellular calcium mobilization and protein kinase C activation leading to neutrophil chemotaxis and degranulation[11Murphy P.M. Neutrophil receptors for interleukin-8 and related CXC chemokines.Semin Hematol. 1997; 34: 311-318Google Scholar]. There is also evidence that IL-8 activates phosphatidyl inositol 3-kinase and small GTPases, which may contribute to increased neutrophil adhesion[11Murphy P.M. Neutrophil receptors for interleukin-8 and related CXC chemokines.Semin Hematol. 1997; 34: 311-318Google Scholar]. Thus, IL-8 does more than simply attract neutrophils. It also activates them and promotes their adhesiveness. Another feature of IL-8 is its affinity for anionic glycosaminoglycans such as heparan sulfate. This may produce an extracellular tissue repository of IL-8 and provide a long-lasting chemotactic stimulus. Although several cell types, including kidney epithelial and mesangial cells, can be induced to release IL-8 in vitro, macrophages remain the most abundant and best established source in vivo. Macrophages isolated from the inflammatory foci of patients with several diseases, such as rheumatoid arthritis and idiopathic pulmonary fibrosis, constitutively express high levels of IL-8 mRNA and secrete large amounts of IL-8. IL-8 mRNA and protein have been identified previously in the glomeruli of patients with GN and in increased amounts in their urine[12Sekikawa T. Kashihara N. Maruyama K. Satoh M. Okamoto K. Kanao K. Maeshima Y. Sugiyama H. Yamasaki Y. Makino H. Expression of interleukin-8 in human glomerulonephritis.Res Comm Mol Pathol Pharmacol. 1998; 99: 217-224Google Scholar],[13Wada T. Yokoyama H. Tomosugi N. Hisada Y. Ohta S. Naito T. Kobayashi K. Mukaida N. Matsushima K. Detection of urinary interleukin-8 in glomerular disease.Kidney Int. 1994; 46: 455-460Google Scholar]. In addition, macrophages isolated from the glomeruli of patients with rapidly progressive GN have been reported to release IL-8 spontaneously[14Matsumoto K. Spontaneous release of interleukin-8 by glomerular macrophages from patients with rapidly progressive crescentic glomerulonephritis [letter].Nephrol Dial Transplant. 1994; 9: 1693Google Scholar]. The ability of neutralizing anti-IL-8 antibodies to block neutrophil infiltration in several models of acute inflammation, including GN[15Wada T. Tomosugi N. Naito T. Yokoyama H. Kobayashi K. Harada A. Mukaida N. Matsushima K. Prevention of proteinuria by the administration of anti-interleukin 8 antibody in experimental acute immune complex-induced glomerulonephritis.J Exp Med. 1994; 180: 1135-1140Google Scholar], provides further evidence of the functional role of IL-8 in vivo. Cockwell and colleagues have also identified IL-8 mRNA in the inflamed glomeruli of patients with ANCA-associated GN at segmental, crescentic and parietal epithelial sites[8Cockwell P. Brooks C.J. Adu D. Savage C.O.S. Interleukin-8: A pathogenetic role in antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis.Kidney Int. 1999; 55: 852-863Google Scholar]. IL-8 protein localized at similar sites as well as to neutrophils within glomerular capillaries. To determine if this expression of IL-8 might be linked to the presence of ANCA, they conducted a series of in vitro studies and found that ANCA stimulates IL-8 production by neutrophils. They also showed that the supernatants of ANCA-stimulated neutrophils inhibit neutrophil chemotaxis towards an IL-8 gradient. From these results, the authors hypothesize that, despite heavy expression of IL-8 by intrinsic glomerular cells, neutrophils are prevented from transmigrating across glomerular capillaries by a counterbalancing autocrine effect resulting from ANCA-induced IL-8 release from neutrophils. This, they propose, holds the neutrophils at the endothelial surface permitting bystander damage to the endothelium by the activated neutrophils. So, given that some as yet unknown stimulus leads to the initiation of vascular damage and to the production of circulating ANCA autoantibodies, what role might ANCA and IL-8 play in the perpetuation or progression of the inflammatory response? The most plausible scenario is that locally activated leukocytes with exposed antigen are engaged by circulating ANCA, which causes degranulation and release of proteases, reactive oxygen species, and lipid mediators. Simultaneous release of IL-8 and possibly other chemokines would set up a paracrine loop attracting and activating more leukocytes, up-regulating their adhesiveness to endothelium and matrix, and thereby amplifying the inflammation and tissue damage. The proposal by Cockwell et al[8Cockwell P. Brooks C.J. Adu D. Savage C.O.S. Interleukin-8: A pathogenetic role in antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis.Kidney Int. 1999; 55: 852-863Google Scholar] that the endovascular release of IL-8 might prevent the transmigration of neutrophils from glomerular capillaries may be an overinterpretation of their in vitro data. It does not take into account the fact that the Duffy antigen on circulating red cells, a non-signaling chemokine receptor, serves as a sink or sponge that soaks up circulating chemokines and favors unidirectional rather than random migration. Nonetheless, the findings in their paper are consistent with the possibility that ANCA contributes to the local release of IL-8 and accounts for the presence of neutrophils in the lesions. However, I expect that similar observations will soon be made with chemokines specific for monocytes and macrophages, which are the predominant inflammatory cell in crescentic GN. Perhaps the most intriguing unanswered question is what initiates the immune response that generates ANCA autoantibodies. Although I tend to favor some pathogenic role for ANCA, I suggest that we should not lose sight of the possibility that they are the consequence and not a cause of the inflammatory response. For example, it is conceivable that the process of glomerular or vascular inflammation initiates the production of anti-MPO or anti-PR3 autoantibodies by exposing MPO or PR3 on the surface of cytokine-primed and/or apoptotic leukocytes, thereby allowing the antigens to be presented to T cells, either directly or after phagocytosis by antigen presenting cells. In the final analysis, given the information available to date, my sense is that ANCA are, in some way, involved in the pathogenesis of necrotizing vasculitis and crescentic GN, but that they are fuel for the fire rather than the spark that ignites the flame. Supported by research grant DK30932 from NIDDK.