By Eugene Friedman, Class of 2012
Faculty Peer Reviewed
The discovery of anti-neutrophil cytoplasmic antibodies (ANCA) was a serendipitous finding. In 1982, Davies and colleagues published a report detailing their discovery of antibodies that specifically localized to the cytoplasm of neutrophils in patients with necrotizing glomerulonephritis and small-vessel vasculitis–antibodies that disappeared after immunosuppressive therapy and reappeared with disease recurrence.  Two years later, Hall and colleagues confirmed this observation , paving the way for the 1985 Lancet article where van der Woude and colleagues proposed that ANCA was a useful marker in the diagnosis of Wegener’s granulomatosis (WG). 
Small-vessel vasculitides such as WG, microscopic polyangiitis, and Churg-Strauss syndrome are serious systemic diseases that require intervention with immunosuppressive drugs, due to the substantial morbidity and mortality associated with crescentic glomerulonephritis and massive pulmonary hemorrhagic alveolar capillaritis. 
Later studies determined that ANCA are found in over 80% of patients with necrotizing small-vessel vasculitis that does not involve immunoglobulin (Ig) deposition in vessel walls.  Further analysis revealed the two major antigen specificities to be soluble lysosomal enzymes myeloperoxidase (MPO)  and proteinase 3 (PR3).  The high prevalence of ANCA in patients with pauci-immune small-vessel vasculitis thus made these antibodies the prime suspects in the pathogenesis of these diseases.
Although the role of ANCA in disease progression has not yet been proven, there is substantial evidence to suggest the capacity of ANCA to promote vascular damage. In-vitro experimental studies have shown that both PR3-ANCA and MPO-ANCA IgG activate neutrophils to release cytotoxic inflammatory mediators.  Both ANCA F(ab’)2 and Fc engagement appear necessary for effective activation of neutrophils by ANCA. The activation of downstream tyrosine kinase and inhibitory G protein pathways results in the activation of the respiratory burst due to the induction of NADP oxidase. [9-10] However, both PR3- and MPO-ANCA are expressed at a relatively low level at the neutrophil cell membrane at rest. They are dramatically upregulated when neutrophils are activated via pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α) , suggesting that environmental influences augment the disease process.
The role of ANCA in the pathogenesis of small-vessel vasculitides is supported by the work of Xiao and colleagues, who developed a mouse model. The passive transfer of anti-MPO IgG from MPO -/- mice that have been immunized with MPO into either immunocompetent or T- and B-cell-deficient mice results in the development of pauci-immune crescentic glomerulonephritis and small-vessel vasculitis that is similar in presentation, but not severity, to human ANCA-associated disease.  Treatment of animals with lipopolysaccaride resulted in a dose-dependent increase in the severity of the ensuing glomerulonephritis, supporting the idea that a synergistic inflammatory process is responsible for the exacerbation of ANCA-related disease.
Interestingly, neutrophils activated by ANCA exhibit impaired surface expression of phosphatidylserine, which promotes non-inflammatory cell removal by macrophages. As a result, neutrophil cell death proceeds through a process resembling necrosis, which by itself results in the release of inflammatory mediators that activate nearby macrophages.  This leads to a further amplification of the disease process, demonstrating how ANCA-mediated vasculitis can cause massive damage.
The development of ANCA has been linked to immunoreactivity against cPR3, a peptide generated by translation of the antisense DNA strand of the PR3 gene. Antibodies against cPR3 have also been found to exhibit activity against PR3.  Interestingly, sequences highly homologous to antisense PR3 have been identified in microbes such as Staphylococcus aureus. This hypothesis was supported by previous evidence that chronic nasal infection with S aureus was associated both with the development and the risk of relapse of WG.  In addition, Ross River virus and Entamoeba histolytica, two organisms also associated with PR3-ANCA, express peptides that are similar to cPR3.  These findings suggest that, as in other autoimmune diseases, ANCA-mediated vasculitis may be the result of molecular mimicry.
In summary, mouse models and molecular evidence support the role of ANCA as primary mediators of pathogenesis in small vessel vasculitis, and implicate neutrophils as the principal effector cells. Amplification of the local innate immune response and a runaway acute inflammatory injury cause the clinical manifestations of the disease. One conceptual difficulty with this model is that ANCA-mediated neutrophil activation requires cell-surface expression of MPO or PR3; however, expression of these molecules has been associated with activated rather than resting neutrophils.  Thus, the model appears to require an outside factor as the initial mediator of neutrophil activation in the early stages of ANCA vasculitis. Some authors have suggested that inflammatory activation by infection may be the trigger that allows the damage to proceed, by showing that ANCA titers [17-18] and neutrophil PR3 expression  are upregulated during infection. However, there is also evidence from small studies showing that PR3 may be expressed in resting neutrophils in a subset of the population , and that the expression of this gene is modulated by epigenetic factors. 
These findings led to the successful treatment of vasculitis by complement immunosuppression with corticosteroids in 1951 and cyclophosphamide in 1971. [22, 23] Because PR3-expression has previously been shown to be upregulated in response to TNF-α signaling , TNF blockade using specific antibody therapy has been investigated as potential adjuvant therapy in acute WG. Booth and colleagues showed in a prospective, open-label study that treatment with infliximab concurrently with prednisolone and cyclophosphamide resulted in a significantly higher rate of remission compared to the control group. However, a later blinded, placebo-controlled trial demonstrated that the addition of etanercept did not significantly increase rates of remission or decrease rates of relapse, and was in fact associated with an increase in the rates of infection and malignancy in the experimental group. 
B-cells have also been shown to play a role in ANCA-associated vasculitis.  In particular, the number of activated B-cells in circulation was associated with disease severity and likelihood of remission.  More recently, two randomized trials have suggested that rituximab, an anti-CD20 monoclonal antibody that effectively depletes the B-cell population, could be an adequate alternative to cyclophosphamide treatment in cases where toxicity or resistance prevented effective treatment. [27-28] These studies have shown that B-cell depletion is an effective method of inducing remission in WG, and does not appear to have the neoplastic and infectious side effect profile of TNF-α blockade, at least in the short term.
While the high cost of antibody-based therapy will likely be a factor in the use of rituximab for induction of remission in WG, the evidence of its effectiveness is testament to the evolution of selective therapy from understanding the molecular pathways of disease.
Eugene Friedman is a 4th year medical student at NYU School of Medicine
Peer reviewed by Michael Pillinger, MD, Associate Professor of Medicine, Division of Rheumatology, NYU Langone Medical Center
Image courtesy of Wikimedia Commons
1. Davies DJ, Moran JE, Niall JF, Ryan GB. Segmental necrotising glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J (Clin Res Ed). 1982;285(6342):606. http://www.ncbi.nlm.nih.gov/pubmed/6297657
2. Hall JB, Wadham BM, Wood CJ, Ashton V, Adam WR. Vasculitis and glomerulonephritis: a subgroup with an antineutrophil cytoplasmic antibody. Aust N Z J Med. 1984;14(3):277-278.
3. van der Woude FJ, Rasmussen N, Lobatto S, et al. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet. 1985;1(8426):425-429. http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(85)91147-X/abstract
4. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum. 1994;37(2):187-192.
5. Jennette JC, Falk RJ. Small-vessel vasculitis. N Engl J Med. 1997;337(21):1512-1523. http://www.nejm.org/doi/full/10.1056/NEJM199711203372106
6. Falk RJ, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med. 1988;318(25):1651-1657. http://www.ncbi.nlm.nih.gov/pubmed/2453802
7. Jennette JC, Hoidal JR, Falk RJ. Specificity of anti-neutrophil cytoplasmic autoantibodies for proteinase 3. Blood. 1990;75(11):2263-2264.
8. Rarok AA, Limburg PC, Kallenberg CG. Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies. J Leukoc Biol. 2003;74(1)3-15. http://www.ncbi.nlm.nih.gov/pubmed/12832437
9. Williams JM, Ben Smith A, Hewins P, et al. Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab’)2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG. J Am Soc Nephrol. 2003;14(3):661-669.
10. Williams JM, Savage CO. Characterization of the regulation and functional consequences of p21ras activation in neutrophils by antineutrophil cytoplasm antibodies. J Am Soc Nephrol. 2005;16(1):90-96.
11. Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci U S A. 1990;87(11):4115-4119. http://www.pnas.org/content/87/11/4115.full.pdf
12. 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(7):955-963.
13. Pendergraft WF 3rd, Preston PA, Shah RR, et al. Autoimmunity is triggered by cPR-3(105-201), a protein complementary to human autoantigen proteinase-3. Nat Med. 2004;10(1):72-79.
14. Stegeman CA, Tervaert JW, Sluiter WJ, Manson WL, de Jong PE, Kallenberg CG. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med. 1994;120(1):12-17.
15. Pendergraft WF 3rd, Pressler BM, Jennette JC, Falk RJ, Preston GA. Autoantigen complementarity: a new theory implicating complementary proteins as initiators of autoimmune disease. J Mol Med. 2005;83(1):12-25. http://www.ncbi.nlm.nih.gov/pubmed/15592920
16. van der Geld YM, Limburg PC, Kallenberg CG. Proteinase 3, Wegener’s autoantigen: from gene to antigen. J Leukoc Biol. 2001;69(2):177-190.
17. George J, Levy Y, Kallenberg CG, Schuenfeld Y. Infections and Wegener’s granulomatosis–a cause and effect relationship? QJM. 1997;90(5):367-373. http://www.ncbi.nlm.nih.gov/pubmed/9205673
18. Pudifin DJ, Duursma J, Gathiram V, Jackson TF. Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody. Clin Exp Immunol. 1994;97(1):48-51.
19. Matsumoto T, Kaneko T, Wada H, et al. Proteinase 3 expression on neutrophil membranes from patients with infectious disease. Shock. 2006;26(2):128-133.
20. Halbwachs-Mecarelli L, Bessou G, Lesavre P, Lopez S, Wittko-Sarsat V. Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool. FEBS Lett. 1995;374(1):29-33.
21. Ciavatta DJ, Yang J, Preston GA, et al. Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis. J Clin Invest. 2010;120(9):3209-3219.
22. Moore PM, Beard EC, Thoburn TW, Williams HL. Idiopathic (lethal) granuloma of the midline facial tissues treated with cortisone: report of a case. Laryngoscope. 1951;61(4):320-331.
23. Novack SN, Pearson CM. Cyclophosphamide therapy in Wegener’s granulomatosis. N Engl J Med. 1971;284(17):938-942.
24. Wegener’s Granulomatosis Etanercept Trial (WGET) Research Group. Etanercept plus standard therapy for Wegener’s granulomatosis. N Engl J Med. 2005;352(4):351-361. http://www.nejm.org/browse?category=research&subtopic=2_1&page=13
25. Pallan L, Savage CO, Harper L. ANCA-associated vasculitis: from bench research to novel treatments. Nat Rev Nephrol. 2009;5(5):278-286. http://www.ncbi.nlm.nih.gov/pubmed/19384329
26. Popa ER, Stegeman CA, Bos NA, Kallenberg CG, Tervaert JW. Differential B- and T-cell activation in Wegener’s granulomatosis. J Allergy Clin Immunol. 1999;103(5 Pt 1):885-894.
27. Jones RB, Tervaert JW, Hauser T, et al. Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis. N Engl J Med. 2010;363(3):211-220. http://www.ncbi.nlm.nih.gov/pubmed/20647198
28. Stone JH, Merkel PA, Spiera R, et al. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010;363(3):221-232.
One comment on “ANCA and Small-Vessel Vasculitis”
This is a fantastic artile, one of the best I’ve seen on clinical correlations. Concise yet complete, this helps explain the current thinking about one of the most complex issues in internal medicine: vasculitis. Great job Eugene.
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