Analysis of the UNC group emphasises the differences in patient selection and assays between it and the Kain studies. The 103 patients were selected from patients attending the UNC vasculitis service and disease activity was scored according to an established clinical index (BVAS) . The patients were segregated into two groups: 58 in remission with BVAS scores of 0; and 45 (including those newly presenting) with BVAS scores greater than 0 and defined as having active disease. This is a very different level of activity from the patients studied by Kain et al. in which those at presentation or in clinical relapse generally had BVAS scores between 10 and 15.
Only 15 of the UNC sera were newly presenting patients, and thus suitable for testing the Vienna group's central proposition; some of these were already on treatment. Sera from seven of these (47%) were positive for antibodies to hLAMP-2 in an ELISA that had been validated using positive and negative control sera supplied by the Vienna group. Thus, the incidence is lower than in the cohorts assayed in Vienna but still much higher than healthy controls [2 of 52 (4%), P = 0.0002, Fisher's Exact Test]. By contrast, all 15 sera were negative when tested by Western blotting and IIF, as were all the other sera that were positive in the UNC LAMP-2 ELISA. Importantly, the Vienna positive control sera were also negative in both assays, which demonstrates that neither detects antibodies to hLAMP-2 in serum. The four Vienna positive controls were also negative in the UNC peptide ELISA. This highlights the lack of concordance between the UNC assays and the difficulty in interpreting results from them, as discussed by others [61▪▪,62▪▪].
The critical question about newly discovered autoantibodies is whether they are pathogenic or simply an epiphenomenon. Rodent models have become the standard strategy for addressing this issue in AAV [15–17]. The two approaches to induce injury are injection of antibodies specific for the proposed target antigen and active immunisation with the antigen or a closely related molecule: both have been used to test the potential pathogenicity of antibodies to LAMP-2. Kain et al. passively immunised Wystar Kyoto rats (WKY) – a rat strain commonly used in vasculitis research – with high titre rabbit IgG to recombinant hLAMP-2 that bound purified rLAMP-2 in ELISA and Western blot and rLAMP-2 in rat liver, kidney and neutrophils by IIF. WKY rats injected intravenously with this IgG had circulating anti-hLAMP-2 antibodies but the concentrations decreased rapidly over 24 h. Rabbit IgG was detected bound to glomerular endothelium 2 h after injection but not at later time points. The injected rats developed glomerulonephritis as evidenced by dipstix positive haematuria, severe proteinuria and development of a piFNGN with crescents in around 25% of glomeruli . None of these effects was seen in rats injected with normal rabbit globulin.
The cross-reactivity between FimH and hLAMP-2 provided another opportunity to test the pathogenicity of anti-LAMP-2 antibodies because the common hLAMP-2/FimH epitope recognised by patients’ autoantibodies is partially conserved in rLAMP-2. WKY rats immunised with recombinant FimH developed antibodies to FimH and eight of the 10 studied developed antibodies that reacted with rat and human LAMP-2. The sera bound to a synthetic peptide P41–49 by dot blot and affinity purified IgG to P41–49 from these sera bound to human glomerular endothelium by IIF. Immunoelectron microscopy confirmed the binding and showed the antibodies to P41–49 bound the same structures within cells as a monoclonal antibody to hLAMP-2. The immunised rats had positive ANCA assays using rat neutrophils and developed piFNGN. This supports the results of the passive immunisation experiments and confirms by a different strategy that anti-LAMP-2 antibodies can be pathogenic and cause piFNGN in rats.
These studies demonstrate that immunization with FimH induces antibodies to rat and human LAMP-2 accompanied by the development pauci-immune FNGN. This proves the molecular mimicry between the two molecules – at least under these experimental conditions – and raises the question whether natural infection with fimbriated bacteria could induce AAV in the same way. Two sets of clinical data are consistent with this: Kain et al. reported that nine of 13 consecutive patients presenting with AAV had had a microbiologically proven infection with a fimbriated organism within the preceding 3 months; and Roth et al.[24▪▪] reported that 12% of a sample of 105 patients with UTIs had positive assays for LAMP-2 in their ELISA. The large prospective multicentre study should determine whether infections with type 1 fimbriated bacteria induce antibodies to hLAMP-2 in man and correlate with the development of AAV (http://http://www.intricate.eu/).
All four published studies show that the frequency of autoantibodies to hLAMP-2 is greatly increased in new onset patients with AAV, and that the autoantibodies are no longer detectable once remission has been achieved. Current controversies concern their absolute frequency, and how closely their presence correlates with disease activity. These controversies are largely attributable to the inadequacies of the current assays for the autoantibodies and will be easily resolved once robust ‘clinical grade’ assays have been developed. However, it is already clear that anti-hLAMP-2 antibodies become undetectable after treatment more quickly than antibodies to MPO and PR3 and so assays for them are unlikely to replace standard ANCA testing for diagnosis (except for ANCA-negative patients). It remains to be seen whether anti-hLAMP-2 antibodies more faithfully reflect disease activity than current assays. If so, their measurement would greatly improve tailoring immunosuppression in the individual patient. The answer will come from large-scale clinical studies.
Papers of particular interest, published within the annual period of review, have been highlighted as:
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 146–147).
1. Davies DJ, Moran JE, Niall JF, et al. Segmental necrotising glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J (Clin Res Ed) 1982; 285:606.
2. 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; 1:425–429.
3. Goldschmeding R, van der Schoot CE, ten Bokkel Huinink D, et al. Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils. J Clin Invest 1989; 84:1577–1587.
4. Falk RJ, Jennette JC. Antineutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988; 318:1651–1657.
5. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: proposal of an international consensus conference. Arthritis Rheum 1994; 37:187–192.
6. Jennette JC, Falk RJ, Bacon PA, et al.
Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum 2013 (in press).
7. Jayne DR, Gaskin G, Rasmussen N, et al. European Vasculitis Study GroupRandomized trial of plasma exchange or high-dosage methylprednisolone as adjunctive therapy for severe renal vasculitis. J Am Soc Nephrol 2007; 18:2180–2188.
8. Pagnoux C, Mahr A, Hamidou MA, et al. French Vasculitis Study GroupAzathioprine or methotrexate maintenance for ANCA-associated vasculitis. N Engl J Med 2008; 359:2790–2803.
9. de Groot K, Harper L, Jayne DR, et al. EUVAS (European Vasculitis Study Group)Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial. Ann Intern Med 2009; 150:670–680.
10. Stone JH, Merkel PA, Spiera R, et al. RAVE-ITN Research GroupRituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med 2010; 363:221–232.
11. Jones RB, Tervaert JW, Hauser T, et al. European Vasculitis Study GroupRituximab versus cyclophosphamide in ANCA-associated renal vasculitis. N Engl J Med 2010; 363:211–220.
12. Flossmann O, Berden A, de Groot K, et al. European Vasculitis Study GroupLong-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis 2011; 70:488–494.
13▪▪. Lyons PA, Rayner TF, Trivedi S, et al. Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med 2012; 367:214–223.
This article presents the results of a large genome wide association study of patients with AAV that show an association with MHC class II genes and proteinase 3 and α1-antitrypsin genes specifically in the case of anti-PR3 associated disease.
14. Kallenberg CG. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis: where to go? Clin Exp Immunol 2011; 164 (Suppl 1):1–3.
15. Heeringa P, Little MA. In vivo approaches to investigate ANCA-associated vasculitis: lessons and limitations. Arthritis Res Ther 2011; 13:204.
16. Coughlan AM, Freeley SJ, Robson MG. Animal models of antineutrophil cytoplasmic antibody-associated vasculitis. Clin Exp Immunol 2012; 169:229–237.
17. Salama AD, Little MA. Animal models of antineutrophil cytoplasm antibody-associated vasculitis. Curr Opin Rheumatol 2012; 24:1–7.
18. Kain R, Matsui K, Exner M, et al. A novel class of autoantigens of antineutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells. J Exp Med 1995; 181:585–597.
19. Kain R, Exner M, Brandes R, et al. Molecular mimicry in pauci-immune crescentic glomerulonephritis. Nat Med 2008; 14:1088–1096.
20▪▪. Kain R, Tadema H, McKinney EF, et al. High prevalence of autoantibodies to hLAMP-2 in anti-neutrophil cytoplasmic antibody-associated vasculitis. J Am Soc Nephrol 2012; 23:556–566.
An important follow-up article confirming the high prevalence of anti-LAMP-2 antibodies in active disease and demonstrating their rapid disappearance after the start of treatment.
21. Etter C, Gaspert A, Regenass S, et al. AntihLAMP2-antibodies and dual positivity for anti-GBM and MPO-ANCA in a patient with relapsing pulmonary-renal syndrome. BMC Nephrol 2011; 12:26.
22. Kawakami T, Ishizu A, Arimura Y, Soma Y. Serum antilysosomal-associated membrane protein-2 antibody levels in cutaneous polyarteritis nodosa. Acta Derm Venereol. 2012. doi: 10.2340/00015555-1418 [Epub ahead of print].
This and the article below provide additional clinical evidence for the with small vessel vasculitis more generally.
23. Kawakami T, Takeuchi S, Arimura Y, Soma Y. Elevated antilysosomal-associated membrane protein-2 antibody levels in patients with adult Henoch-Schönlein purpura. Br J Dermatol 2012; 166:1206–1212.
24▪▪. Roth AJ, Brown MC, Smith RN, et al. Anti-LAMP-2 antibodies are not prevalent in patients with antineutrophil cytoplasmic autoantibody glomerulonephritis. J Am Soc Nephrol 2012; 23:545–555.
An important study using different assays for antibodies to LAMP-2 in a new cohort of patients with AAV and reaching different conclusions to the original studies.
25. Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 2009; 10:623–635.
26. Tanaka Y, Guhde G, Suter A, et al. Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 2000; 406:902–906.
27. Nishino I, Fu J, Tanji K, et al. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 2000; 406:906–910.
28. Cheng Z, Fang Q. Danon disease: focusing on heart. J Hum Genet 2012; 57:407–410.
29. Andrejewski N, Punnonen EL, Guhde G, et al. Normal lysosomal morphology and function in LAMP-1-deficient mice. J Biol Chem 1999; 274:12692–12701.
30. Kostich M, Fire A, Fambrough DM. Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans. J Cell Sci 2000; 113:2595–2606.
31. Wyroba E, Surmacz L, Osinska M, Wiejak J. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues. Eur J Histochem 2007; 51:163–172.
32. Fukuda M. Lysosomal membrane glycoproteins. J Biol Chem 1991; 266:21327–21330.
33. Hatem CL, Gough NR, Fambrough DM. Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmicdomains. J Cell Sci 1995; 108:2093–2100.
34. Gough NR, Hatem CL, Fambrough DM. The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c. DNA Cell Biol 1995; 14:863–867.
35. Zhou D, Li P, Lin Y, et al. Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens. Immunity 2005; 22:571–581.
36. Crotzer VL, Glosson N, Zhou D, et al. LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens. Immunology 2010; 131:318–330.
37. Demirel O, Jan I, Wolters D, et al
. The lysosomal polypeptide transporter TAPL is stabilized by the interaction with LAMP-1 and LAMP-2. J Cell Sci 2012. [Epub ahead of print]
38. Gough NR, Fambrough DM. Different steady state subcellular distributions of the three splice variants of lysosome-associated membrane protein LAMP-2 are determined largely by the COOH-terminal amino acid residue. J Cell Biol 1997; 137:1161–1169.
39▪▪. Wilke S, Krausze J, Büssow K. Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx. BMC Biol 2012; 10:62.
This article shows for the first time the molecular structure of LAMPs’ luminal domain and thus provides a model for the studies of interactions with autoantibodies.
40. Fehrenbacher N, Bastholm L, Kirkegaard-Sørensen T, et al. Sensitization to the lysosomal cell death pathway by oncogene-induced down-regulation of lysosome-associated membrane proteins 1 and 2. Cancer Res 2008; 68:6623–6633.
41. Kreuzaler PA, Staniszewska AD, Li W, et al. Stat3 controls lysosomal-mediated cell death in vivo. Nat Cell Biol 2011; 13:303–309.
42. Huynh KK, Eskelinen EL, Scott CC, et al. LAMP proteins are required for fusion of lysosomes with phagosomes. Embo J 2007; 26:313–324.
43. Beertsen W, Willenborg M, Everts V, et al. Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice. J Immunol 2008; 180:475–482.
44▪▪. Kaushik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol 2012; 22:407–417.
A beautiful summary of current knowledge of chaperone mediated autophagy one of LAMP-2's critical functions.
45. Saftig P, Beertsen W, Eskelinen EL. LAMP-2: a control step for phagosome and autophagosome maturation. Autophagy 2008; 4:510–512.
46. Nimmerjahn F, Milosevic S, Behrends U, et al. Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy. Eur J Immunol 2003; 33:1250–1259.
47. Schmid D, Münz C. Innate and adaptive immunity through autophagy. Immunity 2007; 27:11–21.
48▪. Schneede A, Schmidt CK, Hölttä-Vuori M, et al. Role for LAMP-2 in endosomal cholesterol transport. J Cell Mol Med 2011; 15:280–295.
An important review of LAMP-2's role in cholesterol transport.
49. Saftig P, Eskelinen EL. Live longer with LAMP-2. Nat Med 2008; 14:909–910.
50▪. Huang J, Xu J, Pang S, et al
. Age-related decrease of the LAMP-2 gene expression in human leukocytes. Clin Biochem 2012; 45:1229–1232.
This article characterises the consequences of reduced LAMP-2 leukocyte concentrations with age that might influence their susceptibility to anti-LAMP-2 antibodies.
51. Furuta K, Yang XL, Chen JS, et al. Differential expression of the lysosome-associated membrane proteins in normal human tissues. Arch Biochem Biophys 1999; 365:75–82.
52. Amos B, Lotan R. Modulation of lysosomal-associated membrane glycoproteins during retinoic acid-induced embryonal carcinoma cell differentiation. J Biol Chem 1990; 265:19192–19198.
53. Nabi IR, Dennis JW. The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time. Glycobiology 1998; 8:947–953.
54. Carlsson SR, Fukuda M. The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones. J Biol Chem 1990; 265:20488–20495.
55. Lee N, Wang WC, Fukuda M. Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins. J Biol Chem 1990; 265:20476–20487.
56▪. Pryor PR. Analyzing lysosomes in Live Cells. Methods Enzymol 2012; 505:145–157.
This article summarises methods to visualise lysosomes and reviews the pitfalls when investigating their biology and function.
57. Colley KJ. Golgi localization of glycosyltransferases: more questions than answers. Glycobiology 1997; 7:1–13.
58. Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009; 19:936–949.
59▪. Zuliani L, Graus F, Giometto B, et al. Central nervous system neuronal surface antibody associated syndromes: review and guidelines for recognition. J Neurol Neurosurg Psychiatry 2012; 83:638–645.
This review summarizes autoantibody mediated disorders of the central nervous system (CNS) and provides guidelines and methods to detect the associated antibodies that are applicable also in other autoimmune disorders.
60. Luqmani RA, Bacon PA, Moots RJ, et al. Birmingham Vasculitis Activity Score (BVAS) in systemic necrotizing vasculitis. QJM 1994; 87:671–678.
61▪▪. Fervenza FC, Specks U. Vasculitis: will LAMP enlighten us about ANCA-associated vasculitis? Nat Rev Nephrol 2012; 8:318–320.
This article provides an important critical review of the contrasting results from the two groups analysing antibodies to hLAMP-2 in AAV.
62▪▪. Flint SM, Savage CO. Anti-LAMP-2 autoantibodies in ANCA-associated pauci-immune glomerulonephritis. J Am Soc Nephrol 2012; 23:378–379.
This article provides an important critical review of the contrasting results from the two groups analysing antibodies to hLAMP-2 in AAV.
63. Kerjaschki D, Ullrich R, Exner M, et al. Induction of passive Heymann nephritis with antibodies specific for a synthetic peptide derived from the receptor-associated protein. J Exp Med 1996; 183:2007–2013.