Kain, Renate; Rees, Andrew J.
This year marks the thirtieth anniversary of the discovery of autoantibodies to cytoplasmic components of neutrophilic granulocytes termed anti-neutrophil cytoplasmic antibodies (ANCA)  and it is over 20 years since they were associated with Wegener's Granulomatosis – now called granulomatosis with polyangiitis (GPA) – and microscopic polyangiitis (MPA) . The discovery of myeloperoxidase (MPO) and proteinase-3 (PR3) as their targets [3,4] provided the foundation for current understanding of what became known as ANCA-associated vasculitis (AAV). It also stimulated a period of intensely collaborative research activity that has resulted in the internationally accepted Chapel Hill Consensus Conference Classification of vasculitic syndromes [5,6] and uniform treatment recommendations based on multinational prospective randomised controlled trials were designed [7–11]. Despite these advances, more than 50% of patients with AAV have disease flares within 5 years and mortality remains high . This emphasises the need for specific therapies targeted to underlying immunopathogenesis.
Clinical and experimental studies that are supported by recent genetic evidence [13▪▪] provide overwhelming evidence for the involvement of autoantibodies to MPO and PR3 in the pathogenesis of AAV [reviewed in 14]. Despite this, there is compelling evidence that autoimmunity to MPO and PR3 cannot alone explain the injury in these disorders. At least 10% of patients with apparently identical disease are ANCA negative with no detectable antibodies to MPO or PR3; the presence of antibodies to MPO and PR3 do not correlate with disease activity ; neither antigen is expressed by endothelium which is the primary target of injury; the in-vitro effects of anti-MPO and PR3 antibodies are critically dependent on the simultaneous use of TNF-α or bacterial lipopolysaccharide; and anti-MPO antibodies in experimental models similarly require additional stimuli to induce severe injury [15,16]. These shortcomings have been recently critically reviewed [14,17].
The lack of expression of MPO or PR3 by endothelium prompted the systematic search for autoantibodies that recognise membrane proteins of glomerular endothelium and neutrophils – target and mediator of injury in AAV. This resulted in the original discovery of autoantibodies to human lysosome associated membrane protein 2 (hLAMP-2) . Further studies [19,20▪▪] confirmed their high prevalence in untreated patients presenting with pauci-immune focal necrotizing glomerulonephritis (piFNGN) in the context of AAV and presented evidence for their pathogenicity. These findings have been extended by individual case reports  and studies in cutanous vasculitis and Purpura Henoch-Schönlein [22,23] but have also been challenged [24▪▪], leaving the role of anti-hLAMP-2 antibodies controversial.
All the studies highlight the difficulties in assaying anti-hLAMP-2 antibodies and here we summarise the evidence for their involvement in AAV. In this review, we will describe the structure and function hLAMP-2 emphasising the difficulties of using it as a substrate for assays; critically review the current data on anti-hLAMP-2 antibodies in AAV highlighting the areas of agreement and disagreement; and consider the evidence for the pathogenicity derived from rodent studies.
LAMP-2: MORE THAN A LYSOSOMAL RESIDENT
Lysosome associated membrane protein 2 (LAMP-2) a member of a family of related membrane glycoproteins, that includes LAMP-1, LIMP-2, and DC-LAMP – proteins most abundant in lysosomes but also expressed by late endosomes and on the plasma membrane . LAMP-family proteins are critical for maintaining lysosomal integrity but have other important functions. This is especially true for LAMP-2 and is illustrated by the high death rate in LAMP-2 deficient mice and the development of severe cardiac and muscle disease in the context of ubiquitous abnormalities of lysosome function in survivors  – a phenotype identical to Danon disease, a genetic LAMP-2 deficiency in man [27,28]. By contrast LAMP-1 deficient mice are healthy emphasising the importance of LAMP-2 for normal cellular physiology .
LAMP-2 is highly conserved in avian and mammalian species and related molecules have been described in Caenorhabditis elegans and Paramecium[30,31]. It is a heavily glycosylated 383 amino acid protein that consists of an 11 amino acid cytoplasmic domain, a 24 amino acid transmembrane domain and a heavily glycosylated 324 amino acid luminal (or extracellular) domain. The luminal domain has two similar peptide chains linked by a hinge region  and the degree to which they are glycosylated varies depending on cell type and activation state. LAMP-2 has three splice variants (LAMP-2A, LAMP-2B and LAMP-2C) with identical luminal domains but differences in the transmembranous and cytoplasmic domains affect both their localisation [33,34] and function [35–37]. The amino acid motifs essential for targeting LAMP-2 to lysosomes are conserved in all three isoforms but those responsible for interaction with the plasma membrane and retrieval from it differ. Thus, LAMP-2A and LAMP-2B are detected on the cell surface whereas LAMP-2C is restricted to lysosomes . Consequently, although patients’ autoantibodies recognise the three hLAMP-2 isoforms equally well, binding in vivo is likely to be biased toward those expressed most abundantly on the cell surface.
LAMP-2: A MOLECULE AT THE CHECK-POINT BETWEEN CELL SURVIVAL AND DEATH
The severity of the consequences of genetic deficiency drastically illustrates the importance of LAMP-2 for health. The reasons are rapidly being elucidated and fall into two categories: the control of lysosomal integrity, and the regulation the molecular traffic across lysosomal membranes. LAMP-2, together with LAMP-1, sufficiently coats the lysosomal membrane with glycocalix [39▪▪] and siRNA mediated knock down in vitro and physiologically in vivo results in increased membrane permeability, loss of lysosomal integrity and cell death. LAMP-2 is also essential for fusion of lysosomes with phagosome (and autophagosomes) and contributes to their intracellular movement to the microtubule organising centre . Effects of LAMP-2 deficiency are particularly severe in neutrophils and increase susceptibility to dental plaque accumulation that causes severe periodontitis .
Lysosomal membrane concentrations of LAMP-2 control the activity of chaperone-mediated autophagy (CMA), one of the major defences against cellular stress [44▪▪]. LAMP-2 is also involved in the other major type of autophagy, macrophagy, a more complex process in which autophagocytic vacuoles in the cytoplasm eventually fuse with lysosomes . LAMP-2 has major effects on antigen presentation especially of cytoplasmic antigens and biases the antigen-derived peptides presented by major histocompatibility complex (MHC) Class II molecules to CD4 positive T helper cells [35,36,46,47]. LAMP-2 is essential for exporting cholesterol from late endosomes and lysosomes for esterification in the trans-Golgi network [48▪]. Even modest reductions in LAMP-2 concentrations, such as those found in normal aging, profoundly affect cell function [49,50▪], which raises the important but as yet unanswered question of whether the patients’ autoantibodies also interfere with hLAMP-2 function.
CHARACTERISING AUTOANTIBODIES TO HUMAN LYSOSOME-ASSOCIATED MEMBRANE PROTEIN 2 IN ANCA-ASSOCIATED VASCULITIS
Antibodies to hLAMP-2 were first identified in sera from patients with AAV by their ability to bind protein purified from human glomeruli and neutrophils, which have molecular masses of 130 kDa and 170 and 80–110 kDa, respectively . This reflects the difference in LAMP-2 glycosylation in these two cell types [32,51–55]: in the small sample studied, the autoantibodies bound equally well to LAMP-2 despite these differences.
The influence of glycosylation on antibody binding has been analysed in more detail using recombinant hLAMP-2 expressed in either Escherichia coli or ldlD cells – a Chinese hamster ovary (CHO) cell line in which the extent of glycosylation can be manipulated by varying the culture conditions. Almost all the patients’ autoantibodies bound to both unglycosylated and glycosylated forms of the molecule. This proves that they recognised epitopes on the protein backbone of the hLAMP-2 luminal domain that remain accessible in naturally glycosylated hLAMP-2 – at least to the extent of glycosylation in ldlD cells which is relatively simple and equivalent to that of glomerular hLAMP-2. A few of the patients’ autoantibodies recognised glomerular hLAMP-2 but not neutrophil hLAMP-2 presumably because of its more complex carbohydrate pattern. Exceptional patients had antibodies that only recognise glycosylated hLAMP-2.
Further mapping identified two common epitopes recognized by patients’ IgG that were designated P41–49 and P331–341, based on their position in the amino acid sequence . Importantly, these are not the only epitopes but merely the two most common ones. A very striking feature of the P41–49 epitope is that its amino acid sequence is 100% homologous (and 90% identical) to a sequence in FimH, a bacterial adhesin expressed by type 1 fimbriated bacteria, such as E. coli, and critical for their pathogenicity. Furthermore, the autoantibodies to hLAMP-2 cross-reacted with FimH and immunising rats with FimH induces anti-LAMP-2 antibody synthesis, which demonstrates molecular mimicry between the two molecules. This will be discussed later.
ASSAYING ANTIBODIES TO HUMAN LYSOSOME-ASSOCIATED MEMBRANE PROTEIN 2
Characterising the hLAMP-2 epitopes and how access to them is affected by glycosylation is not of purely academic interest but is critical for developing clinical grade assays for anti-LAMP-2 antibodies, which is proving a real challenge. Purified natural antigen as used in the initial study is appropriately glycosylated but can only be purified in small amounts and so recombinant antigen is the only practical alternative. Recombinant hLAMP-2 expressed in E. coli is unglycosylated and so all peptide epitopes are available but the protein is relatively unstable. Mammalian LAMP-2 is probably more stable but less easy to generate in large quantities and has the additional complication that the glycosylation pattern varies greatly depending on the expression vector and cell type used [19,20▪▪,24▪▪]. A further uncertainty is that varying levels of (transient) transgenic expression driven by powerful promoters to increase expression often introduces aberrant glycosylation patterns not seen under physiological conditions [53,56▪,57,58] with unpredictable effects on antibody binding.
One way of circumventing the problems of purifying recombinant membrane proteins is to target the recombinant antigen to the plasma membrane and then use IIF to detect antibodies that bind to it. This approach has been widely used to assay autoantibodies to cell surface receptors that are naturally targeted to the plasma membrane [59▪]. Expressing hLAMP-2 on the cell surface requires additional steps as the full-length form is targeted to lysosomes and the expressed luminal domain is soluble and not retained within the cell. Stable surface hLAMP-2 expression has been achieved by stably transfecting ldlD cells with hLAMP-2A with a point mutation in the cytoplasmic tail that prevents its retrieval from the plasma membrane. This leads to its unique expression on the cell surface and provides a cell line that can be used to quantify antibodies to hLAMP-2 by IIF without permeabilizing the cells . This eliminates the confounding effects of background staining of IgG from ‘sticky’ sera like those from patients with lupus or RA [59▪].
THE FREQUENCY OF ANTI-HUMAN LYSOSOME-ASSOCIATED MEMBRANE PROTEIN 2 ANTIBODIES IN ANCA-ASSOCIATED VASCULITIS
The initial study  found anti-hLAMP-2 antibodies in 13 of 15 (87%) of the patients but was far too small to estimate their frequency in AAV more generally. This was addressed in a subsequent study  of a cohort of 84 patients presenting with biopsy proven piFNGN. Seventy-eight (93%) of these had autoantibodies to hLAMP-2 detected by ELISA using unglycosylated recombinant hLAMP-2 and the results were confirmed by at least two other assays (Western blotting, IIF and ELISA with glycosylated hLAMP-2). By contrast, sera taken from the same patients after immunosuppression-induced remission remained positive in only six (7%). Anti-hLAMP-2 antibodies were not detected in any of 53 healthy controls and found in only one of 30 controls with other renal diseases.
The high prevalence of antibodies to hLAMP-2 was confirmed in a second study [20▪▪], which showed frequencies of 89, 91 and 80% in three new European patient cohorts of untreated patients with AAV and piFNGN at presentation. Accordingly, they had around twice the frequency of antibodies to MPO and PR3 and were also detected in seven of eight ANCA negative patients. All sera in this study [20▪▪] were tested using three independent assays for anti-hLAMP-2 antibodies – ELISA, immunoblot and IIF and had a combined concordance rate of over 80%.
Autoantibodies to hLAMP-2 have different kinetics to those of anti-MPO and PR3 ANCA and become undetectable quickly after initiation of therapy – a phenomenon described for autoantibodies to other membrane proteins [59▪]. This was shown in a longitudinal study [20▪▪] in which anti-hLAMP-2 antibodies became undetectable within the first month after starting treatment in 42 of 43 patients. None of these patients had a clinical relapse during the subsequent year's follow-up and the anti-hLAMP-2 antibodies remained undetectable throughout, confirming the earlier observation that the antibodies are generally undetectable in the absence of clinical disease activity. This contrasts results with the classical ANCA that commonly continue to be present when patients are clinically in remission.
One consequence of the rapid effect of treatment is that the frequency of anti-hLAMP-2 antibodies is significantly lower in patients presenting with AAV after immunosuppressive therapy had been started [20▪▪]. Anti-hLAMP-2 antibodies were again detected during clinical relapses of disease activity in 16 of 28 (57%) patients with the evolution from negative to positive anti-hLAMP-2 assays being documented in some [20▪▪]. This strongly suggests they correlate with disease activity but more data are needed for proof.
The Vienna studies are remarkably consistent probably because the presence of antibodies to hLAMP-2 is always confirmed by multiple assays. The collective results from three studies [18,19,20▪▪] show that the frequency of anti-hLAMP-2 antibodies lies between 80 and 90% in untreated patients presenting with AAV before steroids and immunosuppressive treatment was started. Overall, 155 of 173 (89%) of patients had anti-hLAMP-2 antibodies.
There is only one other published study [24▪▪] of anti-hLAMP-2 antibodies in AAV and superficially, the results appear to contradict those of Kain et al.. The overall frequency of antibodies to hLAMP-2 in AAV was 21% but this figure combines data from patients with active and quiescent disease [24▪▪]. Comparisons are further complicated by major differences in patient selection (Table 1) and in the assays used (Table 2). Roth et al.[24▪▪] studied two AAV cohorts: 103 sera from patients attending the vasculitis service at University of North Carolina Kidney center (UNC) and analysed by local ELISA, Western blotting and IIF assays; and cohort of 226 patients from the Massachusetts General Hospital Boston. The Boston sera were assayed using a novel ELISA based on a commercially available hLAMP-2 polypeptide that spans just over a quarter of the sequence of the luminal domain. However, IgG binding in the assay was very low and the assay appears not to have been validated using known anti-LAMP-2 positive and negative sera. Accordingly, it is impossible to tell whether it measured authentic anti-hLAMP-2 antibodies or simply nonspecific IgG binding.
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▪▪].
PATHOGENICITY OF ANTI-LAMP-2 ANTIBODIES IN RODENTS
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.
Roth et al. also performed passive immunisation studies in WKY rats but found no evidence either for anti-LAMP-2 antibodies binding in the kidney or for glomerulonephritis. The most likely explanation for the contrasting results is a difference in the antibodies to LAMP-2 used. Kain et al. produced theirs by injecting rabbits with complete recombinant hLAMP-2 whereas Roth et al.[24▪▪] used a 9-mer synthetic peptide corresponding to the hLAMP-2 epitope P41–49 as immunogen. The resulting antibody bound to the immunising peptide and recombinant hLAMP-2 but data on binding to native rLAMP-2 were not presented. There are also two potential problems with the peptide immunogen approach: only six of the nine amino acids in the immunising peptide are conserved between human and rat LAMP-2; and previous experience with Heymann nephritis showed that, even after coupling to KHL and BSA, peptides longer than 12-mers were needed to generate antibodies that bound native antigen sufficiently well to cause injury . Consequently, robust evidence that the anti-hLAMP-2 antibodies bound strongly to native rat LAMP-2 are essential before the results of a negative study such as this can be interpreted.
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.
Conflicts of interest
The authors do not declare any conflict of interest.
The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n° 261382 and n° 238756. The work has been funded by the Vienna Science and Technology Fund (WWTF) through Project LS09-075.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 146–147).
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