Anti-LAMP-2 antibodies are not prevalent in patients with antineutrophil cytoplasmic autoantibody glomerulonephritis

Abstract

Lysosomal membrane protein 2 (LAMP-2) is a target of antineutrophil cytoplasmic autoantibodies (ANCA) in addition to the more commonly known targets proteinase 3 and myeloperoxidase. The prevalence of anti-LAMP-2 antibodies and their relationship to disease in ANCA glomerulonephritis are not well described. We measured anti-LAMP-2 reactivity in 680 sera samples (two academic centers) from patients with ANCA glomerulonephritis (n=329); those with ANCA-negative glomerulonephritis (n=104); those with fimbriated, gram-negative Escherichia coli urinary tract infection (n=104); disease controls (n=19); and healthy volunteers (n=124). With levels in healthy controls used to define a reference range, anti-LAMP-2 reactivity was present in 21% of ANCA sera from two of the centers; reactivity was present in 16% of the control group with urinary tract infection. Western blotting and immunofluorescence microscopy did not verify positivity. Titers of anti-myeloperoxidase and anti-proteinase 3 antibodies were 1500-fold and 10,000-fold higher than anti-LAMP-2 titers, respectively. There was no correlation between anti-LAMP-2 antibodies and disease activity. Furthermore, Wistar Kyoto rats injected with anti-LAMP-2 antibodies did not develop glomerulonephritis. In conclusion, antibodies that react with LAMP-2 may exist at very low titers in a minority of patients with ANCA disease. These data do not support a mechanistic relationship between anti-LAMP-2 antibodies and ANCA glomerulonephritis.

Figure 1.

Substrates used to detect anti–LAMP-2 antibodies included a recombinant/glycosylated protein, a recombinant/nonglycosylated protein, and a synthetic peptide. (A) Schematic of full-length human LAMP-2a protein denoting O-linked hinge region and N-glycosylation sites. (B) Sites of putative pathogenic epitopes are designated in yellow boxes (HGTVTYNGS) (QGKYSTAQDC). For studies at UNC Kidney Center, LAMP-2a cDNA was subcloned, omitting the C-terminal transmembrane domain (T-M) and cytoplasmic tail. (C) Analysis of recombinant protein produced in HEK cells indicated high purity, as assessed by SDS-PAGE. (D) Recombinant protein was recognized by purchased hLAMP polyclonal antibody, assessed by Western blot. (E) Peptide used in studies at UNC Kidney Center. (F) Purity of fast protein liquid chromatography–eluted HGTVTYNGS peptide was indicated by a single peak. (G) Confirmation of peptide composition by mass spectrometry indicated correct mass of 934.943. (H) Schematic of LAMP-2 protein used in Massachusetts General studies produced in wheat germ extract system. (A) Adapted from reference 12, with permission.

Figure 2.

A subgroup of sera from patients was reactive to recombinant LAMP-2 by ELISA but negative by Western blotting. (A) ELISA results indicate a subgroup of ANCA patients’ sera samples were reactive against rLAMP-2 protein, compared with samples from healthy controls, patients with urinary tract infection (UTI), and SLE; ANCA sera from Kain et al., new onset; and never-frozen sera (solid bar indicates 2 SD above the mean of healthy controls). In the ANCA disease group, open squares indicate active disease and filled diamonds indicate patients in remission; in the new-onset group, asterisks indicate samples collected before steroid treatment. (B) Evaluation of serum reactivity by Western blot analysis (M, size marker; lane 1, 1 μg purified rLAMP-2; 1 μg native MPO). MPO-ANCA–positive samples that were also reactive with rLAMP-2 by ELISA did not react with rLAMP-2 (lane 1) but did react with MPO protein (lane 2). Blot 1 was reprobed with a commercial polyclonal anti–LAMP-2 antibody to verify rLAMP-2 protein loading on the gel.

Figure 3.

All sera samples from patients were negative by immunofluorescence staining of cells overexpressing LAMP-2 recombinant protein. (A) Polyclonal anti–LAMP-2 antibody (but not negative control antibody) produced low-level staining in nontransfected HEK, consistent with staining of low-level endogenous LAMP-2 protein (left), whereas HEK cells transfected with LAMP-2 produced intense cytoplasmic staining (right). (B) None of the healthy controls (n=52) or ANCA disease sera (n=103) produced a staining pattern similar to the positive control staining, although some samples had low-intensity staining with other patterns. SLE samples (n=10) often stained cells in a variable pattern, including nuclear staining consistent with antinuclear antibodies, but none stained with a pattern similar to that of the positive control. Representative staining patterns, including low-intensity staining that did not correspond to the positive control pattern, are shown for two healthy controls (Normal), two SLE controls, and four high-titer ANCA disease sera. Note that one MPO-ANCA sample shows an irregular punctuate staining similar to that seen for the control samples, but no samples resembled the positive control. (C) Immunofluorescence assays using rLAMP-2 overexpressing O-linked glycosylation–deficient cells (CHO-LDL-D cells) generously provided by Kain et al. Positive control anti–LAMP-2 antibodies produced intense cytoplasmic staining (left two panels), but ANCA-positive patient sera produced no staining (right panel). (D) Immunofluorescence assay was performed on CHO-LDL-D cells grown in varying conditions to alter the glycosylation pattern of overexpressed rLAMP-2 protein. Of the 103 samples, 3 reacted to the transfected and nontransfected CHO-LDL-D cells, and the pattern did not resemble the positive control.

Figure 4.

There was no significant reactivity of patients' sera against the LAMP-2 synthetic peptide. Positivity was defined as 2 SD above the mean of the healthy controls (1.04). The four positive samples in the total ANCA disease group were all new onset. UTI, urinary tract infection.

Figure 5.

Transfer of anti–LAMP-2 peptide antibodies into rats did not cause GN. (A) Characterization of anti–LAMP-2 antibodies generated in a rabbit for transfer into Wistar Kyoto rats. Antibody strongly reacts with LAMP-2 peptide with some cross-reactivity to FimH peptide. (B) Circulating rabbit IgG detected in rats 1 day after injection. (C) Five days after injection, there was no detectable hematuria or proteinuria.

Figure 6.

Data generated at Massachusetts General, Boston, showed no significant seroreactivity against recombinant/nonglycosylated LAMP-2 protein. Sera samples (blood bank sera [BBS] [n=72], ANCA-negative sera [n=104], MPO-ANCA–positive sera [n=108], and PR3-ANCA–positive sera [n=118]) were evaluated for antibodies against LAMP-2. The distribution and frequency of titers (log10) were defined by regression of optical densities versus antigen concentration, yielding an optical density of 0.4. The curves were linear to an optical density of 0.8. There was no statistical difference in titers among the four groups by both Kruskal-Wallis and ANOVA tests.

Figure 7.

The cytoplasmic staining pattern of LAMP-2 is similar to that of PR3-ANCA. (A) Normal human neutrophils stained with PR3-ANCA in patient’s serum. (B) Normal human neutrophils stained with monoclonal anti–LAMP-2 antibody.