Anti-laminin auto antibodies in ANCA-associated vasculitis

Maurizio Li Vecchi1,, Antonella Radice2, Fabrizio Renda1, Giuseppe Mulé1 and Renato A. Sinico2

1 Istituto di Clinica Medica e Malattie Cardiovascolari, Cattedra di Nefrologia, Università di Palermo, Palermo, and 2 Dipartimenti di Nefrourologia e di Allergologia e Immunologia Clinica, Azienda Ospedaliera Ospedale San Carlo Borromeo, Milano, Italia



   Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Endothelial cell damage occurs during vasculitic processes in vivo. With the alteration of the endothelium, exposure to basement membrane components may occur with induction of humoral immunity.

Methods. In the present study, we evaluated the prevalence of antibodies against the basement membrane antigen laminin (LMN) in patients with ANCA-associated systemic vasculitis (AASV), pathologic controls (systemic lupus erythematosus, mixed cryoglobulinaemia, Henoch–Schönlein purpura, primary glomerulonephritis) and normal individuals.

Results. By ELISA, 21.6% of AASV (16/74) and 10% of pathologic controls (3/30), but only one of the normal controls (2.8%) had these antibodies (P=0.02). When AASV patients were divided into two groups according to diagnosis and ANCA antigen specificity, antibodies to LMN were found in 27.5% of MPO-ANCA positive microscopic polyangiitis patients (11/40) vs only 14.7% of PR3-ANCA positive Wegener granulomatosis patients (5/34). There was no correlation between the presence or titre of anti-LMN antibodies and the main clinical and laboratory parameters.

Conclusion. These results indicate that basement membrane antigens may become immunogenic in patients with AASV, especially in those with MPO-ANCA positivity. These antibodies are most likely the result of endothelial damage secondary to the initial inflammatory process but may well perpetuate further vascular damage in some patients.

Keywords: anti-laminin antibodies; antineutrophil cytoplasmic antibodies; vasculitis



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The vasculitides comprise a spectrum of diseases characterized by inflammation of blood vessels and defined by clinical and histological criteria [1,2]. Vasculitis may constitute the major manifestation of a number of clinical syndromes, or it may represent a relatively minor manifestation of other primary diseases [13].

The basement membrane (BM) of vascular endothelial cells is comprised of connective tissue matrix components, including some collagens and laminin [4]. Laminin (LMN) is a glycoprotein found ubiquitously in BM that promotes the in vitro adhesion of epithelial, endothelial or muscle cells to BM collagen [4,5]. The presence of anti-LMN antibodies has been reported in patients with various diseases involving BM such as Chagas disease [6], American cutaneous leishmaniasis [7], Goodpasture syndrome [8], post-streptococcal glomerulonephritis [9], preeclampsia [10], and Raynaud phenomenon [11].

Endothelial cell damage results during vasculitic process with subsequent exposure of BM and induction of humoral immunity. Therefore, one would also expect the presence of antibodies to BM antigens in patients with ANCA-associated systemic vasculitis (AASV). Indeed, autoantibodies to glomerular basement membrane (GBM) have been reported in patients with AASV, especially in those with MPO specificity [12].

The aim of our study was to determine the prevalence of anti-LMN antibodies in patients with ANCA-positive vasculitis and to correlate the presence of anti-LMN antibodies to the clinical picture and to ANCA antigen specificity.



   Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Our patient population consisted of 74 individuals with biopsy-proven AASV. The patients were classified according to the names and definitions adopted by ‘Chapel Hill consensus conference on the nomenclature of systemic vasculitides’ [13]. There were 34 PR3-ANCA positive Wegener granulomatosis patients and 40 MPO-ANCA positive microscopic polyangiitis (MPA) patients. Sera from 35 healthy individuals, age- and sex-matched with vasculitis patients, served as controls. A further control group consisted of 30 individuals with other immune-mediated diseases (10 with systemic lupus erythematosus [SLE], five with mixed cryoglobulinaemia, five with Henoch–Schönlein purpura, 10 with chronic primary glomerulonephritis).

Methods
Serum samples were taken during an acute phase of the disease. In 49 patients with vasculitis blood was drawn at the time of diagnosis; in the remaining 25 it was drawn during a relapse 1–3 years after diagnosis. Sera were stored frozen in aliquots at -70°C until used.

Detection of ANCA by indirect immunofluorescence
ANCA were detected by indirect immunofluorescence according to the standard procedure delineated at the first ANCA workshop with minor modifications [14,15].

ELISAs for PR3-ANCA and MPO-ANCA
Sera were tested for the presence of PR3-ANCA and MPO-ANCA by ELISA as previously described [16,17].

Anti-GBM assay
Autoantibodies to GBM were detected by ELISA using a commercially available kit (Wieslab, Lund, Sweden) in all the AASV patients positive for anti-LMN antibodies.

Anti-laminin ELISA assay
LMN isolated from Engelbreth-Holm-Swarm sarcoma (Sigma Laboratories, St Louis, MO, USA) was diluted in 0.05 M Ca-carbonate buffer pH 9.6, to a concentration of 5 µg/ml. LMN used in our experiment was pure; it was tested on a polyacrylamide gel. Wells of polystyrene high capacity plates (Greiner, Frickenhausen, Germany) were coated with 100 µl aliquots overnight at 4°C. The plates were washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 and then blocked by incubation with 200 µl of PBS–bovine serum albumin 2% (BSA) for 1 h at 37°C. After three more washes serum samples were diluted 1 : 50 in PBS–BSA (1%) Tween 20 (0.1%) and 100 µl aliquots were incubated in duplicate wells for 1 h at 37°C. After three washes, 100 µl of alkaline phosphatase labelled goat anti-human IgG (KPL, Geithersburg, The Netherlands) diluted 1 : 1300 in PBS–BSA Tween 20 were added to the wells and the plates incubated for 1 h at 37°C. The wells were washed again and the bound IgG detected by incubation with 100 µl/well of 5 mg/ml p-nitrophenyl phosphate disodium (SIGMA) in Mg-carbonate buffer pH 9.8. The optical density (OD) values were measured at 405 nm using a microplate autoreader (Bio Tek instruments, connected with a personal computer Epson model aX2E). Positive and negative controls, selected on the bases of high and low binding in preliminary experiments, were included in each assay. Every serum to be tested was run in both an odd numbered (control) and an even numbered (LMN coated) wells in duplicates, so that each serum could serve as its own control.

The specificity of the assay was determined by inhibition studies. Briefly, a proper dilution (1 : 50 final dilution) of one MPO-ANCA positive serum, selected because of high binding to LMN, was preincubated with an equal volume of varying amounts of MPO or LMN for 1 h at 37°C. This dilution was chosen because the OD values obtained, fell in the middle of the linear part of the serum dilution curve. One hundred microlitre volumes were then assayed in duplicate for LMN binding and inhibition curves were constructed. Controls consisted of aliquots of the same serum preincubated with irrelevant proteins.

Statistical analysis
Because of their skewed distributions, the anti-LMN antibodies values were reported as the median value and interquartile range (25th–75th percentiles). For the same reason a non-parametric statistical analysis (Kruskal–Wallis test) was performed to compare the four groups. A partition value of 0.77 was selected as the upper limit of the normal range of anti-LMN antibodies. This cut-off point was chosen according to the 95th percentile of normal control values. The percentage of patients with abnormal values of anti-LMN antibodies in the Groups A, B, C and D was compared by chi-square analysis. The statistical significance was set at 2 P<0.05.



   Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Sera from 35 healthy control subjects (group A), 30 pathologic controls (group B), 34 patients with PR3 ANCA-positive Wegener granulomatosis (group C) and 40 with MPO ANCA-positive microscopic polyangiitis (group D) were examined for antibodies to LMN. The median values of sera from group A were 0.454 (0.308–0.589), from group B were 0.429 (0.307–0.559), from group C were 0.421 (0.336–0.826), and from group D were 0.472 (0.343–0.638) (Table 1Go and Figure 1Go). The median values obtained were not significantly different from those of sera from normal and pathologic controls. The percentage of patients with abnormal values of anti-LMN antibodies was higher (P=0.02) in the group with ANCA-associated systemic vasculitis (21.6%; 16/74), when compared with the pathologic controls (group B) (10%; 3/30) and with the normal controls (group A) (2.8%; 1/35).


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Table 1. Anti-laminin antibody levels in serum (ELISA)

 


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Fig. 1. Antibodies against laminin in healthy controls (A), pathologic controls (B), patients with PR ANCA positive Wegener's granulomatosis (C) and MPO ANCA-positive microscopic polyangiitis (D). Results are expressed as optical density (OD). Horizontal bars represent the median values. There were no statistical differences in anti-laminin antibodies between the patients with vasculitis and controls. A partition value of 0.77 was slected as the upper limit of the normal range of anti-LMN antibodies. This cut-off point was chosen according to the 95th percentile of normal controls values.

 
When AASV patients were divided into two groups according to diagnosis and ANCA antigen specificity, antibodies to LMN were found in 27.5% of MPO-ANCA positive microscopic polyangiitis patients (11/40) vs only 14.7% of PR3-ANCA positive Wegener granulomatosis patients (5/34), but this difference was not statistically significant. The frequency of anti-LMN antibodies tended to be higher in serum samples taken late in the disease (28% vs 18.4%), but this difference was not statistically significant. The specificity of anti-LMN antibodies was confirmed by fluid-phase inhibition studies.

Significant inhibition (>30%) of binding activity to LMN was achievable only with addition of varying dilution of the specific antigen but not with MPO (Figure 2Go). There was no correlation between the presence or titre of anti-LMN antibodies and the main clinical and laboratory parameters (age, sex, duration of disease, organ-system involvement, serum creatinine, ANCA titre) (data not shown). Anti-GBM antibodies were negative in all tested patients.



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Fig. 2. Fluid-phase inhibition experiment in one selected serum sample with high levels of anti-LMN and anti-MPO antibodies. The addition of laminin ({diamond}) but not of MPO ({blacklozenge}) induces a dose-dependent reduction of binding to laminin.

 



   Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The role of autoantibodies in the pathogenesis of vascular damage in patients with vasculitis is not yet clear. ANCA and anti-endothelial cell antibodies have been detected with high frequency in AASV [18] and recent studies have shown that both ANCA and anti-endothelial cell antibodies can damage vessel wall in vitro [19]. Endothelial cell damage results during the vasculitic process in vivo. With the alteration of the endothelium in vasculitis, there may be exposure to the components of BM with induction of humoral immunity. Indeed, antibodies to collagen, one of the constituents of BM, have been detected in sera of patients with vasculitis and SLE [20].

It is known that BM of vascular endothelial cells is made of tissue matrix components including, in addition to collagen, LMN [21,22]. LMN is large protein with a molecular weight of about 900 KDa, composed of 3 subunits, named alpha, beta, gamma. LMN is a member of a family of proteins that are abundantly present in BM throughout the body, and are a major component of the GBM and the mesangial matrix. There are at least 11 isoforms of LMN. LMN-1, LMN B2 chain, and LMN alpha-1 are present in kidney matrices [2327].

Conflicting results have been published concerning anti-LMN autoantibody in collagen vascular diseases. Some authors have reported significantly elevated levels of anti-LMN antibodies in patients with primary and secondary Raynaud phenomenon [11]. Other authors, using a larger control population, have failed to confirm these results [28]. Little is known about the prevalence of anti-LMN antibodies in patients with AASV. Recently, autoantibodies against other components of the BM, e.g., the so-called Goodpasture antigen, have been demonstrated in this group of patients [12].

In the present study we found that the percentage of patients with abnormal values of anti-LMN antibodies was higher in AASV patients, especially in those with MPO-ANCA positive MPA, when compared with controls. This was not due to cross-reactivity as demonstrated by the results of inhibition studies.

It is possible that antigenic loci in the BM may be exposed during normal or pathologic metabolic processes resulting in production of anti-BM antibodies [29]. The percentage of our patients affected by MPO-ANCA vasculitis with high levels of anti-LMN antibodies is quite similar to the percentage of MPO-ANCA positive vasculitis with anti-GBM autoantibodies [1230]. However, our patients with anti-LMN antibodies were negative on anti-GBM assay. One could hypothesize that MPO-ANCA positive patients are more prone to produce antibodies to BM antigens for an unknown reason (genetic?).

We found no correlation between the presence of anti-LMN antibodies and the clinical picture. A clinical significance for these antibodies seems unlikely. However, the retrospective nature of our study might have influenced these negative results.

If anti-LMN antibodies developed as a result of tissue damage, one would expect them to be a late event in the disease. Indeed, there was a trend towards an increased frequency of anti-LMN antibodies in sera taken late in the disease. However, taking into account that is difficult to determine the precise onset of the disease and the extent of vascular damage, it is not surprising that such correlation was weak.

Our results confirm the absence or low prevalence of anti-LMN antibodies in SLE [20,31]. Patients with SLE develop antibodies against other BM associated antigens, such as collagen type IV and V, but do not demonstrate autoreactivity to LMN [20]. The reason for this is not known; one might assume that anti-LMN antibody production is at least partly genetically controlled.

In conclusion, a significant portion of MPO-ANCA positive microscopic polyangiitis patients had antibodies to constituents of BM (LMN and GBM). This is probably the result of endothelial cell damage secondary to the vasculitic process and not a primary event. The clinical significance, if any, of anti-LMN antibodies remains to be ascertained in a prospective study.



   Notes
 
Correspondence and offprint requests to: Prof. Maurizio Li Vecchi, Istituto di Clinica Medica e Malattie Cardiovascolari, Cattedra di Nefrologia, Via del Vespro, 129, I-90127 Palermo, Italy. Back



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 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 15. 2.99
Accepted in revised form: 12. 4.00





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