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Rheumatology Advance Access originally published online on August 7, 2007
Rheumatology 2007 46(9):1433-1437; doi:10.1093/rheumatology/kem181
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Antibodies against the VRT101 laminin epitope correlate with human SLE disease activity and can be removed by extracorporeal immunoadsorption

H. Amital, M. Heilweil-Harel1, R. Ulmansky1, M. Harlev2, E. Toubi3, A. Hershko1 and Y. Naparstek1,*

Department of Medicine ‘D’, Meir Medical Center, Tel-Aviv University, Kfar-Saba, 1Department of Medicine, Hadassah University Hospital, 2The Authority for Animal Facilities, Hebrew University, Jerusalem and 3Division of Clinical Immunology and Allergy, Bnai-Zion Medical Center, Haifa, Israel.

Correspondence to: H. Amital, MD MHA, Head of Department of Medicine ‘D’, Meir Medical Center, Tshernichovsky 59, Kfar-Saba, 44281, Israel. E-mail: howard.amital{at}clalit.org.il, hamital{at}netvision.net.il


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. We have previously shown that murine pathogenic lupus autoantibodies bind to VRT101, a 21-mer peptide located at the globular part of the laminin-{alpha} chain. In this study, we evaluated whether VRT101 also serves as a target for human lupus antibodies, upholding the hypothesis that VRT101 may serve as a potential target in the therapy of lupus.

Methods. Anti-VRT101 and anti-dsDNA reactivity were measured in the serum of lupus patients and compared with that of healthy individuals and patients with other rheumatic disorders. Statistical correlations between disease activity measured by the SLEDAI-2k scale and compatible serum anti-VRT101 and anti-dsDNA levels were defined. A VRT101-coupled sepharose column was assessed for its efficacy in removing serum anti-VRT101 antibody and its safety in extracorporeal apheresis in sheep.

Results. Anti-VRT101 and anti-dsDNA antibodies were significantly higher in SLE patients compared with patients with other rheumatic conditions. A high degree of correlation was detected between anti-VRT101 levels and the SLEDAI-2k activity in patients with SLE. Immunoadsorption of lupus patients’ sera on the VRT101–sepharose column removed most of the anti-VRT101 antibodies. The column was found to transfer effectively 3l of normal sheep plasma without significant removal of non-specific antibodies or other proteins.

Conclusions. Anti-VRT101 anibodies are abundantly detected in the serum of patients with SLE and correlate with disease activity. Specific removal of serum anti-VRT101 by extracorporeal plasmapheresis with specific immunoadsorption on the VRT101-coupled sepharose columns may serve as a new therapeutic tool for specific immunoadsorption of pathogenic antibodies in SLE patients.

KEY WORDS: SLE, Lupus nephritis, Laminin, Anti-DNA antibodies, Autoantibodies


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Renal involvement in systemic lupus erythematosus (SLE) is a major determinant that affects prognosis and outcome [1]. Many pathogenic pathways have been implicated in the induction of glomerulonephritis in SLE; injury caused by deposition of DNA–anti-DNA immune complexes is commonly mentioned [2, 3]. Immune complex formation and deposition in renal glomeruli, blood vessels, skin, choroid plexus and other tissues probably initiate a virulent inflammatory process [2].

However, it is more commonly appreciated that the pathogenic process in SLE cannot be solely ascribed to the passive deposition of immune complexes or to DNA antibody binding. In spite of the fact that anti-DNA antibodies in SLE are important disease markers it seems that only a minority of them is nephritogenic [4]. Hylkema et al. [5] showed that Farr assay binding of lupus sera was reduced by 80% by prior extensive purification of anti-DNA antibodies with nucleosomes and histones.

Several other intracellular candidate molecules have been proposed as triggers of the renal inflammatory process exerted by binding of autoantibodies. {alpha}-Actinin, an actin-binding protein localized in glomerular podocytes and mesangium, is a possible target recently mentioned [6, 7]. Another molecule is {alpha}-enolase, a glycolytic enzyme, that has been proposed to be a major target of nephritogenic anti-DNA and non-anti-DNA antibodies [8].

Another mechanism that may lead to tissue damage in lupus is the cross reactivity of lupus autoantibodies with extracellular matrix (ECM) antigens. We have recently shown that one of the major antigens to which murine lupus autoantibodies bind, is the 1-laminin VRT101 component, abundantly found in the mesangial ECM [9, 10]. The titres of either anti-ECM or anti-laminin urinary antibodies in SLE patients were also found to correlate with SLE disease activity [10].

We have recently reported that the binding of lupus autoantibodies to ECM could be inhibited in vitro by laminin peptides. The laminin epitope recognized by the lupus antibodies was found to be a 21-mer peptide, designated VRT101, which is located at the globular part of the laminin-{alpha}1 chain [9].

In the current study, we extended our previous investigation of anti-laminin antibodies to the pathogenesis of human lupus nephritis. The VRT101 peptide was not only found to avidly bind human monoclonal anti-DNA antibodies but also to be highly reactive with sera antibodies of SLE patients. Furthermore, immunoadsorption of lupus sera via the VRT101-coupled column abolished most of the VRT101 activity without significant reduction of the total immunoglobulin levels. Specific immunoadsorption on the VRT101-coupled sepharose column may serve as a future therapeutic option for lupus patients.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Synthetic laminin peptides
Synthetic peptides were kindly provided by Schuger et al. [11]. In brief, the peptides were derived from various domains of the {alpha}1-laminin molecule [12, 13]. The VRT101 peptide was prepared by standard solid-phase 9-fluorenylmethoxycarbonyl chemistry and analysed and purified by reverse-phase HPLC and tested by atom-bombardment mass spectrometry. The sequences of the various peptides were discussed previously [9].

Patients serum samples
Blood samples were collected from 95 SLE, 39 antiphospolipid syndrome (APS), 59 systemic sclerosis, 20 primary biliary cirrhosis, 20 myasthenia gravis patients and 157 healthy controls. Informed consent was obtained from all enrollees according to the protocol that was approved by the institute's ethical committee.

Following blood coagulation the samples were centrifuged at a rate of 2500 r.p.m. and the sera were separated from the blood cells, aliquoted and stored at –70°C until use.

The SLE clinical disease activity was assessed by using the SLE disease activity scale-2000 (SLEDAI-2000) [14].

SLE patients monoclonal anti-DNA antibodies
Four human monoclonal IgG anti-DNA antibodies (mAbs), RH14, B3, D5 and DIL-6 were generated by using the human–human hybridoma technique from SLE patients and kindly provided by D. Isenberg (University College, London, UK) [15–17].

ELISA test for anti-VRT 101 levels
Polystyrene 96-well plates (Easy Wash, Costar, Corning, NY, USA). were coated overnight at 4°C with 100 µl/well of 5 µg/ml of different laminin peptides, VRT101 in DDW, or R28, R30, R37, R18, R27, R26 and R35, in carbonate coating buffer (pH 9.6). Wells were washed three times with PBS-0.05% Tween-20 and blocked with 300 µl/well of 1% BSA in PBS for 60 min at room temperature. The plates were washed six times and subsequently incubated with either mAbs at a concentration of 5 µg/ml or with serum samples of the mentioned subjects diluted 1 : 100 with PBS-1% BSA-0.01% Tween-20 for 60 min at room temperature. The plates were rinsed six times with PBS-0.05% Tween-20 and incubated with alkaline phosphatase-conjugated-anti-mouse or anti-human IgG Abs (Sigma) (1 : 2000 in PBS-1%BSA), for 1 h followed by rewashing and addition of 1 mg/ml P-nitrophenylphosphate (pNPP, Sigma), in 0.1 M glycine buffer (pH 9.6). Optical density (OD) was measured at 405 nm by an ELISA reader (Microwell System; Organon Teknika Turnhout, Belgium).

ELISA test for anti-dsDNA levels
Polysterene plates (Immunol II, Dynatech Laboratories, USA) were coated overnight with 100 µl PBS-poly-L-lysine (100 µg/ml) washed three times with distilled water and incubated with 100 µl DNA (100 µg/ml) overnight. Wells were washed three times with PBS-0.05% Tween-20 and blocked with 300 µl/well of 1% BSA in PBS for 60 min at room temperature. The plates were subsequently incubated with the serum samples of the mentioned subjects (1 : 100 in PBS-1% BSA-0.01% Tween-20) for 60 min at room temperature, washed and incubated with alkaline phosphatase-conjugated anti-human IgG Abs and developed with pNPP as described previously.

Filter assay for anti-dsDNA levels
Serum antibody binding to dsDNA was also measured by using the Millipore (Bedford, MA, USA) filter assay [18]. Briefly, 5 µl of serum was diluted in 0.1 ml 0.2 M borate-saline buffer, pH 8.0. 14C-labelled DNA (10 µl, 2800 c.p.m) was added to 100 µl of the diluted serum or to 2.5 µg of mAb. The mixture was incubated for 30 min at 37°C and for additional 60 min at 4°C. The mixture was filtered under reduced pressure using 0.45 µm nitrocellulose filters (Millipore). The filters were washed twice with 3 ml aliquots of borate buffer, dried at room temperature for at least 16 h and counted with toluene-based scintillation fluid in a beta scintillation counter. Results are expressed as the mean of duplicate samples. The samples differed from the mean value by less than 8%.

Specific immunoadsorption of antibodies on VRT101–sepharose columns (Lupusorb)
The VRT101 peptide (1 mg/ml) was conjugated to high performance N-hydroxysuccinimide-activated sepharose beads (Pharmacia, cat. #17-0717-01) according to the manufacturer's instructions. The columns were prepared in GMP protocols by the Fresenius Hemocare Inc. (Seattle, WA, USA). Briefly, the VRT101 peptide was coupled by its mixture with the sepharose beads at 4 day overnight in 1 mM HCL buffer solution, afterwards the columns were blocked by 0.1 M of HCl–Tris (pH 8). The VRT101-coupled sepharose was washed twice by using glycine acetate Tris buffer 0.15M (pH 8.0) followed by HCl–Tris buffer 0.1M (pH 8), and this procedure was repeated six times. Latter measurements demonstrated that the coupling efficiency was 97%. The VRT101 column was termed the Lupusorb column.

Plasma samples were obtained from highly active SLE patients and were passed without further dilution through the VRT101 peptide–sepharose column, at a rate of 0.4 ml/1 ml sepharose/min, simulating the rate of the in vivo apheresis. The column was then eluted with PBS–HCL 0.01 M (pH 2.4) and the eluted samples neutralized with Tris–EDTA 2 M (pH 12). Binding of the serum before and after adsorption as well as the eluted fraction to VRT101 was measured by ELISA.

Extracorporeal apheresis using the Lupusorb column in sheep
A 50 ml Lupusorb column was connected to a Phresenius plasmaphresis machine and was pre-washed with 500 ml PBS. Sheep were anaesthesized, and connected via the jugular and leg veins to the inlet and outlet of the pheresis machine. Three litres of heparnized plasma were run at a rate of 20 ml/min for two and a half hours. At the end of the procedure the column was washed with 500 ml of PBS and a 5 ml sample from the column was eluted as previously described. The eluate was evaluated for the presence of sheep albumin and immunoglobulins.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Binding of human lupus monoclonal Abs to laminin peptides
The laminin-{alpha}1 C-terminal VRT101 peptide was previously shown to bind to antibodies in the sera of lupus mice [9]. To analyse the interaction of pathogenic human lupus antibodies with the laminin molecule we tested the binding of anti-DNA mAbs generated from lupus patients, to peptides that compose the C-terminal of the laminin-{alpha}1chain as well as to the R18 peptide, derived from the N-terminal of the molecule.

DIL-6, a human anti-DNA mAb, which was generated from a patient with active lupus nephritis, reacted selectively and intensely with the VRT101 peptide whereas the B3 mAb originating from a patient with SLE with arthritis had intermediate activity. The human lupus mAb's D5 and RH 14 had negligible reactivity. None of these mAbs bound to other laminin peptides (Fig. 1).


Figure 1
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  		FIG. 1. Binding of human monoclonal antibodies generated from lupus patients to laminin peptides. The binding of 5 µg/ml of the human lupus-derived mAbs (DIL6, B3, D5 and RH14) as well as the mouse anti-DNA/anti-VRT101 C72 mAb to the laminin peptides (VRT101 and R18-R35) was tested by ELISA. Results are the mean of duplicates measured by OD at 405 nm.

 
Binding of sera to the VRT 101 peptide and to dsDNA
In order to assess whether the anti-VRT101 antibodies are specific to SLE patients we analysed the VRT101 binding capacity of 95 patients with active SLE by ELISA and compared their results with those measured in 157 healthy controls.

Sera derived from SLE patients had significantly higher titres of antibodies against the VRT101 peptide compared with healthy donors (Fig. 2). The average binding of SLE patients sera to VRT101 was 1.18 ± 0.06 (OD 405 nm; mean ± S.E) compared with 0.36 ± 0.03 in the healthy group (P < 0.0001). Anti-VRT101 antibodies were significantly higher in SLE patients compared with patients with the APS (P < 0.001). Patients with other autoimmune disorders as well as normal healthy controls had significantly lower anti-VRT101 antibody binding compared with the SLE patients (P < 0.0001). When the cut-off level for anti-VRT101 serum antibody was defined as the mean plus 2 S.D.s of the normal control levels, 57% of the SLE patients compared with 6% of the controls were found to be positive.


Figure 2
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  		FIG. 2. Binding of sera from patients with SLE and other diseases to VRT10l. Serum samples (1 : 100) from patients with SLE, antiphospholipid syndrome (APS), systemic sclerosis (SCL), primary biliary cirrhosis (PBC), myasthenia gravis (MG) and healthy controls were tested for binding to VRT101 by ELISA. Results are the mean of duplicates measured by OD at 405 nm. *P < 0.001.

 
Serum activity level against anti-VRT101 was assessed in eight patients at different periods of their disease, at intervals lasting from 4 months to 2 years, when the degree of activity of their disease varied (Table 1). The SLE disease activity was quantified by the SLEDAI-2000 scale, based on the clinical records of the patients. By using a linear mixed model we assessed the degree by which anti-VRT101 predicts lupus activity levels. Such a correlation was indeed detected and it reached F(1,27.84) = 32.24, leading to a partial correlation of 0.828 (P < 0.001).


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  		TABLE 1. Sequential analysis of anti-VRT101 titres of eight SLE patients during different phases of their disease

 
An illustrative individual case is shown in Fig. 3; consecutive serum titres of anti-VRT101 samples of a single patient with lupus nephritis highly correlated with the disease activity throughout her disease flare and response to cyclophosphamide. Thus, the clinical activity correlation coefficient with the anti-VRT101 antibody was r = 0.96, (P < 0.001) while corresponding levels of anti-DNA antibodies reached only r = 0.3, (P > 0.05). In this patient, whenever a flare was recorded, kidney involvement significantly contributed to the disease severity and to the SLEDAI-2000 scoring.


Figure 3
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  		FIG. 3. VRT101 binding of sequential serum samples of a lupus patient at different phases of her disease during a 5 yr course. The disease activity level was assessed by using the systemic lupus erythematosus disease activity scale (the 2000 version). Serum samples (1 : 100) were tested by ELISA. Results are the mean of duplicates measured by OD at 405 nm.

 
Immunoadsorption of lupus patients’ sera on the VRT101–sepharose column
The VRT101-conjugated sepharose was shown to be highly selective and reduced the anti-VRT101 serum binding more than 85% in the serum of four SLE patients (Fig. 4). This reduction is equivalent to a decrease in the anti-VRT101 antibody concentration of more than 95% according to the titration curve of serum anti-VRT101 binding (data not shown).


Figure 4
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  		FIG. 4. Immunoadsorption of lupus sera on a VRT101-conjugated sepharose column. Sera from four SLE patients and one healthy donor were loaded on the VRT101 sepharose column. Anti-VRT101 levels were measured in the samples prior and following the immunoadsorption by ELISA. Results are the mean of duplicates measured by OD at 405 nm.

 
By using glycine acetate Tris buffer followed by HCl–Tris buffer we eluted the anti- VRT101 antibodies from the VRT101 sepharose column and found reconstitution of most of the anti-VRT101 antibody activity in the serum by ELISA. About 54% of the anti-DNA antibodies activity was also removed by the VRT101 column indicating that about half of the anti-DNA antibodies activity cross-reacts with this laminin peptide (Fig. 5).


Figure 5
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  		FIG. 5. Analysis of lupus serum fractions after immunoadsorption on a VRT101-conjugated sepharose column. Anti-VRT101 and anti-DNA antibody levels were evaluated in serum fractions before and after immunoadsorption on the VRT101 column as well as in the fraction eluted from the column. Anti-VRT was evaluated by ELISA and expressed by OD at 405 nm and anti-DNA is expressed by c.p.m. as measured by the filter assay.

 
Apheresis of sheep serum via the VRT101–sepharose column
To test the specificity and safety of the laminin-coupled column in extracorporeal plasmapheresis, we have performed monthly plasmapharesis in three sheep for two consecutive months.

Three litres of heparinized sheep plasma were easily passed on a 50 ml VRT101 column at a rate of 20 ml/min. Apheresis was performed once a month for 2 months in three sheep. Analysis of the proteins eluted from the column revealed only immunoglobulins and did not reveal any albumin, and the amount of immunoglobulin absorbed on the column represented 0.015% of the total globulin amount transferred on the column. Evaluation of blood counts and liver functions also did not show significant changes after apheresis (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It is quite clear that the pathogenesis of lupus nephritis is complex and multifactorial and cannot be entirely ascribed to the humoral arm of the immune system; however it seems that autoantibodies play a major role in its pathogenesis.

It is widely known that anti-DNA titres are related to the expression and severity of lupus nephritis; however, doubt has been cast on whether anti-DNA antibodies and binding to DNA actually take part in the mediation of disease. Several studies in lupus animal models have shown that induction of nephritis may be dissected from the coexistence of anti-DNA antibodies; Christensen et al. [19] have shown that Toll-like receptor-9-deficient MRL lupus mice lack anti-DNA antibodies but still develop glomerulonephritis. Autoantibodies with diverse affinities, to intracellular antigens other than DNA have also been suggested to participate in the pathogenesis of lupus such as vimentin, {alpha}-actinin [20, 21].

Recently, we have shown that one of the major components to which lupus autoantibodies are directed to is the 1-laminin component of the ECM [10]. Laminin has a major role in the structure of the ECM of the glomerulus. Previous studies had shown that the laminin molecule is essential for the proper arrangement of a single cell epithelium layer adjacent to the glomerular basement membrane and for the formation of the convoluting structure of the glomerular capillaries [22].

Kootstra et al. [23] demonstrated that in the normal glomerulus, laminin epitopes are present only in the mesangial matrix. However, in pathological conditions such as chronic graft-vs-host disease in mice, a murine model for lupus nephritis, laminin molecules have also been detected in immune deposits within the subepithelium and in cases of glomerulosclerosis also in the subendothelium.

In a recent study, we showed that many lupus mAb anti-DNA bind to the laminin peptide VRT101 [9]. Moreover, sera originating from MRL/lpr/lpr and (NZB/NZW)F1 lupus-prone mice intensely reacted with this peptide as their disease progressed. Studying congenic strains carrying lupus susceptibility genes (Sle1–Sle3) from the lupus-prone NZM2410 mouse on the C57BL/6 background has indicated that anti-laminin antibodies induce tissue damage independent of anti-DNA activity. Mice carrying either one or two of the three lupus susceptibility genes had high anti-DNA antibody titres, low anti-VRT101 reactivity and minimal renal disease; however, mice carrying all three susceptibility genes also developed along with high anti-DNA, severe lupus nephritis accompanied by high anti-VRT101 antibodies. Interestingly, only monoclonal anti-DNA antibodies that cross-reacted with the VRT101 epitope exerted glomerulonephritis in a the non-autoimmune RAG 1(–/–) (B6,129.RAG1)-deficient mouse model. Most importantly, treatment of MRL/lpr/lpr mice with the VRT101 peptide prevented the development of nephritis and extended animal longevity similar to therapy with high daily doses of betamethasone [9]. These data imply that the conjunction of anti-VRT101 to anti-DNA activity is highly significant in the pathogenesis of the disease.

In the current study, we showed that human monoclonal and polyclonal anti-DNA antibodies derived from SLE patients cross-react with the VRT101 peptide. Significantly higher titres of anti-VRT101 antibodies were found in SLE and APS patients compared with healthy normal controls whereas patients with systemic sclerosis, primary biliary cirrhosis and myasthenia gravis had similar low binding levels. Anti-VRT101 serum activity was also found to correlate with overall disease activity determined by the SLEDAI-2000 index.

Adsorption of lupus sera to a VRT101-conjugated sepharose gel column has demonstrated that while anti-VRT101 activity was almost completely abolished, anti-DNA activity was only partially affected. Some of the anti-VRT101 immunoadsorbed antibodies eluted from the column also cross-reacted with DNA.

This concept naturally leads to an inevitable clinical application advocating the removal of these pathological antibodies from the peripheral blood. Several forms of immunoadsorptions have been developed for autoimmune diseases. Basically, this modality can be classified according to the type of antibody removal, non-selective or selective. For instance, the Prosorba silica-based system utilizes the non-selective binding of the Staphylococcus aureus protein A to the Fc portion of the immunoglobulins in order to remove them from the blood. This therapeutic modality has been approved by the Food and Drug Administration for the treatment of refractory rheumatoid arthritis and for resistant idiopathic thrombocytopenic purpura [24].

Selective immunoadsorption has been experimentally implemented in myasthenia gravis patients using a peptide originating from the human {alpha}-subunit of the acetylcholine receptor. A study encompassing 22 randomly selected patients showed significant reduction of blocking antibodies with concomitant clinical improvement in more than half of these patients [25]. Another interesting experimental model in which selective removal of pathogenic antibodies was tried is the dilated-type cardiomyopathy. Autoantibodies against the ß1-adrenergic receptor are present in 80% of the patients with idiopathic dilated cardiomyopathy and have been implicated in the pathogenesis of this disorder [26].

In SLE, reduction of anti-DNA levels has been achieved by removal of autoantibodies from the peripheral blood using the LJP394 molecule. This molecule is a selective B lymphocyte immunomodulator that consists of four double-stranded 20-mer oligodeoxynucleotides attached to an inert scaffold composed of a triethylene glycol core [27]. The administration of LJP394 to mice and humans resulted in reduction of serum dsDNA antibodies and dsDNA antibody-producing cells, probably by its binding to circulating antibodies and subsequent clearance of immune complexes [28, 29]. Interestingly, in most patients, treatment with LJP394 induced a significant reduction in anti-DNA levels and decreased the rate of renal flare, yet this agent was not shown to be effective in actual treatment of lupus nephritis. These results imply that non-anti-DNA antibodies may probably have a more significant role than previously acknowledged in the pathogenesis of the lupus nephritis [30].

As the VRT101 has been found to serve as an important target for lupus pathogenic antibodies, and has been shown to suppress nephritis in lupus mice we have prepared a VRT101–sepharose column in GMP conditions and tested its ability to remove anti-VRT101 antibodies. As can be seen in Fig. 5, one passage of lupus plasma on the column removed most of the anti-VRT101 antibodies. Fifty millilitre VRT101–sepharose columns were also used successfully for extracorporeal plasmapheresis in sheep and did not remove non-specifically significant amounts of other immunoglobulins.

In conclusion, anti-laminin antibodies may have a prominent role in the induction of lupus nephritis; although some of these antibodies cross-react with DNA, we have shown by immunoadsorption that the affinity of these antibodies to a laminin-derived peptide designated VRT101 is distinct and independent. These results set a profound basis for a clinical trial assessing the efficacy of extracorporeal-specific immunoadsorption of anti-laminin antibodies in patients with lupus nephritis.

Formula

Y.N. is the chief scientist of Vertomedical Ltd.


   Notes
 
*Y.N. is the incumbent of the Leifermann chair in Osteoporosis and Arthritis of the Hebrew University. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 21 March 2007; revised version accepted 7 June 2007.
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