Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK
1 Department of Internal Medicine, IRCCS Ospedale Maggiore, Via Sforza 35, 20145 Milan, Italy
2 Department of Internal Medicine, IRCCS Istituto Auxologico Italiano, University of Milan, Via L. Ariosto 13, 20145 Milan, Italy.
Correspondence to: P. L. Meroni
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Abstract |
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Keywords: adhesion molecules, anti-endothelial cell antibody, cytokines, systemic lupus erythematosus
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Introduction |
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In vitro studies have demonstrated the ability of AECA to (i) fix complement, at least in systemic lupus erythematosus (SLE) (5), (ii) mediate antibody-dependent cellular cytotoxicity by NK cells (4), and, particularly, (iii) activate endothelial cells (EC) by up-regulating adhesion molecule expression and the secretion of both pro-inflammatory cytokines and chemokines in primary autoimmune vasculitis (6,7) and in scleroderma (8). The demonstration that IgG AECA+ fractions from active SLE and thrombotic thrombocytopenic purpura (TTP) patients display comparable activity suggests that the AECA ability to activate EC might be a general characteristic of these auto-antibodies (911).
However, these studies present several problems. (i) The sera and polyclonal IgG fractions from patients with SLE, for example, might contain auto-antibodies directed against molecules that have been described to adhere to endothelial membrane surfaces such as DNAhistone complexes and phospholipid-binding proteins [such as ß2-glycoprotein I (ß2GPI)] (1215). This makes it difficult to rule out a possible role for other auto-antibodies in activating EC. In addition, sera from patients suffering from active systemic autoimmune diseases may have very high levels of circulating cytokines and it is possible that free or IgG-bound cytokine contamination could explain these data, despite very careful preparation of the polyclonal antibodies (16,17). Finally, the anti-endothelial activity contained in these polyclonal preparations is apparently directed against a variety of constitutive antigens (1821); thus, although the pro-inflammatory effects can be demonstrated, it remains impossible to attribute them to a specific auto-antigen.
The use of single specificity mAb isolated from patients with SLE would solve these problems. The aims of this study were to: (i) obtain mAb which display reactivity against human EC from the B cells of SLE patients, (ii) characterize the endothelial auto-antigen, (iii) investigate whether such a binding could also mediate endothelial activation comparable to that found with the polyclonal AECA preparations and (iv) investigate the signaling pathways responsible for EC activation.
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Methods |
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Cell culture
Cell lines.
Transformed human microvascular endothelial cells (HMEC-1) were a gift from the Centre for Disease Control (Atlanta, GA) (23). The mouse/human heteromyeloma cell line (CB-F7) was a gift from Dr Sigbert Jahn (24). HMEC-1 cells were grown in MCDB 131 medium (Clonetics, San Diego, CA) supplemented with 10 ng/ml epidermal growth factor (Sigma-Aldrich, Gillingham, UK), 1 µg/ml hydrocortisone and 10% FCS (Gibco/BRL Life Technologies, Paisley, UK). All adherent cells were propagated in 75 or 225 cm2 flasks (Corning, High Wycombe, UK). The cells for membrane preparation were mechanically detached to avoid antigen loss by trypsin digestion and processed as described below. All non-adherent cells were propagated in RPMI 1640 (Gibco/BRL Life Technologies).
Primary cultures.
Human umbilical cord vein endothelial cells (HUVEC) were isolated from normal term umbilical cord vein by collagenase perfusion and cultured as previously reported (25).
Production of heterohybridomas.
Heterohybridomas between human peripheral blood lymphocytes (PBL) and CB-F7 were produced according to the technique of Grunow et al. (24). PBL were isolated from SLE patients' blood by differential density centrifugation on lymphoprep (Nycomed, Birmingham, UK). Approximately 107 PBL and myeloma cells were rinsed twice in PBS and fused at 1:1 ratio using 1 ml 42% PEG-1500 (Merck, Lutterworth, UK) containing 15% DMSO (Sigma-Aldrich) in PBS. The fusion mixture was diluted with PBS and the cells centrifuged. The pellet was suspended in RPMI 1640 at a cell density of 106 PBL/ml and 100 µl/well was dispensed into microtiter tissue culture plates (Corning, High Wycombe, UK). After overnight incubation at 37°C, 100 µl double-strength selective medium containing 104 M hypoxanthine, 105 M aminopterin and 1.6x105 M thymidine (Gibco/BRL Life Technologies) was added to each well. After 2 weeks, the hybridomas were initially screened against endothelial cell membrane (ECM) lysates by ELISA. The positive hybridomas were expanded and cloned by limiting dilution. The monoclonality of a positive hybridoma was proven, at least twice, by limiting dilution. Supernatant was produced and concentrated 30- to 100-fold either by membrane filtration using filters (Amicon, Stonehouse, UK) with cut-off values of 100 kDa for IgM and 30 kDa for IgG hybridomas or by ammonium sulfate salt precipitation. IgG antibody preparations were then further purified by Protein G-affinity chromatography (Mab Trap-GII; Pharmacia-Biotech, Uppsala, Sweden) and dialyzed against PBS. Three IgG antibodies were selected for further study: E-3 and C11-2, which were isolated from patients with SLE, and E-1, which was isolated from a normal individual. Only the E-3 mAb displayed a binding activity against ECM lysates by ELISA.
Membrane preparation
Cell membrane preparations from HMEC-1 were isolated by differential centrifugation as described by McCrae et al. (26). In brief, cells were lyzed by freeze fracture followed by centrifugation at 10,000 g for 30 min in media containing the protease inhibitor mix (2 mM EDTA, 100 U/ml aprotinin, 100 µg/ml PMSF, 12.5 µg/ml leupeptin, 10 mM benzamidine, 10 µg/ml soybean trypsin inhibitor and 1 µg/ml pepstatin) as described (26). The pellet was then resuspended in inhibition medium and sonicated followed by centrifugation at 15,000 g for 30 min. Finally, the cytosolic fractions were removed by centrifugation at 4500 g. The protein concentration was determined by the Bradford reaction (27).
Characterization of hybridomas by ELISA
Anti-cell membrane ELISA. ELISA plates (Nunc; Gibco/BRL Life Technologies) were coated overnight at 4°C with 50 µl/well membrane preparations in 0.015 M sodium bicarbonate buffer (pH 9.6) at a protein concentration of 200 µg/ml. The plates were washed 3 times with PBS containing 0.05% Tween 20. After three washes with PBS/Tween, 50 µl/well hybridoma supernatant was added and incubated for 90 min at 37°C. The plates were then washed 3 times in PBS/Tween. Then 50 µl/well peroxidase conjugated rabbit anti-human Ig, specific for IgG or IgM or IgA, and
chains (Dako, Ely, UK) diluted 1/1000 in PBS/Tween containing 5% fat-free milk powder (Marvel Premier Beverages, Knighton Adbaston, UK), was added and incubated for 1 h at 37°C. After three further washes, 50 µl/well o-phenylendiamine (0.5 mg/ml in sodium citrate buffer 101 M, pH 5, plus 0.01% H2O2) was added and incubated for 30 min at 37°C. The enzymatic activity was stopped with 5 M H2SO4 and the absorbance was measured in a multiscan plate reader (Biotec Instruments, Luton, UK).
Anti-cardiolipin (CL) and anti-ß2GPI antibodies.
Anti-CL and anti-ß2GPI were produced as previously reported (28).
Anti-DNA ELISA.
A standard DNA ELISA technique was used to detect anti-DNA antibodies. In brief the plates were coated with 50 µg/ml/well protamine sulfate followed by 1 µg/ml/well calf thymus DNA. After overnight incubation the excess protamine sulfate binding sites were blocked with polyglutamate 10 µg/ml/well. The plates were then blocked with 3% BSA (Sigma-Aldrich) and 0.05% Tween 20 in PBS. The subsequent stages were the same as for the cell membrane ELISA.
Western blotting analysis
Western blotting after discontinuous SDSPAGE (4% stacking gel and 12% resolving gel) was performed using membrane lysates of HMEC-1 preparations applying 8 µg per track. SDSPAGE separated proteins were transferred to nitrocellulose and blocked with 5% fat-free milk PBS with 5% Tween. The nitrocellulose was then blotted with the appropriate mAb at a protein concentration 100 µg/ml for 2 h. After 3 washes with PBS 0.05% Tween, the nitrocellulose was incubated for a further 2 h with a peroxidase-conjugated anti-human IgG or IgM or IgA 1/1000 (Dako, Glostrup, Denmark). After a further 3 washes the blots were developed with chloronapthol.
Flow cytometry analysis
Cytofluorimetric analysis was performed with a FACSCalibur cytometer (Becton Dickinson Biosciences, San Jose, CA). The instrument was set up by using cells cultured in medium and incubated with FITC-conjugated rabbit anti-human IgG (Dako). This sample was employed to regulate the detectors for forward and side scatter. CellQuest software (Becton Dickinson Biosciences) was used to generate the plots.
HMEC-1 were used for cytofluorimetric studies. Cell monolayers were incubated in 1 ml PBS/2% FCS for 2 h at room temperature with human mAb at the final concentration of 100 µg/ml. After extensive washes with D-PBS, the cells were incubated with FITC-conjugated rabbit anti-human IgG for 90 min at room temperature, washed again and detached with 30 µM EDTA-PBS. After washing, recovered cells were resuspended in D-PBS.
AECA assay
AECA were detected by ELISA on confluent living HUVEC as previously described (30). Different final protein concentrations of the mAb preparations were used, ranging from 200 to 3 µg/ml.
Adhesion molecule expression on EC surface
Adhesion molecule cell-surface expression was evaluated by a cell ELISA as previously described (6,9,14,15). Briefly, the assay was performed on confluent HUVEC monolayers in 96-well microtiter plates. Cells were incubated in the presence of 10 U/ml human recombinant IL-1ß (British BioTechnology, Oxford, UK) or medium alone at the final volume of 200 µl as positive or negative control respectively. A 4-h incubation was used for the evaluation of E-selectin expression and a 20-h incubation for ICAM-1 expression. Different final protein concentrations of the purified AECA mAb preparations were used, ranging from 100 to 0.15 µg/ml. After incubation, the cells were washed twice with RPMI 1640/2.5% FCS and incubated for 60 min at room temperature with 100 µl/well murine monoclonal IgG specific for E-selectin or ICAM-1 at a 1/1000 final dilution (Serotec, Kidlington, UK; cat. nos MCA883 and MCA675 respectively). After three more washes, the cells were fixed with 3% paraformaldehyde for 15 min at room temperature. Cells were then washed 3 times and incubated for another 60 min at room temperature with 100 µl peroxidase-conjugated goat anti-mouse IgG (Cappel, Cochranville, PA). After four washes with RPMI 1640/2.5% FCS and one with PBS alone, 100 µl o-phenylendiamine (0.5 mg/ml in Na citrate buffer 101 M, pH 5, plus H2O2 0.01%) was added. The optical density values were evaluated at 450 nm after 30 min incubation by a semiautomatic reader (Platereader; BioRad, Milan, Italy).
Functional adhesion assay
The assay was performed with [51Cr]Na (30 µCi/106 cells; Amersham International, Little Chalfont, UK)-labeled U937 cells as previously described (9,29). Briefly, the monocyte-macrophage cell line was labeled for 1 h at 37°C. Adhesion assays were performed on HUVEC monolayers 24 h pre-incubated with serial protein concentrations of the IgG AECA mAb and the irrelevant control (ranging from 100 to 0.15 µg/ml). Endothelial monolayers incubated with human recombinant IL-1ß (British BioTechnology; 10 U/ml) or with medium alone served as positive or negative control respectively. The endothelial monolayers were extensively washed and radiolabeled U937 cells were added to each well. After 1 h adhesion at 37°C, the non-adherent cells were removed by washes and the adherent cells were lyzed with 0.1% SDS/0.025 M NaOH. Adhesion was quantified as c.p.m. and expressed as percentage of adhered cells referred to total radiolabeled U937 seeded on HUVEC monolayers (100%), as described (6,29). To rule out the potential involvement of U937 FcR in the adhesion to EC monolayers sensitized by the IgG E3, control experiments were carried out in which radiolabeled U937 were pre-incubated with an anti-Fc
RII blocking mAb (50 µg/ml; Medarex, Annandale, NJ) for 30 min at 4°C and then extensively washed. After the treatment, U937 cells were incubated with EC monolayers and the assay was performed as described above.
IL-6 production by EC
Confluent HUVEC monolayers in 96-well plates were incubated for 24 h with serial concentrations of purified AECA mAb at a final volume of 200 µl/well (in culture medium). Plates were then centrifuged at 800 r.p.m. at 4°C for 10 min and the cell-free supernatants used for IL-6 determination. Supernatants from HUVEC incubated with 10 U/ml human recombinant IL-1ß (British Biotechnology) were used as positive controls. The level of IL-6 was measured using an ELISA kit (Amersham International) (and expressed as pg/ml) as previously described (6,9,14,15).
Activation of NF-B
Cells were incubated in M199 containing the AECA mAb at a final concentration of 200 µg/ml at 37°C for appropriate times. The doseresponse curve was constructed over the range 503.125 µg/ml and harvested after 30 min. Samples were prepared at 4°C by rinsing twice with ice-cold HPFEV buffer (50 mM HEPES, 10 mM Na4P2O7, 100 mM NaF, 4 mM EDTA and 2 mM Na3VO4) and then by addition of 300 µl Laemmli sample buffer at 65°C. Samples were then lyzed by passing several times through a 23 gauge needle, boiled (5 min) and analyzed by SDSPAGE followed by Western blotting using a mAb against inhibitor B (I-
B) (kind gift of Professor Ron Hay, St Andrews University, UK). Incubation of EC with 120 µg/ml tumor necrosis factor (TNF)-
(British BioTechnology) for 30 min was used as a positive control.
Measurement of JNK Activation
Cells were incubated in M199 containing the AECA mAb at a final concentration of 200 µg/ml at 37°C for appropriate times. Samples were prepared at 4°C by rinsing twice with ice-cold HPFEV buffer (50 mM HEPES, 10 mM Na4P2O7, 100 mM NaF, 4 mM EDTA and 2 mM Na3VO4) and then by addition of 300 µl Laemmli sample buffer at 65°C. Samples were then lyzed by passing several times through a 23 gauge needle, boiled (5 min) and analyzed by SDSPAGE followed by Western blotting using a polyclonal antibody recognizing the phosphorylated forms of JNK (Promega, Southampton UK). Incubation of EC with 120 U/ml TNF- (British BioTechnology) for 30 min was used as a positive control.
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Results |
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Figure 5 shows that incubation of HUVEC monolayers with E-3 mAb in the presence of IL-1 receptor antagonists (IL-1ra) inhibits ICAM-1 expression by up to 36%.
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NF-B and JNK activation
Figure 7 shows the effect of E-3 mAb on the activation of NF-
B. The level of I-
B substantially decreased by incubation with the AECA E-3 mAb in a way inversely proportional to mAb concentration (Fig. 7A
). The time course shows that I-
B is totally cleared by 15 min and starts to recover by 30 min (Fig. 7B
). Figure 7(C)
shows the relative controls represented by cells incubated with medium alone, with an irrelevant mAb or a positive control (TNF-
). HUVEC activation induced by E3 mAb is comparable to that obtained by TNF-
(120 µg/ml). Incubation of HUVEC with the C11-2 mAb did not affect I-
B expression. The JNK activation pathway was also studied but E-3 failed to activate this pathway (data not shown).
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Discussion |
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Western blotting studies have identified the putative auto-antigen as having a mol. wt of 42 kDa. The exact nature of the epitope recognized by E-3 remains unclear and is under investigation in our laboratory.
The AECA E3 mAb induces a pro-adhesive and a pro-inflammatory endothelial phenotype in vitro similar to that previously reported with polyclonal IgG fractions from SLE sera (9,10). In this regard, E3 AECA mAb appears to be representative of the anti-endothelial antibodies spontaneously occurring in SLE and suggested to play a role in sustaining vessel wall inflammation (9,10).
These findings are comparable to those found with AECA from other autoimmune vasculitis, i.e. Wegener's granulomatosis, micropolyarteritis, Takayasu's arteritis, and from scleroderma and TTP (68,11). E-3 also behaves in a similar manner to a mouse anti-EC mAb directed against a 68 kDa auto-antigen on the surface of EC and derived from B cells of mice with an experimentally induced AECA-associated autoimmune vasculitis and to human mAb obtained from Takayasu's arteritis patients (7,29). Thus, it appears that the ability to modulate EC activation in vitro is a common characteristic of AECA present in different diseases that share in common an immune-mediated endothelial inflammation.
The endothelial stimulation is dose dependent and the effective concentrations are comparable to those previously reported for a murine mAb reacting with HUVEC (29) and for human AECA mAb from Takayasu's patients (7). The effective concentrations are comparable with those that demonstrate binding to whole live EC except in the case of E-selectin, which was up-regulated at concentrations lower than either ICAM-1 or IL-6. However, the dose that up-regulates E-selectin is comparable with that which up-regulates U937 adhesion. In this regard, it is useful to point out that we found that E-3-induced IL-1ß secretion is able to further sustain endothelial activation through an autocrine loop, as previously shown for other polyclonal AECA (8,10,31). Thus, it is likely that low mAb amounts, even lower than those able to give the optimal antibody binding, might support E-selectin up-regulation either directly or through the autocrine effects of cytokines produced by the EC. An autocrine mechanism is supported by the studies of Carvalho et al. who showed that polyclonal SLE AECA IgG induce the release of at least two mediators, one as yet unidentified and the other IL-1, which, over different time courses, mediate EC activation (10). The possibility that E-3 mediates its effects by stimulating multiple autocrine factors may explain the different doseresponse thresholds to E-3 of activation markers such ICAM-1 and E-Selectin.
Previous studies have shown activation of EC in response to cytokines such as TNF- requires activation of both JNKMAP kinases and NF-
B, although it is thought that these are on parallel pathways (33,34). This data suggests that activation of one pathways can occur in response to an appropriate stimulus without the recruitment of the other, and this activation is likely to lead to up-regulation of adhesion molecules and cytokine production.
Although the exact identity of the antigen recognized by E-3 remains unclear, the whole live cell binding data support the hypothesis that the antibody recognizes a cell-surface antigen. Auto-antibodies reactive with non-constitutive EC antigens have been described previously as pro-inflammatory. It has been demonstrated that antibodies reactive with ß2GPI are pro-inflammatory, and stimulate both cytokine production and adhesion molecule expression (14,15,35,36). Chan et al. (13) have demonstrated that anti-DNA antibodies are capable of exerting pro-inflammatory effects on EC. Both of these antigens are planted antigens in that they are not normally expressed on EC surfaces. The lack of reactivity of E-3 against ß2GPI and DNA and its preliminary characterization suggest it appears to react with a constitutive antigen of EC membranes as described for the murine mAb derived from an AECA-associated experimental model of vasculitis (29).
Furthermore, this is the first demonstration that an AECA mAb from a patient with lupus can strongly activate EC and suggests E-3 mediates its effects by transmembrane signaling, the usual route for activation of NF-B. Although comparable data have been recently reported with human AECA mAb obtained from Takayasu's patients (7), further work is required with other pro-inflammatory AECA mAb to determine whether this is a common activation pathway.
Previous studies, which have demonstrated a pro- inflammatory effect of AECA, are subject to two major criticisms. First, that contamination by bacterial lipopolysaccharide could induce the same effect; however, treatment of the mAb with Polymixin B failed to alter the pattern of cytokine and adhesion molecule expression, thus eliminating this explanation for these data. The second criticism is that even purified IgG preparations may be contaminated by cytokines, particularly as they have been isolated from patients who have high levels of circulating cytokines (16,17). This criticism cannot be applied to mAb that have been in culture for as long as 2 years since the original isolation. Furthermore, other mAb from normal individuals and patients failed to have the same effect. Thus, the only interpretation of these data is that E-3 is a pro-inflammatory auto-antibody.
This in vitro study using a human mAb derived from a patient with SLE strongly supports the pro-inflammatory activity of AECA. In concert with the large body of evidence correlating their occurrence with disease symptoms and severity, and the in vitro pro-inflammatory effects of patients' sera, these data point in favor of a potential pathogenetic role for AECA in SLE.
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Acknowledgments |
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Abbreviations |
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AECA anti-endothelial cell antibodies |
ß2GPI ß2-glycoprotein I |
CL cardiolipin |
EC endothelial cell |
ECM endothelial cell membrane |
HMEC-1 human microvascular endothelial cell line |
HUVEC human umbilical cord vein endothelial cell |
IL-1ra IL-1 receptor agonist |
PBL peripheral blood lymphocyte |
SLE systemic lupus erythematosus |
TNF tumor necrosis factor |
TTP thrombotic thrombocytopenic purpura |
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Notes |
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Received 21 August 2000, accepted 6 December 2000.
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References |
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