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(The FASEB Journal. 2003;17:136-143.)
© 2003 FASEB

Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice

STEPHANIE PLANQUE*, YONG-XIN ZHOU*, YASUHIRO NISHIYAMA*, MEENAL SINHA*, MAUREEN O’CONNOR-MCCOURT{dagger}, FRANK C. ARNETT* and SUDHIR PAUL*1

* Chemical Immunology and Therapeutics Research Center, Departments of Pathology and Internal Medicine, University of Texas-Houston Medical School, Houston, Texas, USA; and
{dagger} Biotechnology Research Institute, National Research Council Canada, Montreal, Canada

1Correspondence: Department of Pathology, MSB 2.250, 6431 Fannin, University of Texas-Houston Medical School, Houston, TX 77030, USA. E-mail: Sudhir.Paul{at}uth.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autoantibodies to the recombinant extracellular domain of epidermal growth factor receptor (exEGFR) were detected by ELISA in the serum of Fas-defective old MRL/MpJ/lpr and C3H/HeJ/gld mice, but not young mice from these strains, or nonautoimmune young and old BALB/c, MRL/MpJ/++, and C3H/HeJ/MMTV mice. Compared with control human subjects without autoimmune disease, the frequency of exEGFR-binding autoantibodies was increased in scleroderma (systemic sclerosis) patients and to a lesser extent in lupus patients. Phage autoantibodies (Fv fragments) isolated from a lupus library by selection on a linear epitope of EGFR (residues 294–310) displayed the ability to bind exEGFR. Treatment of EGFR-expressing A431 cells with autoantibodies purified by affinity chromatography on immobilized exEGFR resulted in specific staining of the cells. Short-lived but strong inhibition of cellular DNA synthesis was observed in the presence of the autoantibodies. We concluded that autoantibody responses to EGFR hold the potential of fulfilling a pathogenic role in autoimmune disease.—Planque, S., Zhou, Y.-X., Nishiyama, Y., Sinha, M., O’Connor-McCourt, M., Arnett, F.C., Paul, S. Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice.


Key Words: Fas defects • EGFR • antibody library


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE EPIDERMAL GROWTH factor receptor (EGFR) is a 170 kDa transmembrane protein belonging to the tyrosine kinase type I family. As EGFR is overexpressed in many tumors, antibodies capable of interfering with EGFR-stimulated cell proliferation have been proposed as reagents for cancer therapy (1) . However, EGFR is also found in noncancerous cells. The importance of EGFR as a mediator of cell growth in the fetus and in early development is unquestioned (2 , 3) . For instance, newborn EGFR-knockout mice display profound abnormalities in multiple organ systems (4) . The physiological functions of the receptor in adults, on the other hand, are not well established, but a pleiotropic role is likely. EGFR has been implicated as a regulator of cell growth in certain adult tissues, including airway remodeling in response to stress (5) , gastrointestinal mucosal cell regeneration in ulcers (6) , angiogenesis (7) , and menstrual cycle associated events in the ovary and uterus (8 , 9) . Perturbations of EGFR expression have been noted in patients with systemic sclerosis (SSc) (10) , psoriasis (11) , asthma (5) , and dementia (12) .

Here, we describe for the first time the existence of autoantibodies to the extracellular domain of EGFR (exEGFR) in Fas-defective mice and in subpopulations of patients with SSc and systemic lupus erythematosus (SLE). Short-lived but strong inhibition of cellular DNA synthesis in the presence of the autoantibodies was observed, indicating their potential to influence cellular growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Polyclonal antibodies
Sera from young mice (5–6 wk) and old mice (24–32 wk) of the following strains were obtained by retrorbital bleeding: MRL/MpJ-Faslpr (MRL/lpr), MRL/MpJ/++ (Mrl/++), C3H/HeJ/gld (C3H/gld), C3H/HeJ/MMTV (C3H/++), and BALB/c (Jackson Laboratories, Bar Harbor, ME). Serum samples from SLE (n=34) and SSc patients (n=42) were obtained by the Division of Rheumatology, University of Texas-Houston Medical School during the course of other institutionally approved research studies. The SLE family member group was composed of healthy first-degree relatives of SLE patients identified by one of us (Arnett) in unrelated genetic studies. Rheumatoid arthritis patients (RA; n=10) were from the rheumatology clinic at the University of Nebraska Medical Center. Patient selection was based on the classification criteria of The American College of Rheumatology for SSc (13) , SLE (14) , and RA (15) . Thirty age- and gender-matched individuals without a history of autoimmune disease served as controls (10 whites, blacks, and Hispanics each). Purification of IgG was by affinity chromatography on immobilized protein G-Sepharose (16) . Affinity purified antibodies to exEGFR were obtained by fractionation of IgG [10 mg in 10 mM sodium phosphate, pH 7.4, 0.137 M NaCl, 2.7 mM KCl (PBS); SLE IgG pooled from patients 657, 670, 1017, 1016; SSc IgG pooled from patients 95, 111] on 0.15 mg exEGFR immobilized via NH2 groups on an Affi-Gel 10 column (Bio-Rad; 0.4 mL settled gel, coupling efficiency ~75%). After removal of unbound IgG with PBS, bound protein was eluted with 6 vol of 0.1 M glycine-HCl, pH 2.7, 0.02% NaN3 and neutralized with 1 M Tris-HCl, pH 9.0. Estimation of IgG was done using NanoOrange protein quantitation kit (Molecular Probes, Eugene, OR).

Recombinant Fv
EGFR-binding Fv constructs were isolated from a single chain Fv phage library derived from two SLE patients cloned in pHEN2 vector (library size 1.4x107) (17) . Phage particles displaying Fv-g3 fusion proteins (5x1012 colony forming units) were packaged from TG1 cells, precipitated with polyethylene glycol, and subjected to selection on a biotinylated synthetic peptide corresponding to residues 294–310 of EGFR (Bt-MEEDGVRKCKKCEGPCR; Bt-EGFRpep). The peptide was synthesized by the Protein Core Lab at the Baylor College of Medicine, and its identity was confirmed by mass spectrometry. Bt-EGFRpep (5 µg) was incubated with the phages for 2 h [25°C, 0.5 mL, 20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 2% skim milk; phages were preadsorbed on UltraLink Streptavidin gel (Pierce, Rockford, IL) in 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 2% skim milk followed by incubation for 30 min with 1 mM diisopropylfluorophosphate]. Bound phages were immobilized by shaking for 1 h with 350 µl streptavidin gel, and the gel was poured into a column (0.7x6 mm) and washed with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl until A280 returned to <0.01. Bound phages were eluted with 0.1 M glycine-HCl, pH 2.7, followed by 0.1 M triethylamine, pH 12 (collected in appropriate volumes of 2 M Tris base and acid, respectively, to neutralize the eluates). The first low pH fraction was discarded, and 128 individual clones were grown in 24-well plates from 20 mL of the low pH eluate and 24 mL of the high pH eluate for screening of EGFR binding by soluble Fv. To this end, HB2151 cells were infected with 0.5 mL eluate phages and induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (23°C) and the medium was collected by centrifugation. Fv from periplasmic extracts was purified by metal affinity chromatography to >95% homogeneity by SDS-polyacrylamide gel electrophoresis (8–25% Phast gels, Pharmacia; the constructs contain a carboxyl-terminal his6 tag) (18) . Before ELISA, purified Fv was dialyzed against PBS. Expression levels determined by immunoblotting for the c-myc tag were 0.26–19 mg/L in medium and 0.25–16 mg/L in periplasmic extracts. cDNA sequences were analyzed for germline gene origin and family and subgroup assignment using NCBI IgBlast and Kabat databases (http://www.ncbi.nlm.nih.gov/BLAST; http://immuno.bme.nwu.edu; ref 19 ).

ELISA and immunoblotting
Ninety-six-well plates (Becton-Dickinson, Franklin Lakes, NJ) were coated with 100 µl of 2 µg/mL recombinant exEGFR (20) in 100 mM NaHCO3, pH 8.6, for 1 h, and the plates were washed with wash buffer (10 mM sodium phosphate, 0.137 M NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 7.4), blocked with 200 µl blocking buffer (5% BSA in wash buffer, 1 h), and incubated with test samples containing antibodies for 2 h in this buffer (37°C). After additional washing (3x), bound murine IgG was detected using 100 µl of a goat anti-mouse IgG-peroxidase conjugate (Fc specific, 1:500 antibody buffer, 1 h (Sigma Chemical Company, St. Louis, MO). Detection of bound human IgG was with 100 µl goat anti-human IgG (Fc specific, 1:1000, Sigma) followed by 100 µl of a rabbit anti-goat IgG-peroxidase conjugate (whole molecule, 1:1000, Sigma, 1 h). Bound Fv was detected similarly, using monoclonal mouse anti-c-myc (1:500; delipidated ascites from hybridoma 9E10, ATCC) and goat anti-mouse IgG-peroxidase (Fc specific, Sigma). Peroxidase activity was measured at A490 with an ELISA plate reader (Bio-Rad) (18) . ELISA for Bt-EGFRpep binding was performed using Maxisorp plates (Nunc, Rockchester, NY) coated with 10 µg/mL streptavidin (Sigma) followed by 5 µg/mL Bt-EGFRpep (1 h). Certain sera and IgG samples displayed binding to BSA-blocked wells in the absence of immobilized exEGFR (BSA-coated wells). To eliminate nonspecific binding effects from the data, values of exEGFR binding reported here were computed as: (A490exEGFR coated–A490BSA coated). Antibody binding to immobilized calmodulin (Sigma) and the extracellular domain of tumor necrosis factor receptor type 1 (TNFR1; residues 22–211, ref 21 ; R&D Systems, Minneapolis, MN) was measured essentially as described for exEGFR.

exEGFR run on SDS gels was electroblotted onto nitrocellulose and stained with affinity purified human anti-EGFR antibodies followed by goat anti-human IgG and peroxidase conjugated rabbit anti-goat IgG (18) . As positive controls, the exEGFR was stained with a monoclonal anti-EGFR antibody (clone c225; Labvision, Fremont, CA) or polyclonal murine antibodies to the synthetic EGFR peptide composed of residues 294–310, followed by peroxidase conjugated goat anti-mouse IgG. The anti-peptide antibodies were raised by immunization of 4- to 5-wk-old MRL/lpr mice with 100 µg of EGFR(294–310) conjugated to keyhole limpet hemocyanin (KLH) in Freund’s complete adjuvant (i.p.) followed by four boosters in Freund’s incomplete adjuvant at 14 day intervals (i.p.), with the hyperimmune serum obtained from blood drawn 7 days after the last booster. Cys residues of the peptide were conjugated to Lys sidechains using {gamma}-maleimidobutryic acid N-hydroxysuccinimide ester (Sigma) according to Sambrook et al. (22) . The degree of conjugation determined from measurement of free peptide SH groups using Ellman’s reagent was 279.1 mol peptide/mol KLH.

Antibody staining of cells
A431 (ATCC, CRL1555) cells were grown on Lab-Tek slides chamber (2x105 cells; Nunc) until 50–60% confluence had been reached, fixed for 10 min with 3% formaldehyde in PBS, washed three times with PBS, and then treated with 5% goat serum (Sigma; in PBS, 1 h). Autoantibodies purified by affinity chromatography on immobilized exEGFR were incubated with the cells in PBS containing 1% goat serum (1 h), followed by three washes, incubation with goat anti-human IgG conjugated to fluorescein isothiocyanate (1 h; Fc specific, 1:64; Sigma), and three washes and microscopic examination under ultraviolet illumination (Nikon Labphot-2; {lambda}ex 450–590 nm; equipped with video capture camera, Optronics Engineering, Goleta, CA). Controls included incubation of the cells with 1) binding buffer; 2) nonimmune human antibodies instead of anti-EGFR autoantibodies (unbound fraction obtained by chromatography of IgG from a control healthy subject devoid of exEGFR binding activity on immobilized exEGFR), and 3) anti-EGFR antibodies previously treated with with exEFGR (10 µM; 1 h). Staining of cells with monoclonal mouse anti-EGFR (clone c225) was done using FITC-conjugated Fab’2 fragments of goat anti mouse IgG + IgM (Jackson Immunoresearch, West Grove, PA). Control mouse monoclonal IgG from a myeloma cell line (UPC10) was from Sigma.

DNA synthesis
A431 cells grown in T-75 flasks were treated with 0.25% trypsin/0.03% EDTA (Sigma) in DMEM to detach the cells, fetal bovine serum (FBS) was added to 10% vol/vol, the cells washed once and then allowed to attach to 96-well plates (Nunc; 8000 cells/well) for 24 h in culture medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 U/mL Nystatin) (37°C, 5% CO2). Incubation was continued in 50 µl fresh culture medium containing human autoantibodies purified by affinity chromatography on exEGFR. Bromodeoxyuridine (BrdU) was added to 10 µM in each well 5 h before fixation with ethanol and measurement of BrdU incorporation by an ELISA Kit (Roche Molecular Biochemicals, Indianapolis, IN). Controls included incubation of the cells with equivalent concentrations of nonimmune antibodies (unbound fraction obtained by chromatography of IgG without exEGFR binding on the immobilized exEGFR column), incubation in culture medium without antibodies, and incubation with antibodies but without BrdU (background ELISA reaction).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Murine autoantibodies to exEGFR
Recombinant human exEGFR (residues 1–621) used as antigen in the present study is an electrophoretically homogeneous protein (85 kDa) with intact EGF binding activity (20) . Pooled sera from autoimmune mouse strains with defects in the Fas ligand gene (C3H/gld) and the Fas receptor gene (MRL/lpr) were analyzed for binding to immobilized exEGFR (Fig. 1 ). Sera from nonautoimmune wild-type strains MRL/++ and C3H/++ and from BALB/c mice served as the controls. Sera from young autoimmune mouse sera did not express binding activity, whereas the binding was evident in older autoimmune mice (24–32 wk). No binding to exEGFR by sera from young (4–6 wk) as well as old (24–32 wk) nonautoimmune mice was detected [MRL/++; C3H/++ (not shown) and BALB/c mice (not shown)]. Individual sera (1:50 dilution) from all five MRL/lpr and seven C3H/gld mice analyzed displayed detectable exEGFR binding activity (mean A490 ± SE: 0.22 ± 0.04 and 0.23 ± 0.08, respectively).



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  		Figure 1. Autoantibodies to exEGFR in old Fas deficient mice. Equivalent volumes of sera from the indicated mouse strains were pooled and analyzed for binding to immobilized exEGFR by ELISA. A: MRL/MpJ-Faslpr; B: MRL/MpJ/++; C: C3H/HeJ/gld.

Human autoantibodies to exEGFR
Equivalently diluted sera from nonautoimmune individuals, nonsymptomatic first degree relatives of SLE patients (Fam-SLE) and patients with SLE, SSc, and RA were assayed for exEGFR binding antibodies (Fig. 2 ). Readily detectable binding activity by several sera was evident. Most striking was the increased binding in the SSc group (P<0.0001, 2-tailed Mann-Whitney rank order U test; P=0.014, 2-tailed Student’s t test; vs. nonautoimmune control group). Significantly increased binding was also evident according to the U test in the SLE and Fam-SLE groups (P<0.05) but not the RA group. According to the t test, the binding was statistically indistinguishable in these groups compared to the nonautoimmune group (P>0.1). One outlier nonautoimmune subject displayed reproducibly elevated EGFR binding activity (A490~2.0). The nonautoimmune group was composed of 10 black, white, and Hispanic individuals each. No differences of EGFR binding were evident in these subgroups (P>0.1). A review of the clinical histories of the SSc and SLE subpopulation positive for exEGFR autoantibodies did not identify any clear relationship between the binding activity and clinical symptoms. [14 SSc and 5 SLE patients with A490 values > mean + 3 SD (mean, 0.064; SD, 0.044; outlier nonautoimmune subject was ignored in these computations)].



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  		Figure 2. Autoantibodies to exEGFR in humans with autoimmune disease. Each data point represents serum (1:250) from different subject (means of 2 closely agreeing replicates). Indicated P values are vs. control non-autoimmune subjects (C; Mann-Whitney U test, 2-tailed). SSc, systemic sclerosis; SLE, lupus; Fam-SLE, nondiseased family members of SLE patients; RA, rheumatoid arthritis patients.

Confirmatory evidence that antibodies are responsible for the binding activity was obtained by analysis of IgG purified from the sera of four patients by protein G-Sepharose chromatography. Concentration-dependent exEGFR binding by the IgG was evident (Fig. 3 ). Binding of equivalently purified IgG from a nonimmune subject was not detected. Further chromatography of the IgG on immobilized exEGFR resulted in near complete recovery of the ELISA reactivity in the acid eluates from the column (63–99% recovery from 2 column runs each of SLE IgG and SSc IgG) and considerably increased exEGFR binding activity (average 66-fold increase of A490 ELISA signal per unit protein compared to the unfractionated IgG; Fig. 4 ). The IgG fractions purified by affinity chromatography on immobilized exEGFR displayed no detectable binding to TNFR1 or calmodulin (A490<0.1). SDS-PAGE and immunoblotting confirmed the reactivity of affinity purified autoantibodies with exEGFR (Fig. 4) .



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  		Figure 3. Concentration-dependent exEGFR binding by purified autoimmune IgG. IgG samples purified by protein G-Sepharose chromatography from two SSc patients (A) and two SLE patients (B) were analyzed. Values are means of 3 replicates ± SD.



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  		Figure 4. Affinity purification of the autoantibodies on immobilized exEGFR. Pooled IgG (10 mg) from SSc (patients 95, 111) or SLE patients (patients 657, 670, 1017, 1018,) was allowed to bind immobilized exEGFR gel (16 h, 4°C), the gel packed into a column, and the flow-through fractions and pH 2.7 eluate were analyzed for exEGFR binding by ELISA (1:20 dilution). Data are expressed as A490 ELISA values normalized for IgG concentration. A490 values corrected for background A490 values were SSc exEGFR 0.30, SSc protein G 0.22, SLE exEGFR 0.18, SLE protein G 0.322 (exEGFR bars, affinity purification on immobilized exEGFR, protein G bars, total IgG). Bottom: immunoblot of SDS gels showing exEGFR stained with murine anti-exEGFR(294–310) (lane 1, 0.2 µM), lupus affinity purified acid eluate from the exEGFR column (lane 2, 0.8 µM), and the unbound IgG from the column (lane 3, 0.8 µM).

Lupus Fv-exEGFR binding
exEGFR-binding Fv constructs were isolated by selecting an SLE phage display library on a biotinylated synthetic peptide corresponding to residues 294–310 of EGFR (Bt-EGFRpep). Antibodies to this peptide raised by experimental immunization are capable of binding full-length EGFR expressed on the cell surface (23 , 24) . Of 128 Fv clones screened following selection on immobilized Bt-EGFRpep, the culture media from 13 clones were positive for Bt-EGFRpep binding, and from 6 clones, for both Bt-EGFRpep as well exEGFR binding (A490 3 SD>control culture medium from cells harboring vector without Fv inserts; A490 range 0.27–1.13). Two Fv constructs were purified from the periplasmic extract by metal affinity chromatography. Concentration-dependent binding of these Fv constructs by immobilized exEGFR and immobilized Bt-EGFR (294–310) was observed (Fig. 5 ).



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  		Figure 5. Bt-EGFR(94–310) (A) and exEGFR (B) binding by recombinant lupus Fv constructs. See Materials and Methods for details.

Analyses of cDNA sequences indicated extensive mutations in the VL genes of the Fv constructs compared to the germline counterparts (Table 1 ; GenBank accession numbers clone SP1A2, AF294557; clone SP7D2, AF329456). Ten replacement mutations are evident in the VL domain of Fv SP1A2, of which nine are located in the CDRs. In this clone, the ratio of replacement to silent mutations was overwhelmingly greater in the VL CDRs compared to the FRs, suggesting that it has been subjected to V region affinity maturation. Mutations in the VL domain of clone Fv SP7D2 appear to be distributed randomly, judged by their near-equivalent presence in the CDRs and FRs. No mutations were evident in the VH CDRs of either clone.


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  		Table 1. Fv mutation status and germline origina

Binding to cellular EGFR
exEGFR binding antibodies were prepared by affinity chromatography of pooled SSc and SLE IgG. Binding of anti-exEGFR autoantibodies by EGFR-expressing A431 cells (squamous epithelial cell line) was determined by immunofluorescent staining. Both sets of autoantibodies (SSc and SLE) stained the cells specifically, judged by comparison with the unbound antibodies (flow-through) recovered by passing IgG from a healthy control subject through immobilized exEGFR. Moreover, the staining was reduced by preincubation of the autoantibody preparation with soluble exEGFR. Representative micrographs are presented in Fig. 6 . Diffuse as well as speckled staining of the cells by the autoantibodies was evident. The pattern of autoantibody staining was distinct from that observed with a commercially available monoclonal anti-EGFR antibody (c225).



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  		Figure 6. Binding of autoantibodies by cellular EGFR. A: SSc IgG; B: SSc IgG preincubated with 10 µM exEGFR; C: SLE IgG; D: nonimmune human IgG; E: mouse monoclonal anti-EGFR antibody (c225); F: nonimmune mouse monoclonal IgG (UPC10). Human IgG, 1 µM; mouse monoclonal antibodies, 0.7 µM. SSc and SLE IgG prepared by chromatography on immobilized protein G and immobilized exEGFR (x100).

Effects on A431 cells
The effect of exEGFR-specific autoantibodies on BrdU incorporation by A431 cells was analyzed. This cell line is susceptible to the growth inhibitory effects of certain monoclonal anti-EGFR antibodies (25) . FBS present in these cultures is the source of the ligand (EGF/TGF-ß) responsible for stimulation of EGFR. Time-dependent inhibition of BrdU incorporation by SSc and SLE autoantibodies was evident (Fig. 7 ). The inhibitory effect after 10 h incubation with the autoantibodies was highly reproducible in each of four experiments (P<0.03 for SSc and SLE preparations vs. nonimmune IgG; two-tailed unpaired t test). After 20–24 h, the level of inhibition was consistently lower, and statistically significant inhibition was observed only in two experiments using SLE autoantibodies and none of the experiments using SSc autoantibodies. No loss of cell viability was observed following 24 h incubation with the SSc and SLE IgG as determined by staining with trypan blue (>85% viability). The concentration dependence of the inhibitory effect was studied using SSc autoantibodies. At autoantibody concentrations of 0.1 and 1 µM (10 h treatment), 12.2 ± 2.7 and 26.0 ± 7.8% inhibition was evident, respectively.



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  		Figure 7. Effect of anti-EGFR autoantibodies on DNA synthesis by A431 cells. Cells (8000/well) were grown in the presence of 1 µM pooled SSc IgG, SLE IgG and nonimmune IgG. BrdU was added 5 h before termination of cultures. Values are means of 3 wells relative to incorporation observed in nonimmune IgG treated cells (A450 at 5, 10 and 20 h: 0.52, 0.61 and 0.65 respectively; SD <0.14 in each case). SSc and SLE IgG prepared by chromatography on immobilized protein G and immobilized exEGFR. P < 0.03 for SLE and SSc IgG at 10 h and SLE IgG at 20 h; vs. nonimmune IgG.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Disturbances in signal transduction mediated by EGFR have been implicated previously in several disorders, including SSc, psoriasis, and asthma (5 , 10 , 11) . To our knowledge, the present report is the first indication that the receptor is subject to autoantibody-induced dysfunction. The autoantibodies were identified and measured based on their ability to recognize the exEGFR in ELISA studies. Cells known to express EGFR (A431 cell line) were also stained specifically by autoantibodies from SSc and SLE patients isolated by affinity chromatography on immobilized exEGFR. We concluded that the autoantibodies recognize the native cellular form of EGFR. Affinity- purified IgG preparations failed to bind TNFR1 and calmodulin under conditions in which EGFR binding was readily detectable. The fine specificity of the autoantibodies remains to be studied, but it may be concluded that the EGFR binding is not an expression of a polyreactive binding pattern.

Reduced apoptosis due to defects in the Fas receptor gene and the Fas ligand gene results in uncontrolled lymphoproliferation and increased autoantibody production, eventually resulting in spontaneous appearance of SLE-like autoimmune disease in the lpr and gld mouse strains. Aged Fas receptor-defective lpr mice and Fas ligand-defective gld mice were positive for exEGFR binding autoantibodies, whereas the control wild-type mice were not. The disease-association of the human autoantibodies to exEGFR is statistically unambiguous, particularly for SSc patients. Rank order binding activities were significantly increased in SLE patients and in nonsymptomatic relatives of SLE patients, but the mean binding values in these groups were statistically indistinguishable from nonautoimmune controls. Autoimmune recombinant Fv constructs with EGFR binding activities were cloned to permit future studies of autoantibody functional effects. The two EGFR-binding Fv constructs are derived from distinct germline genes and express varying levels of mutations. Combinatorial pairing of VL/VH domains occurs randomly in the library, without assurance about the representation of natural VL-VH pairs (26) . However, the individual VL/VH domains often bind antigens independently. Moreover, the antigen binding activity of the individual V domains is sufficient to impart high affinity antigen activity to the Fv construct, even when paired with a promiscuous partner (27 , 28) . Thus, the presence of mutations localized mainly in the VL CDRs (clone SP1A2, Table 1 ) supports the hypothesis of V region affinity maturation-driven development of EGFR binding activity.

EGFR plays a pleiotropic role in cell fate determination, differentiation, proliferation, and apoptosis (29 , 30) . As the initial step in delineating the functional role of the autoantibodies, their effect on DNA synthesis in A431 cell cultures was studied. These cells are susceptible to the growth inhibitory and proapoptotic effects of monoclonal anti-EGFR antibodies under consideration for passive immunotherapy of cancer (25 , 31) . Short-lived but strong inhibition of DNA synthesis by the SSc and SLE autoantibodies was observed, supporting the possibility that the autoantibodies may exert important biological effects. However, several caveats can be seen in further interpretation of this finding. Polyclonal antibodies are composed of individual species that may differ with respect to epitope specificity and other immunochemical characteristics, and different individuals within the disease groups studied here may mount distinct patterns of autoantibody responses. There is no assurance, therefore, that inhibition of DNA synthesis is a uniform autoantibody effect. Similarly, the mechanism of reduced DNA synthesis is open to question. No inhibition of 125I-EGF binding by EGFR expressed on A431 cells was evident in the presence of EGFR binding autoantibodies purified from a patient with lupus, and EGF-induced tyrosine kinase activity of cellular EGFR was also unaffected by the autoantibodies, determined by measuring receptor autophosphorylation (Planque and Paul, unpublished results). In addition to the EGF binding site, the extracellular domain of EGFR contains binding sites for the ligands TGF-ß, amphiregulin, heparin-binding EGF-like growth factor, epiregulin, and betacellulin (29) ; the ability of the autoantibodies to recognize these sites remains to be studied. Similarly, EGFR signal transduction may entail multiple, independent pathways (e.g., EGFR directly binds and activates calmodulin, independent of its tyrosine kinase activity; ref 32 ), opening additional routes that may mediate autoantibody effects. Additional mechanisms subject to interference by autoantibodies are an accelerated removal of surface expressed-EGFR due to internalization of antibody-EGFR complexes; antibody effects on EGFR dimerization and activation; allosteric regulation of EGFR by antibody binding at an epitope distant from the ligand binding site; and stimulation of compensatory EGFR synthesis. The foregoing phenomena have been documented using monoclonal antibodies to the extracellular domain of EGFR (25 , 31 , 33 34 35) , and EGFR overexpression is observed in certain diseases (see next paragraph). Furthermore, our DNA synthesis studies were restricted to a tumor cell line (A431), and differences in autoantibody effects on various cell types can not be excluded, as the magnitude of EGFR expression in various cells can vary dramatically. Thus, no firm conclusions can be made at this stage about the precise functional consequences of the autoantibodies in SSc and SLE. Availability of homogeneous and renewable recombinant autoantibodies described in the present study, however, can be anticipated to help delineate the role of the autoantibodies in future studies.

SSc is characterized by extensive fibrosis of skin and visceral organs. Fibroblasts in SSc patients display an activated phenotype and produce abundant quantities of various collagens and other extracellular matrix components. Autoantibodies to the matrix components are generally infrequent, but recently autoantibodies to fibrillin-1, the major component of microfibrils, have been described in the majority of SSc patients (36) . Fibrillin-1 is well known to contain domains bearing certain sequence identities to EGF, which serves as a ligand for EGFR (37) . However, there is no noticeable sequence similarity between EGFR itself and fibrillin-1 (assessed with the program BLOSUM62, Blastp). Overexpression of EGFR has been observed previously in SSc patients (10) , which may be linked to increased output of connective tissue components from fibroblasts (38) . There is no evidence of altered EGFR expression levels in SLE, but increased expression of the protein has been reported in renal damage due to other causes (39) . It remains to be determined whether autoantibody formation results from loss of tolerance due to overexpression of EGFR. Conversely, EGFR overexpression may be a compensatory phenomenon induced by the autoantibodies.

Identification of autoantibodies to EGFR serves as another example of the interesting link between tumor immunology and autoimmune disease (reviewed in ref (40) . Because of their growth inhibitory properties, certain monoclonal anti-EGFR antibodies are described to induce tumor remission alone or in combination with other anti-tumor agents (25 , 31 , 41) . If EGFR is assumed to help maintain the balance between cell death and cell growth/tissue regeneration in adults, the antibodies carry the risk of inducing autoimmune dysfunction. Conversely, demonstration of autoantibody responses to EGFR in the present study may be interpreted as showing the potential of the immune system in modulating susceptibility to cancer. Definitive conclusions about this point, however, are beyond the scope of the present report. Polyclonal immune responses in disease can be composed of antibodies with diverse biological actions on different types of cancer cells, potentially including growth-inhbitory as well as growth-stimulating antibodies. Previous epidemiological studies have noted an increased predisposition toward certain types of cancers in SSc patients (e.g., ref (42) .


   ACKNOWLEDGMENTS
 
This work was supported by USPHS Grants HL-44126, AI-31268, and CA-80312 and a Specialized Center of Research Grant in scleroderma (P50AR44888).

Received for publication November 11, 2001. Accepted for publication September 29, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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S. Planque, H. Taguchi, G. Burr, G. Bhatia, S. Karle, Y.-X. Zhou, Y. Nishiyama, and S. Paul
Broadly Distributed Chemical Reactivity of Natural Antibodies Expressed in Coordination with Specific Antigen Binding Activity
J. Biol. Chem., May 23, 2003; 278(22): 20436 - 20443.
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