BULLOUS SYSTEMIC lupus erythematosus

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1 OBSERVATION Bullous Systemic Lupus Erythematosus With Autoantibodies Recognizing Multiple Skin Basement Membrane Components, Bullous Pemphigoid Antigen 1, Laminin-5, Laminin-6, and Type VII Collagen Lawrence S. Chan, MD; Jean-Christophe Lapiere, MD; Mei Chen, PhD; Tom Traczyk, BS; Anthony J. Mancini, MD; Amy S. Paller, MD; David T. Woodley, MD; M. Peter Marinkovich, MD Background: Bullous systemic lupus erythematosus is a generalized subepidermal blistering skin eruption in patients suffering from systemic lupus erythematosus. Type VII collagen was initially identified as the target antigen. Observation: We studied an unusual patient who had bullous systemic lupus erythematosus. The patient fulfilled the criteria of systemic lupus with an antinuclear antibody titer of 1:5120. Immunopathological testing revealed in vivo deposition of all subclasses, secretory 1, and both light chains at the patient s skin basement membrane. The in vivo bound and were localized at the hemidesmosomes and lamina densa. The patient s and circulating autoantibodies labeled both the epidermal roof and the dermal floor of saltsplit skin and recognized the hemidesmosomal protein BP230 as well as the full-length native form and the recombinant noncollagenous domain 1 of type VII collagen (anchoring fibril). In addition, the patient s autoantibodies recognized the anchoring filament proteins laminin-5 and laminin-6 ( 3 chain and 2 chain). Conclusions: We conclude that patients with bullous systemic lupus erythematosus may have autoantibodies to multiple basement membrane components critical for epidermal-dermal junctional adhesion. Possible pathogenic mechanisms in this patient s clinical diseases include provocation of organ-specific disease (bullous disease) by systemic autoimmunity (lupus) and the epitope spreading immune phenomenon. Arch Dermatol. 1999;135: From the Medicine Service, Section of Dermatology, Lakeside Division, Veterans Affairs Chicago Health Care System (Dr Chan), and the Departments of Dermatology (Drs Chan, Lapiere, Chen, Mancini, Paller, and Woodley and Mr Traczyk) and Pediatrics (Drs Mancini and Paller), Northwestern University Medical School, Chicago, Ill; the Department of Dermatology, Stanford University School of Medicine, Stanford (Dr Marinkovich), and the Dermatology Service, Palo Alto Veterans Affairs Health Care System, Palo Alto (Dr Marinkovich), Calif. BULLOUS SYSTEMIC lupus erythematosus (BSLE) is an autoantibody-mediated blistering skin disease occurring in patients with systemic lupus erythematosus (SLE). 1-9 In addition to having clinical and laboratory findings that fulfill the American Rheumatism Association diagnostic critieria of SLE, 2 patients with BSLE have a generalized subepidermal blistering skin disease with immunoglobulin deposition along the skin basement membrane zone (BMZ). 1,3-9 Some patients had detectable circulating autoantibodies that labeled the dermal floor of chemically separated normal skin substrate, indicating that the target antigen was located below the middle of the lamina lucida Some patients with BSLE had autoantibodies that recognized the major anchoring fibril component type VII collagen The antigenic domains recognized by BSLE autoantibodies were within the noncollagenous (NC1) domain, identical to the antigenic domains recognized by autoantibodies from patients with epidermolysis bullosa acquisita. 14 More recently, other investigators have reported indirect immunofluorescence findings suggesting that type VII collagen may not be the exclusive target antigen in BSLE. 15 In this article, we report an unusual patient with BSLE whose autoantibodies labeled both the epidermal roof and the dermal floor of chemically separated normal skin substrate. We took the opportunity to delineate the multiple skin BMZ components recognized by this patient s autoantibodies and to discuss the possible mechanisms responsible for such unusual immune responses. REPORT OF A CASE A 15-year-old female patient was admitted to Children s Memorial Hospital, Chicago, Ill, for a persistent generalized bullous eruption. Two months prior to admission, the patient was diagnosed with dermatitis herpetiformis and was treated with dapsone resulting in partial clearing of the skin lesions. The patient reported painful ulcers in her mouth and on her lips, dysphagia, arthralgia, malaise, lethargy, 569

2 METHODS AND RESULTS HISTOPATHOLOGICAL FINDINGS A skin biopsy specimen from a blister fixed in formalin, processed in paraffin, and stained with hematoxylin-eosin revealed a subepidermal blister with intact epidermis. Epidermal necrosis and acantholysis were not observed. An inflammatory cell infiltrate in the papillary dermis and the blister cavity included predominantly neutrophils (99%) and trace eosinophils (1%). Moderately dense mononuclear cell perivascular infiltrates were noted on upper dermis (data not shown). IMMUNOFLUORESCENCE STUDIES Direct immunofluorescence microscopy was performed on 6-µm-thick cryosections of the patient s perilesional skin biopsy specimen, with fluorescein-conjugated goat antihuman,, IgM, C3, and fibrinogen (Immco, Buffalo, NY). 16 Direct immunofluorescence microscopy was also performed with monoclonal antibodies against human 1, 2, 3, 4, 1, secretory component (Sigma-Aldrich, St Louis, Mo), and 2 (Southern Biotechnology, Birmingham, Ala), followed by fluorescein-conjugated goat antimouse (Kirkegaard & Perry, Gaithersburg, Md). In addition, direct immunofluorescence microscopy was performed with fluoresceinconjugated goat antihuman immunoglobulin light chain and light chain (Kirkegaard & Perry). The in vivo bound immune deposits at the patient s skin BMZ consist of,, and C3. The subclass studies detected in vivo bound BMZ immunoglobulins of all subclasses (1-4), secretory 1, and both light chains ( and ). 2 deposits were not detected (data not shown). These findings indicated that the patient s anti-bmz autoantibodies were polyclonal in nature. Indirect immunofluorescence microscopy was performed on EDTA-split normal human skin substrate by first incubating with the patient s serum on 6-µm-thick cryosections, followed by incubation with fluoresceinconjugated goat antihuman. 17 Controls included serum samples from patients with bullous pemphigoid, epidermolysis bullosa acquisita, and healthy individuals. Whereas the control from the patient with bullous pemphigoid labeled the epidermal roof and the control from the patient with epidermolysis bullosa acquisita labeled the dermal floor, the from this patient with BSLE labeled both sides. Identical findings were observed for the patient s autoantibodies (data not shown). Normal serum does not label either side. IMMUNOELECTRON MICROSCOPIC STUDIES Direct immunoelectron microscopy was performed on 14-µm-thick normal skin and periblistered skin specimens from the patient using a modified peroxidase-antiperoxidase method. 17 and antibodies were detected at the hemidesmosomes and lamina densa areas of the patient s skin BMZ but not at the BMZ of the healthy skin (Figure 2). ANTIBODIES Polyclonal antihuman collagen VII antibody was prepared by immunizing rabbits with a full-length, eukaryoticexpressed, 145-kd NC1 recombinant protein. 18 Human anti- 3 chain (laminin-5/laminin-6) antibody was obtained from serum of a patient with cicatricial pemphigoid. 19 (Polyclonal antirat laminin-5 antibody J-18 was supplied by J. C. R. Jones, PhD, Northwestern University, Chicago, Ill. 20 Polyclonal rabbit antibody to a glutathione S-transferase fusion protein of human BP180 NC16A domain was supplied by G. Giudice, PhD, Medical College of Wisconsin, Milwaukee. 21 ) and sharp pain in the right side of her back. Examination revealed numerous tense bullae on her entire skin surface (Figure 1) and multiple hyperpigmented macules and patches. There were some milia in the patient s extremities, but frank scarring was not observed. Abnormal laboratory findings included hemoglobin, 86 g/l (reference range [RR], g/l); serum urea nitrogen, 11.1 mmol/l (RR, mmol/l), creatinine, 159 µmol/l (1.8 mg/dl) (RR, 27- µmol/l [ mg/ dl]); C3, 0.71 g/l (RR, g/l), C4, 0.12 g/l (RR, g/l), positive autoantibodies including antinuclear antibodies (titer 1:5120, speckled), anti-sm, anti- SSB/La, anti-dsdna, anti-rnp, and direct Coombs test. In addition, radiographic evidence of pleural effusion, and echocardiographic evidence of pericardial effusion were documented. The patient was successfully treated with systemic corticosteroids, azathioprine, and dapsone. COMMENT We detail the findings in a patient with BSLE whose autoantibodies recognized BPAg1, laminin-5, laminin-6, and type VII collagen. This is the first report of such a clinical case in which BMZ components other than type VII collagen have been definitively recognized by the autoantibodies of a patient with BSLE. The phenomenon of the association of SLE, a systemic autoimmune disease, with autoantibodymediated subepidermal blistering skin disease, an organspecific autoimmune disease, can be explained by 2 possible immune mechanisms. The first is that the organspecific autoimmune disease is provoked by systemic autoimmunity. 26 This mechanism has been documented in a spontaneous mouse model of chronic inflammatory joint disease strikingly similar to the human disease rheumatoid arthritis. 26 Crossing the nonobese diabetes mouse strain with a T-cell receptor transgenic line generated offspring that were affected by a rheumatoid arthritis like syndrome, without the need of specific induction by external injection of joint-specific antigen. 26 While our patient s clinical manifestations could conform to this scenario, it would be difficult to explain the complete pathogenesis by this mechanism. That is, this patient would need to carry T-cell receptor genes capable of recognizing 4 different BMZ components: laminin-5, laminin-6, bullous pemphigoid antigen 1, and type VII col- 570

3 PROTEIN SUBSTRATES PREPARATION Substrates containing the full-length native form of type VII collagen were prepared by concentrating cultureconditioned medium of WISH cells. 18 Recombinant NC1 domain of type VII collagen was prepared from cultureconditioned medium of 293 cells transfected with the fulllength complementary DNA-encoding human type VII collagen NC1 domain. 18 Substrates containing heterotrimers of human laminin-5/laminin-6 were prepared from a primary healthy human keratinocyte culture as described. 22 Total human epidermal cell extracts were prepared from human keratinocytes. 16 Glutathione S-transferase fusion protein of the human BP180 NC16A domain was supplied by G. Giudice, PhD (Medical College of Wisconsin). 23 IMMUNOBLOT STUDIES The above substrates containing BMZ proteins were mixed with sample buffer, loaded onto a 4% loading gel over a running gel (6%, 8%, or 10 %), and vertically separated by a sodium dodecyl sulfate polyacrylamide gel electrophoresis separating system (Novex, La Jolla, Calif) under reducing conditions. 16 The separated proteins were then horizontally transferred to a supported nitrocellulose membrane (Bio-Rad, Hercules, Calif). 19 After the transfer, efficiency was examined by a reversible Ponceau S stain (Sigma-Aldrich); the membranes were cut into strips and blocked by 5% nonfat powdered milk. The membranes were first incubated with primary antibodies overnight at 4 C, followed by incubation at room temperature for 1 hour with peroxidase-conjugated goat antibodies to rabbit, human chain, human chain, human Ig light chain, and human Ig light chain (Kirkegaard & Perry). The immunoreactions were visualized with peroxidase substrate 4-chloro-1-naphthol (Bio-Rad). 19 The patient s serum revealed both and autoantibodies that recognized the 230-kd bullous pemphigoid antigen 1, but not the 180-kd bullous pemphigoid antigen 2 (Figure 3). Furthermore, the patient s serum did not contain or autoantibodies that recognized the recombinant human BP180 NC16A domain, whereas a rabbit antibody and control serum from a human patient with bullous pemphigoid labeled it (data not shown). In addition, the patient s serum contained autoantibodies that recognized laminin-5 2 chain and 3 chain (Figure 4). The 3 chain has been identified as the major laminin-5 (and laminin-6) subunit recognized by autoantibodies from a subset of patients with cicatricial pemphigoid. 19,24 Moreover, the patient s serum contained both and autoantibodies that recognized the full-length 290-kd native-type VII collagen and the full-length 145-kd recombinant NC1 domain (Figure 5). The autoantibodies recognizing the NC1 domain consisted of both and light chains (data not shown). ENZYME-LINKED IMMUNOSORBENT ASSAY Enzyme-linked immunosorbent assay testing of the patient s serum on purified recombinant human type VII collagen NC1 domain was carried out. 18 The patient s antibodies specifically reacted with NC1 domain of type VII collagen in a titratable manner (data not shown). IMMUNOPRECIPITATION Immunoprecipitation studies were performed with conditioned media from sulfur 35 ( 35 S) methionine/cysteine metabolically labeled normal human keratinocyte cultures containing laminin-5 and laminin-6 proteins. 19,25 Control antibodies included normal human serum and polyclonal antibodies to laminin-5. 19,25 The patient s serum contained autoantibodies that coprecipitated laminin-5 proteins with polyclonal anti laminin-5 antibody. Normal human serum does not precipitate these proteins (data not shown). lagen. It would also be difficult to explain how the immune system can recognize bullous pemphigoid antigen 1, an intracellular protein, but not bullous pemphigoid antigen 2, an extracellular protein that has been documented to be pathogenic in a passive transfer mouse model. 21 However, the exclusion of bullous pemphigoid antigen 2 as a target antigen of this patient s autoantibodies was based solely on detectability of her circulating autoantibodies. It is possible that the patient generated anti-bp180 autoantibodies that were all bound to skin BMZ and were not available in her circulation for detection, as only a small percentage of patients with bullous pemphigoid have circulating antibodies against BP180 detectable by immunoblotting and immunoprecipitation studies. A recent study reported that the skininfiltrating, predominantly CD4 + T cells from patients with SLE recognize a relatively limited epitope and are likely expanded by antigen-driven stimulation, 27 thus suggesting a potential role of these T cells in inducing organspecific immunobullous diseases against skin BMZ components. The second possible mechanism may involve an epitope-spreading phenomenon. 28 Epitope spreading describes an immunologic event in which a primary autoimmune or inflammatory process causes tissue injury, releasing previously sequestered antigenic epitopes, and leading to a secondary autoimmune response to the new antigenic epitope. There are many antibody-mediated blistering skin diseases in which epitope spreading may play a role in the initiation or progression of the disease. 29 Chen et al 30 recently found that the NC1 domain of type VII collagen forms binding with the 3 chain of laminin-5. One could envision that an inflammatory process initially involving the NC1 domain of type VII collagen can easily cause injury to the adjacent component laminin-5. Thus, by the mechanism of epitope spreading, the primary autoimmune reaction against type VII collagen can lead to secondary autoimmune reactions against laminin-5 and other adjacent BMZ components. In this patient, the history suggested that the systemic autoimmune (SLE) component and the organ-specific blistering skin disease developed simultaneously. The concurrent development of systemic and organ-specific diseases, at first glance, may not lend strong support for an essential role of epitope spreading. This phenomenon requires a sequence of events that involves injuries in- 571

4 kd M Figure 3. The patient s serum contains both and autoantibodies that recognize the bullous pemphigoid antigen 1 (BP230) but not BP180. Cultured human keratinocyte extracts separated by a 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis separating system and transferred to nitrocellulose membranes were incubated with positive bullous pemphigoid control serum samples (lanes 1 and 2), serum of our patient with bullous systemic lupus erythematosus (lanes 3 and 5), or normal human serum (lanes 4 and 6), followed by incubation with peroxidase-conjugated goat antibodies to human chain (lanes 1-4) or chain (lanes 5 and 6). M indicates molecular-weight standards. Figure 1. The clinical manifestations of bullous systemic lupus erythematosus. The patient exhibited large bullae on her chest (top) and face (bottom). kd Figure 2. The ultrastructural localization of in vivo bound immunoglobulins to hemidesmosomes and lamina densa. The in vivo bound (top) and (bottom) autoantibodies in the patient s skin were detected both in the hemidesmosomes and in the lamina densa and sublamina densa areas by direct immunoelectron microscopy using a peroxidase-antiperoxidase method. The bar indicates 1 µm and applies to both panels. M Figure 4. The patient s serum contains autoantibodies that recognize laminin-5 and laminin-6 3 and 2 chains. Human keratinocyte matrix preparations were separated by an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis separating system under reducing conditions, transferred to nitrocellulose paper, then reacted with polyclonal rabbit antibody to laminin-5 J-18 (lane 1), a human autoantibody that recognized 3 chain of laminin-5 and laminin-6 (lane 2), our patient s serum (lanes 3 and 6), the serum of a patient with epidermolysis bullosa acquisita (lane 4), and normal human serum (lanes 5 and 7), followed by incubation with peroxidase-conjugated goat antibodies to rabbit (lane 1), human chain (lanes 2-5), and human chain (lanes 6-7). M indicates molecular-weight standards. duced by chronic inflammation, release of sequestered antigen, exposure of new antigen to antigenpresenting cells and helper T cells and B cells, and activation of autoreactive T cells and B cells. Nevertheless, it is certainly possible that this patient had a longstanding subclinical SLE, which caused tissue injury and exposed BMZ components to autoreactive lymphocytes prior to her clinical manifestations of blistering skin disease. A third explanation is the involvement of both of the above mechanisms. Following the primary autoimmune reaction against type VII collagen as a result of provocation by SLE, epitope spreading plays a role in the induction of secondary autoimmune reactions against laminin-5, laminin-6, and BPAg1. Regardless of the actual mechanism involved in the initiation and progression of the organ-specific autoimmune subepi- 572

5 Recombinant NC1 dermal blistering skin disease, this unusual case of BSLE demonstrates the dynamic interplay between systemic autoimmunity and organ-specific skin diseases. Accepted for publication October 8, This work was supported in part by a Clinical Investigator Award, K08 AR01961 (Dr Chan), a project grant, P01 AR44012 (Dr Marinkovich), and a research grant, R01 AR33625 (Dr Woodley), National Institutes of Health, Bethesda, Md; a Merit Review Research Grant, Veterans Affairs Research Committee, Livermore (Dr Chan), and by the Office of Research and Development, Palo Alto Veterans Affairs Health Care System, Palo Alto (Dr Marinkovich), Calif. Presented at the International Investigative Dermatology Meeting, Cologne, Germany, May 9, Reprints: Lawrence S. Chan, MD, Department of Dermatology, Northwestern University Medical School, Tarry 4-721, Mail Code T225, 300 E Superior St, Chicago, IL ( larrychan@nwu.edu). REFERENCES WISH Cell Medium kd kd Pedro DS, Dahl MV. 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The eukaryotic expressed recombinant type VII collagen NC1 protein (lanes 1-6) and WISH-cell conditioned medium that contains native type VII collagen (lanes 7-11) were vertically separated in an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis separating system under reducing conditions, horizontally transferred to nitrocellulose paper, then reacted with a rabbit anti-nc1 antibody (lanes 1 and 11), a control serum positive for epidermolysis bullosa acquisita (lane 2), our patient s serum (lanes 3,5,8,10), and normal control serum samples (lanes 4,6,7,9). The immunoreactions were visualized with peroxidase-conjugated goat antibodies to rabbit (lanes 1 and 11), human chain (lanes 2-4, 9,10), and human chain (lanes 5-8). M indicates molecular-weight standards Camisa C, Sharma HM. Vesiculobullous systemic lupus erythematosus: report of two cases and a review of the literature. 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