- Email: [email protected]
Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid
Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid Zelmira Lazarova, MD,a Cassian Sitaru, MD,b Detlef Zillikens, MD,a and Kim B. Yancey, MDb Milwaukee, Wisconsin, and Wurzburg, Germany Background: Anti-epiligrin cicatricial pemphigoid (AECP) is a subepidermal blistering disease characterized by circulating anti-basement membrane autoantibodies to laminin 5. Objective: To evaluate the relative sensitivity of immunoblotting and immunoprecipitation techniques for the detection of anti-laminin 5 antibodies, comparative studies using reference laminin 5 antiserum as well as sera from patients with AECP, other immunobullous diseases, and normal volunteers were performed. Methods: Equivalent amounts of protein from five different substrates were studied by immunoblotting; immunoprecipitation experiments examined biosynthetically radiolabeled human keratinocyte (HK) extracts. Results: HK extracellular matrix (ECM) was the most sensitive substrate for detection of antibodies to laminin 5; extracts of HKs, A-431 cells and HaCat cells represented alternative test substrates (though the later required higher amounts of protein input). Sera from patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. Immunoprecipitation studies found that a 10,000-fold dilution of reference laminin 5 antiserum retained the ability to identify laminin 5. Maximal dilutions of sera from AECP patients retaining the ability to immunoprecipitate laminin 5 ranged from 500 to 5,000. Conclusion: Immunoprecipitation was the most sensitive technique for detection of anti-laminin 5 antibodies, while immunoblotting of HK ECM or HK extracts represented practical alternatives. ( J Am Acad Dermatol 2004;51:886-92.)
A
nti-epiligrin cicatricial pemphigoid (AECP) is a chronic, subepidermal autoimmune blistering disease of mucous membranes and skin.1,2 Patients with this form of cicatricial pemphigoid (CP) have circulating IgG autoantibodies against laminin 5 that serve as a specific marker for this disorder.3,4 Prior studies showed that experimental and patient anti-laminin 5 IgG are
From the Departments of Dermatology, Medical College of Wisconsina and the University of Wurzburg.b Supported by a grant from the National Institutes of Health (R01 AR048982-01A1) to K. B. Y. Conflicts of interest: None identified. Accepted for publication June 1, 2004. Reprint requests: Zelmira Lazarova, MD, Department of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Road, MFRC Building, Room 4066, Milwaukee, WI 532264801. E-mail: [email protected]. 0190-9622/$30.00 ª 2004 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2004.06.004
886
pathogenic in animal models, indicating the central role of such antibodies in disease pathophysiology.5-7 Longitudinal studies of a cohort of 35 patients with AECP suggested that this form of CP is associated with an increased relative risk for solid cancers.8,9 This potential association warrants further study, including the evaluation of such risk in patients with other forms of CP and/or other immunobullous diseases. To achieve the goal, sensitive and practical ways to establish the diagnosis of AECP are needed. Current criteria for the diagnosis of AECP include: (1) the presence of subepidermal blisters that are primarily localized to mucosal surfaces; (2) continuous deposits of IgG (6C3) in the epidermal basement membrane (BM) of normal appearing perilesional skin; (3) circulating IgG anti-basement BM antibodies that bind the dermal side of 1M NaCl split skin; and (4) circulating IgG autoantibodies to laminin 5.10 Hence, in addition to traditional clinical and immunopathologic criteria used to establish the diagnosis of CP, the designation AECP has been
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
restricted to patients with demonstrable autoantibodies to human laminin 5. These stringent criteria were established to distinguish patients with this form of CP from individuals with other subepidermal blistering disorders also characterized by IgG autoantibodies directed against the dermal side of 1M NaCl split skin (eg, mucosal predominant epidermolysis bullosa acquisita [EBA], p200 pemphigoid, and other yet to be characterized immunobullous diseases). Historically, anti-laminin 5 autoantibodies were identified in immunoprecipitation studies of biosynthetically radiolabeled human keratinocyte (HK) extracts or culture media. Such investigative techniques are largely restricted to selected research laboratories.1,2 Immunoblotting of various substrates represents an alternate approach for the identification of anti-laminin 5 autoantibodies in patients with AECP.3,4 To compare the relative sensitivity of various immunochemical procedures as well as the reactivity of laminin 5 within different immunoblot substrates, a series of comparative studies using reference anti-laminin 5 antiserum as well as sera from patients with AECP, other blistering diseases, and normal volunteers were performed.
MATERIALS AND METHODS Patients Three AECP patients were included in this study; all patients satisfied the diagnostic criteria for this form of CP. Control samples included sera from normal volunteers (n = 11) as well as representative patients with bullous pemphigoid (BP) or EBA. All serum samples from patients with BP immunoprecipitated bullous pemphigoid antigen 1 (BPAG1) from extracts of radiolabeled HKs. All serum samples from patients with EBA immunoblotted type VII collagen in extracts of human lamina densa/ dermis. Indirect immunofluorescence microscopy Indirect immunofluorescence (IF) microscopy of 1M NaCl split normal human skin was performed as previously described.11 Studies included sera from patients and normal volunteers as well as wellcharacterized reference rabbit laminin 5 antiserum and normal rabbit serum.5 Second-step antibodies included fluorescein isothiocyanate-conjugated goat F(ab9)2 anti-human IgG and goat F(ab9)2 anti-rabbit IgG (Biosource Inc., Burlingame, Calif). Cell cultures HKs were isolated from the foreskins of newborns and cultured in serum-free media (Invitrogen Life Technologies, Carlsbad, Calif). A-431 cells
Lazarova et al 887
were obtained from American Type Culture Collection (Rockville, Md); HaCat cells were a gift of Dr. Norbert Fusenig, German Cancer Research Center, Heidelberg, Germany. Both cell lines were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (all from Invitrogen Life Technologies, Carlsbad, Calif). Immunoblot studies Patients’ and control sera were characterized for their reactivity to five standard immunoblot substrates: (1) extracts of cultured HKs (HK extract); (2) HK extracellular matrix (ECM); (3) extracts of the A431 human epidermoid carcinoma cell line (A-431 extract); (4) extracts of the HaCat transformed HK cell line (HaCat extract); and (5) extracts of human epidermal sheets (ES extract). These immunoblot substrates were prepared as follows. Confluent monolayer cultures of HKs were extracted with Tris-buffered saline containing 1% sodium dodecyl sulfate (SDS), 5% b-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM each of leupeptin, antipain, chymostatin, and pepstatin A to yield HK extracts.5 HK ECM was isolated by sequential extraction of HK monolayers with 1% Triton X-100 in phosphate-buffered saline, 2 M urea in 1 M NaCl, and 8 M urea as previously described.5,12 A-431 cell extracts were prepared by treating cell monolayers with Tris-buffered saline containing 1.5% SDS, 5% b-mercaptoethanol, 2 mM PMSF, 2 mM CaCl2, and 5 mM each of antipain, chymostatin, and pepstatin A.13 HaCat cells were grown to near confluency, then the culture medium was removed, and the attached cells were lysed with 20 mM NH4OH. Residual HaCat cell proteins were solubilized in Laemmli sample buffer to yield HaCat cell extract.14 ES extracts were prepared by incubating human foreskins in 1 M NaCl containing 2 mM EDTA and 2 mM PMSF for 48 hours at 48C to yield epidermal sheets as described previously.15 ES were subsequently extracted with the same buffer used to prepare A-431 cell extracts.13 The protein concentration of each antigenic substrate was determined using the Bio-Rad protein assay detection kit (BioRad, Hercules, Calif). One hundred twenty g of each antigenic substrate was reduced, separated on 6% Tris-glycine preparative minigels (Invitrogen Corp., Grand Island, NY), electrophoretically transferred to nitrocellulose membranes (Invitrogen Corp.), and immunoblotted with serial dilutions of reference laminin 5 antiserum as well as sera from patients and controls.3,15,16 Alkaline phosphataseconjugated goat F(ab9)2 anti-human IgG and alkaline phosphatase-conjugated goat F(ab9)2 anti-rabbit IgG
888 Lazarova et al
(Biosource Inc.) in dilutions of 1:1000 were used as second-step antibodies in immunoblotting experiments. Reaction products were identified with the Bio-Rad alkaline phosphatase developing kit using a standardized development time of 3 minutes. The end-titer of various sera was defined as the greatest dilution showing reactivity against all subunits of laminin 5. Immunoprecipitation studies Subconfluent monolayers of HKs were biosyn35 thetically radiolabeled with S-methionine (Amersham Biosciences Corp., Arlington, Ill). Radiolabeled HK extracts were processed and studied as described previously using reference laminin 5 antiserum and normal rabbit serum as well as sera from patients and controls.5,17,18 Resulting samples were applied to 6% Tris-glycine minigels (Invitrogen Corp.) exposed to the film for a week and evaluated. Note, in contrast to prior studies, these immunoprecipitation experiments examined laminin 5 in HK extracts rather than that secreted into HK culture media. Accordingly, the molecular weights of laminin 5 polypeptides identified in these studies were different from those previously reported.19
RESULTS Indirect IF microscopy To provide perspective about comparative immunoblot and immunoprecipitation studies, the titers of anti-BM antibodies in reference laminin 5 antiserum as well as sera from patients and controls were defined against 1M NaCl split skin. These studies found that the titers of reference rabbit laminin 5 antiserum and normal rabbit serum were 20,480 (dermal-sided binding) and 0, respectively. IgG from three reference patients with AECP bound the dermal side of 1M NaCl split skin with titers of 40 (Patient 1), 80 (Patient 2), and 160 (Patient 3); normal human serum (negative control) tested in parallel showed no reactivity to this tissue substrate. Comparative immunoblot studies To define the relative sensitivity of five different immunoblot substrates for detection of anti-laminin 5 antibodies, equivalent amounts of protein (specifically, 120 g) were applied to Tris-glycine minigels, transferred to nitrocellulose paper, and studied in a series of comparative immunoblot studies using serial dilutions of a high-titered reference laminin 5 antiserum (and normal rabbit serum, negative control). These studies found that reference laminin 5 antiserum only displayed strong immunoblot reactivity to HK ECM (titer
J AM ACAD DERMATOL DECEMBER 2004
2560), HK extract (titer 320), and A-431 extract (titer 160) (Fig 1). Matching side-by-side immunoblot studies of HaCat extract and ES extract were negative (Fig 1). A 20-fold increase in protein load (ie, 2.4 mg of HaCat cell extract per minigel) yielded substrates on which reference laminin 5 antiserum immunoblotted all subunits of this laminin isoform (titer 160). Interestingly, a 50-fold increase in protein load of ES extract (ie, 6 mg per minigel) yielded immunoblot substrates that were devoid of reactivity when tested with reference laminin 5 antiserum (and normal rabbit serum, negative control) at dilutions of 40 or greater. Control immunoblot studies of serum from a reference patient with BP demonstrated that this preparation of ES extract contained BPAG1 and bullous pemphigoid antigen 2 (BPAG2) of appropriate immunoreactivity and molecular weight (ie, 230 and 180 kds, respectively) (data not shown). Following the demonstration that HK ECM represented the optimal (ie, most sensitive) substrate for detection of anti-laminin 5 IgG in reference laminin 5 antiserum, serial dilutions of sera from three representative patients with AECP were tested against this substrate by immunoblotting to determine their end titers. In these studies, IgG from all AECP patients specifically immunoblotted the unprocessed (200 kd) and processed (165 kd) a subunits of laminin 5 in keeping with prior studies.3,4 The end titers of these sera were 320, 1280, and 2560 for Patients 1, 2, and 3, respectively (Fig 2). These end titers exceeded those identified for the same sera in indirect IF microscopy studies of 1 M NaCl split skin. In these immunoblot studies of HK ECM, strong and specific reactivity (ie, bright immunoreactive bands with minimal levels of background staining) were consistently observed when patients’ sera were tested at a dilution of 40. Equivalent dilutions of normal human sera showed no reactivity to HK ECM in control studies conducted in parallel. These experimental conditions required modest amounts of patient sera, control sera, and HK ECM. Moreover, there was a general correlation between the titer of these patients’ IgG autoantibodies against laminin 5 in HK ECM by immunoblotting and 1M NaCl split skin by indirect IF microscopy. Comparative immunoprecipitation studies Immunoprecipitation studies typically use a small amount of patient (or control) serum (eg, 10 L) and a standard amount of biosynthetically radiolabeled keratinocyte proteins in a fixed volume (eg, 0.5 mL) to form model immune complexes that are subsequently precipitated, recovered, applied to Trisglycine minigels, and studied by fluorography.
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
Lazarova et al 889
Fig 1. Comparative immunoblot studies of 5 antigen preparations demonstrated that HK ECM was the optimal substrate for detection of antibodies against laminin 5. One hundred twenty g of HK ECM, HK extract, A-431 extract, HaCat extract, and ES extract were reduced, separated on minigels, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with serial dilutions of reference laminin 5 antiserum (1:40 to 1:81,920 lanes 1 to 12, respectively). Alkaline phosphatase-conjugated goat F(ab9)2 anti-rabbit IgG (dilution 1:1000) was used as a second-step antibody. Reference laminin 5 antiserum immunoblotted all unprocessed and processed subunits of laminin 5 (denoted by ticks, left margin). The end titer of the reference antiserum against all laminin 5 subunits in HK ECM was 2560 (panel A, lane 7, arrow). End titers of the reference antiserum against HK and A-431 extracts were 320 (panel B, lane 4, arrow) and 160 (panel C, lane 3, arrow), respectively. Under these conditions (ie, 120 g of immunoblot substrate), reference antiserum showed no specific reactivity to HaCat (panel D) or ES extracts (panel E). In each set of these immunoblot experiments, lane 13 (negative control) corresponded to the reactivity of normal rabbit serum tested at a dilution of 1:40, the highest concentration of antiserum used in these experiments.
Immunoprecipitation experiments are typically conducted under conditions that allow antigenantibody interactions to occur under relatively native (ie, nondenaturing) conditions thus allowing detection of conformational as well as linear epitopes (the former being absent in standard immunoblot experiments). Under conditions previously used in studies of sera from patients with various immunobullous diseases including AECP, patient serum samples were tested at dilutions of 1:50. To assess the relative sensitivity of this immunochemical procedure, various dilutions (specifically, 1:50, 1:100, 1:500, 1:5000, 1:10,000, and 1:50,000) of reference laminin 5 antiserum, normal rabbit serum (negative
control), patients’ sera, and normal human serum (negative controls) were studied in comparative immunoprecipitation experiments utilizing a fixed amount of biosynthetically radiolabeled HK extracts. These studies showed that a 1:10,000 dilution of reference laminin 5 antiserum retained the ability to immunoprecipitate this protein from HK extracts (Fig 3, A). Maximal dilutions of sera from representative AECP patients that retained the ability to immunoprecipitate laminin 5 in these comparative experiments were 1:500 for Patients 1 and 2 and 1:5000 for Patient 3 (Fig 3, B). Again, there was a general correlation between the relative ability of these patients’ sera to immunoprecipitate HK-derived
890 Lazarova et al
J AM ACAD DERMATOL DECEMBER 2004
Fig 2. Sera from representative patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. One hundred twenty g of HK ECM was studied by immunoblot as shown in Fig 1; serial dilutions of patient sera (1:40 to 1:10,240) were tested simultaneously. All AECP sera immunoblotted the 200 kd (unprocessed) and 165 kd (processed) a subunits of laminin 5. The end titers of sera from Patients 1, 2, and 3 in these immunoblot experiments were 320, 1280, and 2560, respectively (denoted by arrows). Strong and specific reactivity (ie, bright immunoreactive bands with minimal levels of background staining) were consistently observed when patients’ sera were tested at a dilution of 1:40; an equivalent dilution of normal human serum showed no reactivity to HK ECM in control studies conducted in parallel (negative control, lane 28). Reference laminin 5 antiserum (positive control, lane 30) immunoblotted all subunits of laminin 5; normal rabbit serum (lane 29) showed no reactivity to these polypeptides.
laminin 5 and bind the dermal side of 1M NaCl split skin by indirect IF microscopy.
DISCUSSION Over the past 15 years, studies from different laboratories have shown that autoantibodies from patients with CP target different autoantigens in epithelial BMs.1,20-24 Accordingly, CP is currently thought to represent a disease phenotype rather than a single nosologic entity. AECP represents a well-characterized disease that has been estimated to comprise 5%-15 % of patients with CP. Recent studies have shown that patients with AECP have an increased relative risk for malignant solid tumors.8,9 This relative risk is considerable and approximates the incidence of solid tumors among adults with dermatomyositis.8 To date, AECP-associated solid tumors have included adenocarcinomas of the lung, stomach, colon, and uterus.8 Most cancers in patients with AECP proved to be fatal within a relatively short interval after their identification.8 Accordingly, the identification of patients with AECP has assumed greater significance. This consideration has direct relevance to this study since patients with AECP cannot be differentiated from patients with other types of CP by routine clinical, histologic, or immu-
nopathologic studies. To date, the diagnosis of AECP has been restricted to patients with demonstrable circulating autoantibodies directed against laminin 5. In this study, various immunochemical techniques used to detect anti-laminin 5 autoantibodies were compared directly to establish their relative sensitivity and practical utility. The first investigative technique used for the detection and characterization of autoantibodies from patients with AECP was immunoprecipitation studies of biosynthetically radiolabeled HK extracts and/or culture media.1,2 Studies of reference laminin 5 antiserum as well as patient and control sera demonstrated that this method is indeed the most sensitive technique for the detection of antibodies directed against laminin 5. Despite this sensitivity, immunoprecipitation techniques remain technically demanding, expensive, labor intensive, and largely dependent upon the use of biosynthetically radiolabeled cell-derived materials. These considerations limit the availability of immunoprecipitation techniques to a relatively small number of research laboratories. Accordingly, a number of groups have developed and applied various immunoblotting protocols for the detection of anti-laminin 5 autoantibodies. Since there is no standard antigen
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
Lazarova et al 891
Fig 3. Immunoprecipitation studies of biosynthetically radiolabeled HK extracts represented the most sensitive method for detection of antibodies directed against laminin 5. A, Immunoprecipitation studies of biosynthetically radiolabeled HK extracts using reference laminin 5 antiserum (panel A, lanes l-6, serum dilutions 1:50 to 1:50,000, see Materials and Methods) identified laminin 5 at dilutions as great as 1:10,000 (lane 5); normal rabbit serum (control) did not immunoprecipitate laminin 5 (or other polypeptides) at the highest concentration tested (i.e., 1:50, lane 7). B, Sera from patients with AECP (Patient 1, lanes 1-6; Patient 2, lanes 7-12; Patient 3, lanes 13-18) recognized the same set of polypeptides as the reference laminin 5 antiserum. None of these polypeptides were immunoprecipitated by normal human serum (lane 19). Maximal dilutions of patient sera that retained the ability to immunoprecipitate laminin 5 were as follows: 1:500 for Patients 1 and 2 (lanes 3 and 9) and 1:5000 for Patient 3 (lane 16) as denoted by arrows. Molecular weights of the laminin 5 polypeptides in this complex were 190, 170, 140 and 130 kdDs.
substrate for such immunoblot experiments, this study sought to compare directly the relative utility of different preparations for detection of antibodies against laminin 5. Comparative studies of 5 different immunoblot substrates found that HK ECM was clearly the preferred substrate for detection of antibodies directed against laminin 5. A practical alternate substrate for detection of anti-laminin 5 antibodies was extracts of cultured HKs. HK extracts were somewhat easier to prepare than HK ECM, and when applied to minigels at higher protein loads, provided a favorable substrate for detection of antibodies against laminin 5 as well as other keratinocyte autoantigens such as BPAG1 and BPAG2. Comparative immunoblot studies showed that extracts of HKs were superior to extracts derived from A-431 or HaCat cell lines. Interestingly, though epidermal sheets were shown to be a source of the keratinocyte autoantigens BPAG1 and BPAG2, comparative immunoblot experiments found that this antigen source lacked sufficient amounts of laminin 5 to be of practical utility for the routine detection of autoantibodies against this laminin isoform. This finding was of relevance in that extracts of epidermal sheets have been used for the identification and characterization of autoantibodies from patients with immunobullous diseases.
In summary, the detection of autoantibodies against laminin 5 can be performed using a series of routine and specialized tests. Frontline screening measures should rely upon indirect immunofluorescent microscopy testing of 1M NaCl split skin since patients with AECP have demonstrable IgG autoantibodies directed against the dermal side of this tissue substrate. To distinguish patients with AECP from other patients with IgG anti-BM autoantibodies directed against the dermal side of 1 M NaCl split skin, immunoblot studies of HK ECM or HK extracts can be used to identify autoantibodies against laminin 5 subunits. Rabbit antilaminin 5 IgG is now commercially available (Biodesign International, Saco, Me) for use as a reference positive control in such immunoblot studies. While this antibody identifies all unprocessed and processed subunits of this laminin isoform in HK ECM, its relative sensitivity is 5-fold less than our laboratory’s reference laminin 5 antiserum (data not shown). Interestingly, another recent study found that immunoblotting of purified laminin 5 was a valid way to identify patients with AECP.25 Detection of low titer anti-laminin 5 autoantibodies as well as autoantibodies uniquely reactive with conformational determinants on this heterotrimer (ie, epitopes not displayed under the
892 Lazarova et al
denaturing conditions of standard immunoblot studies) should be submitted to reference laboratories for immunoprecipitation studies of biosynthetically radiolabeled HK extracts and/or media. Such laboratories often have available an array of test substrates as well as monoclonal antibodies directed against human IgG (especially, IgG4) that can be used to further enhance the identification of patients with AECP.26 Characterizing the specific reactivity of autoantibodies from patients with CP allows such patients to be segregated into subgroups that can be studied longitudinally to determine if such patients display differences in their relative extent of disease, prognosis, associations, or response to therapy. REFERENCES 1. Domloge-Hultsch N, Gammon WR, Briggaman RA, Gil SG, Carter WG, Yancey KB. Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease. J Clin Invest 1992;90:1628-33. 2. Domloge-Hultsch N, Anhalt GJ, Gammon WR, Lazarova Z, Briggaman R, Welch M, et al. Anti-epiligrin cicatricial pemphigoid. A subepithelial bullous disorder. Arch Dermatol 1994; 130:1521-9. 3. Kirtschig G, Marinkovich MP, Burgeson RE, Yancey KB. Anti-basement membrane autoantibodies in patients with anti-epiligrin cicatricial pemphigoid bind the alpha subunit of laminin 5. J Invest Dermatol 1995;105:543-8. 4. Lazarova Z, Hsu R, Yee C, Yancey KB. Anti-epiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5 (alpha3beta3gamma2). Br J Dermatol 1998;139:791-7. 5. Lazarova Z, Yee C, Darling T, Briggaman RA, Yancey KB. Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice. J Clin Invest 1996;98:1509-18. 6. Lazarova Z, Hsu R, Yee C, Yancey KB. Human anti-laminin 5 autoantibodies induce subepidermal blisters in an experimental human skin graft model. J Invest Dermatol 2000;114: 178-84. 7. Lazarova Z, Hsu R, Briggaman RA, Yancey KB. Fab fragments directed against laminin 5 induce subepidermal blisters in neonatal mice. Clin Immunol 2000;95:26-32. 8. Egan CA, Lazarova Z, Darling TN, Yee C, Cote T, Yancey KB. Anti-epiligrin cicatricial pemphigoid and relative risk for cancer. Lancet 2001;357:1850-1. 9. Egan CA, Lazarova Z, Darling TN, Yee C, Yancey KB. Antiepiligrin cicatricial pemphigoid: clinical findings, immunopathogenesis, and significant associations. Medicine (Baltimore) 2003;82:177-86. 10. Egan CA, Yancey KB. The clinical and immunopathological manifestations of anti-epiligrin cicatricial pemphigoid, a recently defined subepithelial autoimmune blistering disease. Eur J Dermatol 2000;10:585-9.
J AM ACAD DERMATOL DECEMBER 2004
11. Lazarova Z, Yancey KB. Reactivity of autoantibodies from patients with defined subepidermal bullous diseases against 1 mol/L salt-split skin. Specificity, sensitivity, and practical considerations. J Am Acad Dermatol 1996;35:398-403. 12. Carter WG, Ryan MC, Gahr PJ. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell 1991;65:599-610. 13. Lee CW. An extract of A-431 cells contains major tissue antigens of autoimmune bullous diseases. Br J Dermatol 2000;143:821-3. 14. Leverkus M, Bhol K, Hirako Y, Pas H, Sitaru C, Baier G, et al. Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230. Br J Dermatol 2001;145:998-1004. 15. Labib RS, Anhalt GJ, Patel HP, Mutasim DF, Diaz LA. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol 1986;136: 1231-5. 16. Lazarova Z, Yee C, Lazar J, Yancey KB. IgG autoantibodies in patients with anti-epiligrin cicatricial pemphigoid recognize the G domain of the laminin 5 alpha-subunit. Clin Immunol 2001;101:100-5. 17. Stanley JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981;24:897-903. 18. Basset-Seguin N, Caughman SW, Yancey KB. A-431 cells and human keratinocytes synthesize and secrete the third component of complement. J Invest Dermatol 1990;95:621-5. 19. Marinkovich MP, Lunstrum GP, Burgeson RE. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 1992;267:17900-6. 20. Bernard P, Prost C, Durepaire N, Basset-Seguin N, Didierjean L, Saurat JH. The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen. J Invest Dermatol 1992;99:174-9. 21. Shimizu H, Masunaga T, Ishiko A, Matsumura K, Hashimoto T, Nishikawa T, et al. Autoantibodies from patients with cicatricial pemphigoid target different sites in epidermal basement membrane. J Invest Dermatol 1995;104:370-3. 22. Ghohestani RF, Nicolas JF, Rousselle P, Claudy AL. Identification of a 168-kDa mucosal antigen in a subset of patients with cicatricial pemphigoid. J Invest Dermatol 1996;107:136-9. 23. Tyagi S, Bhol K, Natarajan K, Livir-Rallatos C, Foster CS, Ahmed AR. Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization. Proc Natl Acad Sci U S A 1996;93:14714-9. 24. Chan LS, Majmudar AA, Tran HH, Meier F, Schaumburg-Lever G, Chen M, et al. Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid. J Invest Dermatol 1997;108:848-53. 25. Hisamatsu Y, Nishiyama T, Amano S, Matsui C, Ghohestani R, Hashimoto T. Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid. J Dermatol Sci 2003;33:113-9. 26. Hsu R, Lazarova Z, Yee C, Yancey KB. Noncomplement fixing, IgG4 autoantibodies predominate in patients with anti-epiligrin cicatricial pemphigoid. J Invest Dermatol 1997;109:557-61.
A
nti-epiligrin cicatricial pemphigoid (AECP) is a chronic, subepidermal autoimmune blistering disease of mucous membranes and skin.1,2 Patients with this form of cicatricial pemphigoid (CP) have circulating IgG autoantibodies against laminin 5 that serve as a specific marker for this disorder.3,4 Prior studies showed that experimental and patient anti-laminin 5 IgG are
From the Departments of Dermatology, Medical College of Wisconsina and the University of Wurzburg.b Supported by a grant from the National Institutes of Health (R01 AR048982-01A1) to K. B. Y. Conflicts of interest: None identified. Accepted for publication June 1, 2004. Reprint requests: Zelmira Lazarova, MD, Department of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Road, MFRC Building, Room 4066, Milwaukee, WI 532264801. E-mail: [email protected]. 0190-9622/$30.00 ª 2004 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2004.06.004
886
pathogenic in animal models, indicating the central role of such antibodies in disease pathophysiology.5-7 Longitudinal studies of a cohort of 35 patients with AECP suggested that this form of CP is associated with an increased relative risk for solid cancers.8,9 This potential association warrants further study, including the evaluation of such risk in patients with other forms of CP and/or other immunobullous diseases. To achieve the goal, sensitive and practical ways to establish the diagnosis of AECP are needed. Current criteria for the diagnosis of AECP include: (1) the presence of subepidermal blisters that are primarily localized to mucosal surfaces; (2) continuous deposits of IgG (6C3) in the epidermal basement membrane (BM) of normal appearing perilesional skin; (3) circulating IgG anti-basement BM antibodies that bind the dermal side of 1M NaCl split skin; and (4) circulating IgG autoantibodies to laminin 5.10 Hence, in addition to traditional clinical and immunopathologic criteria used to establish the diagnosis of CP, the designation AECP has been
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
restricted to patients with demonstrable autoantibodies to human laminin 5. These stringent criteria were established to distinguish patients with this form of CP from individuals with other subepidermal blistering disorders also characterized by IgG autoantibodies directed against the dermal side of 1M NaCl split skin (eg, mucosal predominant epidermolysis bullosa acquisita [EBA], p200 pemphigoid, and other yet to be characterized immunobullous diseases). Historically, anti-laminin 5 autoantibodies were identified in immunoprecipitation studies of biosynthetically radiolabeled human keratinocyte (HK) extracts or culture media. Such investigative techniques are largely restricted to selected research laboratories.1,2 Immunoblotting of various substrates represents an alternate approach for the identification of anti-laminin 5 autoantibodies in patients with AECP.3,4 To compare the relative sensitivity of various immunochemical procedures as well as the reactivity of laminin 5 within different immunoblot substrates, a series of comparative studies using reference anti-laminin 5 antiserum as well as sera from patients with AECP, other blistering diseases, and normal volunteers were performed.
MATERIALS AND METHODS Patients Three AECP patients were included in this study; all patients satisfied the diagnostic criteria for this form of CP. Control samples included sera from normal volunteers (n = 11) as well as representative patients with bullous pemphigoid (BP) or EBA. All serum samples from patients with BP immunoprecipitated bullous pemphigoid antigen 1 (BPAG1) from extracts of radiolabeled HKs. All serum samples from patients with EBA immunoblotted type VII collagen in extracts of human lamina densa/ dermis. Indirect immunofluorescence microscopy Indirect immunofluorescence (IF) microscopy of 1M NaCl split normal human skin was performed as previously described.11 Studies included sera from patients and normal volunteers as well as wellcharacterized reference rabbit laminin 5 antiserum and normal rabbit serum.5 Second-step antibodies included fluorescein isothiocyanate-conjugated goat F(ab9)2 anti-human IgG and goat F(ab9)2 anti-rabbit IgG (Biosource Inc., Burlingame, Calif). Cell cultures HKs were isolated from the foreskins of newborns and cultured in serum-free media (Invitrogen Life Technologies, Carlsbad, Calif). A-431 cells
Lazarova et al 887
were obtained from American Type Culture Collection (Rockville, Md); HaCat cells were a gift of Dr. Norbert Fusenig, German Cancer Research Center, Heidelberg, Germany. Both cell lines were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (all from Invitrogen Life Technologies, Carlsbad, Calif). Immunoblot studies Patients’ and control sera were characterized for their reactivity to five standard immunoblot substrates: (1) extracts of cultured HKs (HK extract); (2) HK extracellular matrix (ECM); (3) extracts of the A431 human epidermoid carcinoma cell line (A-431 extract); (4) extracts of the HaCat transformed HK cell line (HaCat extract); and (5) extracts of human epidermal sheets (ES extract). These immunoblot substrates were prepared as follows. Confluent monolayer cultures of HKs were extracted with Tris-buffered saline containing 1% sodium dodecyl sulfate (SDS), 5% b-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM each of leupeptin, antipain, chymostatin, and pepstatin A to yield HK extracts.5 HK ECM was isolated by sequential extraction of HK monolayers with 1% Triton X-100 in phosphate-buffered saline, 2 M urea in 1 M NaCl, and 8 M urea as previously described.5,12 A-431 cell extracts were prepared by treating cell monolayers with Tris-buffered saline containing 1.5% SDS, 5% b-mercaptoethanol, 2 mM PMSF, 2 mM CaCl2, and 5 mM each of antipain, chymostatin, and pepstatin A.13 HaCat cells were grown to near confluency, then the culture medium was removed, and the attached cells were lysed with 20 mM NH4OH. Residual HaCat cell proteins were solubilized in Laemmli sample buffer to yield HaCat cell extract.14 ES extracts were prepared by incubating human foreskins in 1 M NaCl containing 2 mM EDTA and 2 mM PMSF for 48 hours at 48C to yield epidermal sheets as described previously.15 ES were subsequently extracted with the same buffer used to prepare A-431 cell extracts.13 The protein concentration of each antigenic substrate was determined using the Bio-Rad protein assay detection kit (BioRad, Hercules, Calif). One hundred twenty g of each antigenic substrate was reduced, separated on 6% Tris-glycine preparative minigels (Invitrogen Corp., Grand Island, NY), electrophoretically transferred to nitrocellulose membranes (Invitrogen Corp.), and immunoblotted with serial dilutions of reference laminin 5 antiserum as well as sera from patients and controls.3,15,16 Alkaline phosphataseconjugated goat F(ab9)2 anti-human IgG and alkaline phosphatase-conjugated goat F(ab9)2 anti-rabbit IgG
888 Lazarova et al
(Biosource Inc.) in dilutions of 1:1000 were used as second-step antibodies in immunoblotting experiments. Reaction products were identified with the Bio-Rad alkaline phosphatase developing kit using a standardized development time of 3 minutes. The end-titer of various sera was defined as the greatest dilution showing reactivity against all subunits of laminin 5. Immunoprecipitation studies Subconfluent monolayers of HKs were biosyn35 thetically radiolabeled with S-methionine (Amersham Biosciences Corp., Arlington, Ill). Radiolabeled HK extracts were processed and studied as described previously using reference laminin 5 antiserum and normal rabbit serum as well as sera from patients and controls.5,17,18 Resulting samples were applied to 6% Tris-glycine minigels (Invitrogen Corp.) exposed to the film for a week and evaluated. Note, in contrast to prior studies, these immunoprecipitation experiments examined laminin 5 in HK extracts rather than that secreted into HK culture media. Accordingly, the molecular weights of laminin 5 polypeptides identified in these studies were different from those previously reported.19
RESULTS Indirect IF microscopy To provide perspective about comparative immunoblot and immunoprecipitation studies, the titers of anti-BM antibodies in reference laminin 5 antiserum as well as sera from patients and controls were defined against 1M NaCl split skin. These studies found that the titers of reference rabbit laminin 5 antiserum and normal rabbit serum were 20,480 (dermal-sided binding) and 0, respectively. IgG from three reference patients with AECP bound the dermal side of 1M NaCl split skin with titers of 40 (Patient 1), 80 (Patient 2), and 160 (Patient 3); normal human serum (negative control) tested in parallel showed no reactivity to this tissue substrate. Comparative immunoblot studies To define the relative sensitivity of five different immunoblot substrates for detection of anti-laminin 5 antibodies, equivalent amounts of protein (specifically, 120 g) were applied to Tris-glycine minigels, transferred to nitrocellulose paper, and studied in a series of comparative immunoblot studies using serial dilutions of a high-titered reference laminin 5 antiserum (and normal rabbit serum, negative control). These studies found that reference laminin 5 antiserum only displayed strong immunoblot reactivity to HK ECM (titer
J AM ACAD DERMATOL DECEMBER 2004
2560), HK extract (titer 320), and A-431 extract (titer 160) (Fig 1). Matching side-by-side immunoblot studies of HaCat extract and ES extract were negative (Fig 1). A 20-fold increase in protein load (ie, 2.4 mg of HaCat cell extract per minigel) yielded substrates on which reference laminin 5 antiserum immunoblotted all subunits of this laminin isoform (titer 160). Interestingly, a 50-fold increase in protein load of ES extract (ie, 6 mg per minigel) yielded immunoblot substrates that were devoid of reactivity when tested with reference laminin 5 antiserum (and normal rabbit serum, negative control) at dilutions of 40 or greater. Control immunoblot studies of serum from a reference patient with BP demonstrated that this preparation of ES extract contained BPAG1 and bullous pemphigoid antigen 2 (BPAG2) of appropriate immunoreactivity and molecular weight (ie, 230 and 180 kds, respectively) (data not shown). Following the demonstration that HK ECM represented the optimal (ie, most sensitive) substrate for detection of anti-laminin 5 IgG in reference laminin 5 antiserum, serial dilutions of sera from three representative patients with AECP were tested against this substrate by immunoblotting to determine their end titers. In these studies, IgG from all AECP patients specifically immunoblotted the unprocessed (200 kd) and processed (165 kd) a subunits of laminin 5 in keeping with prior studies.3,4 The end titers of these sera were 320, 1280, and 2560 for Patients 1, 2, and 3, respectively (Fig 2). These end titers exceeded those identified for the same sera in indirect IF microscopy studies of 1 M NaCl split skin. In these immunoblot studies of HK ECM, strong and specific reactivity (ie, bright immunoreactive bands with minimal levels of background staining) were consistently observed when patients’ sera were tested at a dilution of 40. Equivalent dilutions of normal human sera showed no reactivity to HK ECM in control studies conducted in parallel. These experimental conditions required modest amounts of patient sera, control sera, and HK ECM. Moreover, there was a general correlation between the titer of these patients’ IgG autoantibodies against laminin 5 in HK ECM by immunoblotting and 1M NaCl split skin by indirect IF microscopy. Comparative immunoprecipitation studies Immunoprecipitation studies typically use a small amount of patient (or control) serum (eg, 10 L) and a standard amount of biosynthetically radiolabeled keratinocyte proteins in a fixed volume (eg, 0.5 mL) to form model immune complexes that are subsequently precipitated, recovered, applied to Trisglycine minigels, and studied by fluorography.
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
Lazarova et al 889
Fig 1. Comparative immunoblot studies of 5 antigen preparations demonstrated that HK ECM was the optimal substrate for detection of antibodies against laminin 5. One hundred twenty g of HK ECM, HK extract, A-431 extract, HaCat extract, and ES extract were reduced, separated on minigels, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with serial dilutions of reference laminin 5 antiserum (1:40 to 1:81,920 lanes 1 to 12, respectively). Alkaline phosphatase-conjugated goat F(ab9)2 anti-rabbit IgG (dilution 1:1000) was used as a second-step antibody. Reference laminin 5 antiserum immunoblotted all unprocessed and processed subunits of laminin 5 (denoted by ticks, left margin). The end titer of the reference antiserum against all laminin 5 subunits in HK ECM was 2560 (panel A, lane 7, arrow). End titers of the reference antiserum against HK and A-431 extracts were 320 (panel B, lane 4, arrow) and 160 (panel C, lane 3, arrow), respectively. Under these conditions (ie, 120 g of immunoblot substrate), reference antiserum showed no specific reactivity to HaCat (panel D) or ES extracts (panel E). In each set of these immunoblot experiments, lane 13 (negative control) corresponded to the reactivity of normal rabbit serum tested at a dilution of 1:40, the highest concentration of antiserum used in these experiments.
Immunoprecipitation experiments are typically conducted under conditions that allow antigenantibody interactions to occur under relatively native (ie, nondenaturing) conditions thus allowing detection of conformational as well as linear epitopes (the former being absent in standard immunoblot experiments). Under conditions previously used in studies of sera from patients with various immunobullous diseases including AECP, patient serum samples were tested at dilutions of 1:50. To assess the relative sensitivity of this immunochemical procedure, various dilutions (specifically, 1:50, 1:100, 1:500, 1:5000, 1:10,000, and 1:50,000) of reference laminin 5 antiserum, normal rabbit serum (negative
control), patients’ sera, and normal human serum (negative controls) were studied in comparative immunoprecipitation experiments utilizing a fixed amount of biosynthetically radiolabeled HK extracts. These studies showed that a 1:10,000 dilution of reference laminin 5 antiserum retained the ability to immunoprecipitate this protein from HK extracts (Fig 3, A). Maximal dilutions of sera from representative AECP patients that retained the ability to immunoprecipitate laminin 5 in these comparative experiments were 1:500 for Patients 1 and 2 and 1:5000 for Patient 3 (Fig 3, B). Again, there was a general correlation between the relative ability of these patients’ sera to immunoprecipitate HK-derived
890 Lazarova et al
J AM ACAD DERMATOL DECEMBER 2004
Fig 2. Sera from representative patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. One hundred twenty g of HK ECM was studied by immunoblot as shown in Fig 1; serial dilutions of patient sera (1:40 to 1:10,240) were tested simultaneously. All AECP sera immunoblotted the 200 kd (unprocessed) and 165 kd (processed) a subunits of laminin 5. The end titers of sera from Patients 1, 2, and 3 in these immunoblot experiments were 320, 1280, and 2560, respectively (denoted by arrows). Strong and specific reactivity (ie, bright immunoreactive bands with minimal levels of background staining) were consistently observed when patients’ sera were tested at a dilution of 1:40; an equivalent dilution of normal human serum showed no reactivity to HK ECM in control studies conducted in parallel (negative control, lane 28). Reference laminin 5 antiserum (positive control, lane 30) immunoblotted all subunits of laminin 5; normal rabbit serum (lane 29) showed no reactivity to these polypeptides.
laminin 5 and bind the dermal side of 1M NaCl split skin by indirect IF microscopy.
DISCUSSION Over the past 15 years, studies from different laboratories have shown that autoantibodies from patients with CP target different autoantigens in epithelial BMs.1,20-24 Accordingly, CP is currently thought to represent a disease phenotype rather than a single nosologic entity. AECP represents a well-characterized disease that has been estimated to comprise 5%-15 % of patients with CP. Recent studies have shown that patients with AECP have an increased relative risk for malignant solid tumors.8,9 This relative risk is considerable and approximates the incidence of solid tumors among adults with dermatomyositis.8 To date, AECP-associated solid tumors have included adenocarcinomas of the lung, stomach, colon, and uterus.8 Most cancers in patients with AECP proved to be fatal within a relatively short interval after their identification.8 Accordingly, the identification of patients with AECP has assumed greater significance. This consideration has direct relevance to this study since patients with AECP cannot be differentiated from patients with other types of CP by routine clinical, histologic, or immu-
nopathologic studies. To date, the diagnosis of AECP has been restricted to patients with demonstrable circulating autoantibodies directed against laminin 5. In this study, various immunochemical techniques used to detect anti-laminin 5 autoantibodies were compared directly to establish their relative sensitivity and practical utility. The first investigative technique used for the detection and characterization of autoantibodies from patients with AECP was immunoprecipitation studies of biosynthetically radiolabeled HK extracts and/or culture media.1,2 Studies of reference laminin 5 antiserum as well as patient and control sera demonstrated that this method is indeed the most sensitive technique for the detection of antibodies directed against laminin 5. Despite this sensitivity, immunoprecipitation techniques remain technically demanding, expensive, labor intensive, and largely dependent upon the use of biosynthetically radiolabeled cell-derived materials. These considerations limit the availability of immunoprecipitation techniques to a relatively small number of research laboratories. Accordingly, a number of groups have developed and applied various immunoblotting protocols for the detection of anti-laminin 5 autoantibodies. Since there is no standard antigen
J AM ACAD DERMATOL VOLUME 51, NUMBER 6
Lazarova et al 891
Fig 3. Immunoprecipitation studies of biosynthetically radiolabeled HK extracts represented the most sensitive method for detection of antibodies directed against laminin 5. A, Immunoprecipitation studies of biosynthetically radiolabeled HK extracts using reference laminin 5 antiserum (panel A, lanes l-6, serum dilutions 1:50 to 1:50,000, see Materials and Methods) identified laminin 5 at dilutions as great as 1:10,000 (lane 5); normal rabbit serum (control) did not immunoprecipitate laminin 5 (or other polypeptides) at the highest concentration tested (i.e., 1:50, lane 7). B, Sera from patients with AECP (Patient 1, lanes 1-6; Patient 2, lanes 7-12; Patient 3, lanes 13-18) recognized the same set of polypeptides as the reference laminin 5 antiserum. None of these polypeptides were immunoprecipitated by normal human serum (lane 19). Maximal dilutions of patient sera that retained the ability to immunoprecipitate laminin 5 were as follows: 1:500 for Patients 1 and 2 (lanes 3 and 9) and 1:5000 for Patient 3 (lane 16) as denoted by arrows. Molecular weights of the laminin 5 polypeptides in this complex were 190, 170, 140 and 130 kdDs.
substrate for such immunoblot experiments, this study sought to compare directly the relative utility of different preparations for detection of antibodies against laminin 5. Comparative studies of 5 different immunoblot substrates found that HK ECM was clearly the preferred substrate for detection of antibodies directed against laminin 5. A practical alternate substrate for detection of anti-laminin 5 antibodies was extracts of cultured HKs. HK extracts were somewhat easier to prepare than HK ECM, and when applied to minigels at higher protein loads, provided a favorable substrate for detection of antibodies against laminin 5 as well as other keratinocyte autoantigens such as BPAG1 and BPAG2. Comparative immunoblot studies showed that extracts of HKs were superior to extracts derived from A-431 or HaCat cell lines. Interestingly, though epidermal sheets were shown to be a source of the keratinocyte autoantigens BPAG1 and BPAG2, comparative immunoblot experiments found that this antigen source lacked sufficient amounts of laminin 5 to be of practical utility for the routine detection of autoantibodies against this laminin isoform. This finding was of relevance in that extracts of epidermal sheets have been used for the identification and characterization of autoantibodies from patients with immunobullous diseases.
In summary, the detection of autoantibodies against laminin 5 can be performed using a series of routine and specialized tests. Frontline screening measures should rely upon indirect immunofluorescent microscopy testing of 1M NaCl split skin since patients with AECP have demonstrable IgG autoantibodies directed against the dermal side of this tissue substrate. To distinguish patients with AECP from other patients with IgG anti-BM autoantibodies directed against the dermal side of 1 M NaCl split skin, immunoblot studies of HK ECM or HK extracts can be used to identify autoantibodies against laminin 5 subunits. Rabbit antilaminin 5 IgG is now commercially available (Biodesign International, Saco, Me) for use as a reference positive control in such immunoblot studies. While this antibody identifies all unprocessed and processed subunits of this laminin isoform in HK ECM, its relative sensitivity is 5-fold less than our laboratory’s reference laminin 5 antiserum (data not shown). Interestingly, another recent study found that immunoblotting of purified laminin 5 was a valid way to identify patients with AECP.25 Detection of low titer anti-laminin 5 autoantibodies as well as autoantibodies uniquely reactive with conformational determinants on this heterotrimer (ie, epitopes not displayed under the
892 Lazarova et al
denaturing conditions of standard immunoblot studies) should be submitted to reference laboratories for immunoprecipitation studies of biosynthetically radiolabeled HK extracts and/or media. Such laboratories often have available an array of test substrates as well as monoclonal antibodies directed against human IgG (especially, IgG4) that can be used to further enhance the identification of patients with AECP.26 Characterizing the specific reactivity of autoantibodies from patients with CP allows such patients to be segregated into subgroups that can be studied longitudinally to determine if such patients display differences in their relative extent of disease, prognosis, associations, or response to therapy. REFERENCES 1. Domloge-Hultsch N, Gammon WR, Briggaman RA, Gil SG, Carter WG, Yancey KB. Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease. J Clin Invest 1992;90:1628-33. 2. Domloge-Hultsch N, Anhalt GJ, Gammon WR, Lazarova Z, Briggaman R, Welch M, et al. Anti-epiligrin cicatricial pemphigoid. A subepithelial bullous disorder. Arch Dermatol 1994; 130:1521-9. 3. Kirtschig G, Marinkovich MP, Burgeson RE, Yancey KB. Anti-basement membrane autoantibodies in patients with anti-epiligrin cicatricial pemphigoid bind the alpha subunit of laminin 5. J Invest Dermatol 1995;105:543-8. 4. Lazarova Z, Hsu R, Yee C, Yancey KB. Anti-epiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5 (alpha3beta3gamma2). Br J Dermatol 1998;139:791-7. 5. Lazarova Z, Yee C, Darling T, Briggaman RA, Yancey KB. Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice. J Clin Invest 1996;98:1509-18. 6. Lazarova Z, Hsu R, Yee C, Yancey KB. Human anti-laminin 5 autoantibodies induce subepidermal blisters in an experimental human skin graft model. J Invest Dermatol 2000;114: 178-84. 7. Lazarova Z, Hsu R, Briggaman RA, Yancey KB. Fab fragments directed against laminin 5 induce subepidermal blisters in neonatal mice. Clin Immunol 2000;95:26-32. 8. Egan CA, Lazarova Z, Darling TN, Yee C, Cote T, Yancey KB. Anti-epiligrin cicatricial pemphigoid and relative risk for cancer. Lancet 2001;357:1850-1. 9. Egan CA, Lazarova Z, Darling TN, Yee C, Yancey KB. Antiepiligrin cicatricial pemphigoid: clinical findings, immunopathogenesis, and significant associations. Medicine (Baltimore) 2003;82:177-86. 10. Egan CA, Yancey KB. The clinical and immunopathological manifestations of anti-epiligrin cicatricial pemphigoid, a recently defined subepithelial autoimmune blistering disease. Eur J Dermatol 2000;10:585-9.
J AM ACAD DERMATOL DECEMBER 2004
11. Lazarova Z, Yancey KB. Reactivity of autoantibodies from patients with defined subepidermal bullous diseases against 1 mol/L salt-split skin. Specificity, sensitivity, and practical considerations. J Am Acad Dermatol 1996;35:398-403. 12. Carter WG, Ryan MC, Gahr PJ. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell 1991;65:599-610. 13. Lee CW. An extract of A-431 cells contains major tissue antigens of autoimmune bullous diseases. Br J Dermatol 2000;143:821-3. 14. Leverkus M, Bhol K, Hirako Y, Pas H, Sitaru C, Baier G, et al. Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230. Br J Dermatol 2001;145:998-1004. 15. Labib RS, Anhalt GJ, Patel HP, Mutasim DF, Diaz LA. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol 1986;136: 1231-5. 16. Lazarova Z, Yee C, Lazar J, Yancey KB. IgG autoantibodies in patients with anti-epiligrin cicatricial pemphigoid recognize the G domain of the laminin 5 alpha-subunit. Clin Immunol 2001;101:100-5. 17. Stanley JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981;24:897-903. 18. Basset-Seguin N, Caughman SW, Yancey KB. A-431 cells and human keratinocytes synthesize and secrete the third component of complement. J Invest Dermatol 1990;95:621-5. 19. Marinkovich MP, Lunstrum GP, Burgeson RE. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 1992;267:17900-6. 20. Bernard P, Prost C, Durepaire N, Basset-Seguin N, Didierjean L, Saurat JH. The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen. J Invest Dermatol 1992;99:174-9. 21. Shimizu H, Masunaga T, Ishiko A, Matsumura K, Hashimoto T, Nishikawa T, et al. Autoantibodies from patients with cicatricial pemphigoid target different sites in epidermal basement membrane. J Invest Dermatol 1995;104:370-3. 22. Ghohestani RF, Nicolas JF, Rousselle P, Claudy AL. Identification of a 168-kDa mucosal antigen in a subset of patients with cicatricial pemphigoid. J Invest Dermatol 1996;107:136-9. 23. Tyagi S, Bhol K, Natarajan K, Livir-Rallatos C, Foster CS, Ahmed AR. Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization. Proc Natl Acad Sci U S A 1996;93:14714-9. 24. Chan LS, Majmudar AA, Tran HH, Meier F, Schaumburg-Lever G, Chen M, et al. Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid. J Invest Dermatol 1997;108:848-53. 25. Hisamatsu Y, Nishiyama T, Amano S, Matsui C, Ghohestani R, Hashimoto T. Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid. J Dermatol Sci 2003;33:113-9. 26. Hsu R, Lazarova Z, Yee C, Yancey KB. Noncomplement fixing, IgG4 autoantibodies predominate in patients with anti-epiligrin cicatricial pemphigoid. J Invest Dermatol 1997;109:557-61.