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Takashi Hashimoto, M.D., Department of Dermatology, Kurume University, School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Email: email@example.com
Anti-laminin γ1 pemphigoid is an autoimmune subepidermal bullous disease first described in 1996, and has been distinct from previously known subepidermal blistering diseases, such as bullous pemphigoid and epidermolysis bullosa acquisita. Circulating autoantibodies of the patients do not react to any known autoantigen of the skin, but react to a 200-kDa molecule (p200) from dermal extracts. The identity of p200 was unmasked as laminin γ1, an extracellular matrix glycoprotein composing several forms of laminin heterotrimers. We renamed this disease from the previously used anti-p200 pemphigoid to anti-laminin γ1 pemphigoid, a new entity of an autoimmune bullous disease. In this decade, we have experienced over 70 cases of this disease. Although the number of the cases of anti-laminin γ1 pemphigoid is half as many as the number of definitely diagnosed cases of epidermolysis bullosa acquisita in the same duration, a considerable number of the cases could be clinically misdiagnosed as epidermolysis bullosa acquisita. Unveiling the pathogenicity and development of a useful diagnostic method is necessary for appropriate management of this new disease.
Investigation into autoimmune bullous diseases is at the frontier of the research field of autoimmune diseases. A series of outstanding studies have contributed to a better understanding of their pathology. In addition, major autoantigens of autoimmune bullous diseases have been identified in the last few decades of the 20th century.1–4 Nevertheless, some autoantigens are still mysterious in these serious and sometimes even life-threatening diseases. Anti-laminin γ1 pemphigoid has been at the head of the list.5 This is a new autoimmune subepidermal bullous disease, which has been characterized by autoantibodies against a 200-kDa protein (p200) of the dermal–epidermal junction. Despite attempts, the identity of p200 had remained an enigma.
Unveiling the pathogenicity and development of a useful diagnostic method are necessary for appropriate treatment of new diseases. We have contributed to a comprehensive review in a recent issue of the Journal.5 In the present review, we will highlight the biological aspects of this disease chronologically, and describe our new work suggesting that laminin γ1 is the autoantigen in patients with anti-laminin γ1 pemphigoid. Thus, we herein describe this disease, which was formerly called anti-p200 pemphigoid, as a new entity, “anti-laminin γ1 pemphigoid”.6
Discovery of anti-laminin γ1 pemphigoid
The first cases
In 1996, we reported two cases of a novel disease with autoantibodies to a basement membrane zone antigen that was different from the targets, which had been described so far.7,8
One patient, a 72-year-old woman with a 20-year history of psoriasis, developed widespread tense blisters on both intact skin and on psoriatic plaques, with pruritic erythematous lesions resembling bullous pemphigoid. There was no mucosal involvement.7 Another patient, a 54-year-old man without any history of psoriasis, showed widespread tense bullae and severe mucous membrane involvement.8 In both patients, direct immunofluorescence microscopy showed linear immunoglobulin (Ig)G and C3 deposition at the basement membrane zone. The patients’ IgG autoantibodies bound exclusively to the dermal side of salt-split normal human skin. Indirect immunogold electron microscopy showed a marked deposition of IgG at the lower lamina lucida and minimal deposition at the hemidesmosomes. Immunoblot analysis identified a unique 200-kDa autoantigen (p200) in dermal extracts. The autoantibody did not recognize bullous pemphigoid antigens, the autoantigen of epidermolysis bullosa acquisita, or laminin 332 (formerly laminin 5).
These are the first cases of anti-laminin γ1 pemphigoid, differentiated from preexisting autoimmune bullous diseases by its unique autoantigen with a molecular weight of 200 kDa.
Typical patients of anti-laminin γ1 pemphigoid are approximately 50–70 years old, somewhat younger than most patients of bullous pemphigoid, and often suffering from preexisting psoriasis. The patients develop tense blisters and urticarial eruptions, sometimes with itches, closely resembling those of bullous pemphigoid. In indirect immunofluorescence microscopy on 1 mol/L NaCl-split skin, anti-laminin γ1 pemphigoid immunoglobulin (Ig)G reacts with the dermal side of basement membrane, whereas bullous pemphigoid IgG shows reactivity with its epidermal side. Both anti-laminin 332 mucous membrane pemphigoid IgG and epidermolysis bullosa acquisita IgG show reactivity with the dermal side of basement membrane in salt-split skin. However, mucous membrane pemphigoid mainly affects ocular and oral areas, and develops clearly demarcated bullae and blisters without surrounding urticarial eruptions. Typical epidermolysis bullosa acquisita patients also develop non-inflamed mechanical bullae that heal with atrophic scarring, although clinically indistinguishable from anti-laminin γ1 pemphigoid or bullous pemphigoid in many cases.5,9
Histologically, skin biopsy specimen from an early erythematous lesion show a linear infiltrate of neutrophils, and sometimes eosinophils, along the dermoepidermal junction, as discussed later.5,9,10
We have diagnosed more than 70 cases of anti-laminin γ1 pemphigoid. The number of cases is just half as many as the number of cases of epidermolysis bullosa acquisita, which we have definitely diagnosed by immunoblotting of human dermal extracts during the same time. One may assume that a fair share of subepidermal autoimmune bullous disease had been diagnosed by only the result of immunofluorescence of salt-split skin showing circulating autoantibody against the dermal side of the basement membrane zone, not by further confirmation with immunoblot study. Therefore, a considerable number of the cases of anti-laminin γ1 pemphigoid could have been clinically misdiagnosed as epidermolysis bullosa acquisita. We should make an effort to develop a useful diagnostic method for appropriate treatment of this new disease.
Characterization of p200
The early suspects
The identity of p200 has been unresolved for more than a decade. According to molecular weight, the first suspects of the 200-kDa antigen were β1 or γ1 components of laminins, such as laminin 111 (formerly, laminin 1), laminin 211 (laminin 2) and laminin 311 (laminin 6).7–9 This idea was supported by the result of immunoelectronmicroscopic studies showing that p200 localizes to the lower potion of lamina lucida outside desmosomal plaques. In our early immunoblot studies of human dermal extracts, the anti-human placental laminin polyclonal antibodies reacted with an approximately 200-kDa protein, which corresponded to β1 and γ1 chains and showed a just similar band to the p200 bands detected by the patients’ sera.7–9
Nevertheless, by immunoblot analyses of human placental laminin, which should be mainly composed of laminin 211 and 221, no patients’ sera showed any reactivity in a reduced condition. Further, by immunoblot analyses with purified laminin 332 and 311, none of the patients’ sera showed specific reactivity. Also, sera from all the patients showed no reactivity with dermal vessels, the basement membrane zone of kidney or lung on indirect immunofluorescence, although β1 and γ1 chains are the essential components of blood vessel walls as laminin 411 (laminin 8).7–9
This is how those first suspects were released, at least for 10 years. However, there is no statute of limitations in the field of science. The suspects were confronted with irrefutable evidence later.
In 2003, Shimanovich et al.11 approached p200 by biochemical methods. This study suggested that the p200 is an acidic non-collagenous N-linked glycoprotein of the cutaneous basement membrane. Two-dimensional gel electrophoresis demonstrated that p200 is a protein with an isoelectric point of 5.4–5.6. Mass spectrometry identified the α3 chain of type VI collagen and filamin A as the major components of the spot. However, no patients’ serum was reactive with either purified human type VI collagen or recombinant full-length human filamin A.
Bruckner-Tuderman’ s group had precisely investigated the identity of p200.12 In 2008, Hofmann et al. reported that p200 is synthesized by both keratinocytes and fibroblasts, and evaluated whether nidogen-2 is p200, because it is a 200-kDa protein and a participant of the dermal–epidermal junction in the basement membrane zone, where it connects laminins with type IV collagen. Indirect immunofluorescence showed that anti-p200 serum and nidogen-2 antibodies label the dermal side of the blister in human salt-split skin. Immunoblot study of human fibroblast extracts showed that both anti-p200 serum and polyclonal nidogen-2 antibodies recognize a protein of identical size. Ultimately, however, patients’ sera showed no reactivity with recombinant human nidogen-2.
Laminin γ1 is p200
Recently, we also tried to identify the 200-kDa autoantigen of this disease by proteomic strategy.6 We performed 2-D gel electrophoresis of dermal extracts and immunoblotting with patients’ sera, followed by mass spectrometry analysis of a unique protein band. The protein band corresponded to laminin γ1. Anti-laminin γ1 monoclonal antibody (mAb) reacted with the anti-p200 immunoprecipitates by immunoblotting. Anti-p200 sera from the 32 patients showed 90% reactivity to the recombinant products of laminin γ1 (Fig. 1). None of the healthy control sera reacted with laminin γ1. Therefore, we concluded that laminin γ1 is the autoantigen in this disease, which we had temporally called anti-p200 pemphigoid.
Our proteomic method has detected and characterized the two other candidates for p200 (Teruki Dainichi, unpubl. data, 2008). The band on 2-D gel electrophoresis of approximately 200-kDa and pI 6.1 region corresponded to the α3 chain of type VI collagen, although its band migrated to a little more basic side than that detected in the previous study and the unprocessed α3 chain of type VI collagen is estimated to be MW 345 471 and pI 6.40 from primary structure. Another unique band located at approximately 200-kDa and pI 5.1 corresponded to the α1 chain of type XIV collagen, calculated to be MW 194 744 and pI 5.16. Type VI collagen is a microfibril collagen, consists of three different chains and mutations in the genes cause Bethlem myopathy and Ullrich congenital muscular dystrophy. Type XIV collagen is a fibril-associated collagen composed of only one type of chain. Both type VI and type XIV collagen are resistant or partially sensitive to bacterial collagenase.
However, immunoblot study of dermal extracts showed that neither anti-collagen VI α3 mAb nor collagen XIV antiserum detected a specific band similar to the p200 band. By immunoblot study of purified collagen VI, none of the patients’ sera showed specific reactivity. Immunofluorescence study did not show any specific reaction of anti-collagen VI α3 mAb nor collagen XIV antiserum to the basement membrane zone of the skin. Therefore, there are poor evidences showing that these other candidates, α3 chain of type VI collagen and α1 chain of type XIV collagen, are p200. It is possible that some patients’ sera show bystander reactivity to these collagen components and the p200 band in immunoblotting of dermal extracts may correspond to heterogeneous protein, although laminin γ1 certainly is a major component.
Pathogenesis and laminin γ1
Laminin γ1 in the skin
Laminin γ1 is a 200-kDa N-linked glycoprotein,13 that is present in the cutaneous basement membrane zone, as a component of different forms of laminin heterotrimers, such as laminin 311/321 (laminin 6/7) and 511 (laminin 10), which contributes to dermal–epidermal adhesion outside hemidesmosomes (Fig. 2). The globular domains of α chain and C-terminal structure of γ1 chains are responsible for laminin–integrin molecular interaction. On the other hand, there is a nidogen-binding site on the arm part of laminin γ1. Interaction of laminin γ1 with the nidogen anchors laminin γ1 to the type IV collagen in the basement membrane zone.14,15 Functional inhibition of laminin γ1 by a nidogen-binding laminin γ1 chain-blocking fragment resulted in complete suppression of basement membrane formation in a 3-D co-culture of human skin keratinocytes and fibroblasts.16 Whether patients’ sera react with nidogen-binding sites on the laminin γ1 molecule needs further clarification.
By immunoblotting, reactivity of a patient’s serum with p200 was competitively inhibited by adding anti-laminin γ1 C-terminus mAb, of which the recognizing epitope is localized to the 246 C-terminal amino acids within the coiled-coil domain.6 Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin γ1.6 Most laminin γ1-positive sera showed reactivity with the recombinant laminin γ1 C-terminal E8 fragment. The nine C-terminal residues are known to be critically involved in laminin recognition by integrins. Particularly, the glutamic acid residue at the third position from the C-terminus of laminin γ1 is required for integrin binding by laminin trimers.17 One therefore may speculate that anti-laminin γ1 autoantibodies modify the laminin–integrin interaction by interfering with the intrinsic binding site used for connecting these molecules in patients’ skin in a direct or indirect manner.
The patients with anti-laminin γ1 pemphigoid develop skin blisters but show no pathological changes in other organs, although laminin γ1 is widely expressed in basement membrane zones over the body.18 Indeed, in immunofluorescence on human skin sections, most patients’ sera show no visible reactivity with blood vessel walls, except for a particular case.5,6,9 We speculated the mechanism of this antigenic discrepancy among each organ as follows. The epitope of the autoantigen, suggested to be located at the C-terminus of laminin γ1, may have a different 3-D structure in each laminin trimer among organs, or even in a posttranslational modification level of the single chain before folding. We evaluated the reactivity of the patients’ sera and purified IgG to the dermal extracts, blood vessel extracts and purified laminin 111 from the JAR cell line, in immunoblot study (Fig. 3).6 Reactivity of patients’ sera and purified IgG to dermal laminin γ1 was higher than reactivity to blood vessel laminin γ1 under reducing conditions. These results suggest that laminin γ1 in the epidermal basement membrane zone may have different posttranslational modifications, such as glycosylation, compared with laminin γ1 expressed in blood vessels (Fig. 4). Differences in posttranslational modification may allow further possible explanations for the organ specificity of the disease.
Laminin and psoriasis
As with one of the first cases of anti-laminin γ1 pemphigoid,7 a fair share of the cases of anti-laminin γ1 pemphigoid are associated with psoriasis.5,9 Indeed, almost one-third of the 61 cases we have experienced were associated with psoriasis (Takako Tsuji, unpubl. data, 2008). This proportion is as high as the coincidence of psoriasis in any other known autoimmune bullous diseases such as bullous pemphigoid, which has occasionally developed in psoriatic patients.19
Previous immunohistochemical study showed that the expression of the α1 chain in the basal lamina of active psoriasis vulgaris is discontinuous or normal, the expression of the α2 chain is weak and irregular in the lesions, while the expression of the β1 and γ1 laminin chains and collagen IV is normal.20,21 Nevertheless, one can imagine that qualitative or quantitative change of laminin γ1 expression, which is unique in psoriatic skin but could not be detected by previous immunohistochemical studies, may be involved in the production of anti-laminin γ1 autoantibodies in psoriatic patients, and followed by the development of anti-laminin γ1 pemphigoid (Fig. 5).
In psoriasis, production of many cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1 and γ-interferon is upregulated, and these cytokines are also capable of regulating expression of matrix metalloproteinase (MMP). Several types of MMP degrade laminins.22 A previous report showed that laminin γ1 from laminin 511 can be degraded by MMP-9,23 which is highly expressed in the psoriatic skin lesions.22 Increase of laminin γ1 fragments that are processed by MMP-9 in psoriatic lesions may initiate the production of anti-laminin γ1 autoantibody, which binds to the essential site for molecular interaction in the basement membrane zone of the skin.
Several lines have suggested that expression of integrins is modified in the basement membrane zone of psoriatic skin.24 Changes in integrin expression in basal keratinocytes may affect the laminin–integrin interaction, and the molecular kinetics and metabolism of laminins in the basement membrane zone. These changes may be involved in anti-laminin autoantibody production.
Thus, pathogenic interaction between psoriasis and anti-laminin γ1 pemphigoid should be worth investigating for elucidation of the molecular mechanism in both diseases.
Laminin and neutrophils
In some patients of anti-laminin γ1 pemphigoid, histopathological findings in lesional skin associate with superficial infiltration of various numbers of eosinophils, resulting in a microscopic picture reminiscent of bullous pemphigoid. However, typical histological features of anti-laminin γ1 pemphigoid show superficial inflammatory infiltrate of neutrophils, even with occasional formation of microabscesses at the tips of dermal papillae.5,10
Interleukin-8 is a major chemotactic cytokine for neutrophils and has been implicated in the inflammatory process of bullous pemphigoid25 and psoriasis.26 Kitajima’ s group examined whether autoantibodies from a patient with p200 pemphigoid can upregulate the release of IL-8 from normal human epidermal keratinocytes in vitro.27 No response was seen in the release of IL-8 from keratinocytes treated with purified IgG from a single case of anti-p200 pemphigoid in their system. It is not an unexpected result, because laminin is not a membranous protein such as bullous pemphigoid (BP) 180 protein, but rather a secretory protein.
On the other hand, integrin is a receptor for several kinds of biological activity, such as cell proliferation, cell shape and polarity, and cytokine production of epithelial cells.28 For example, α3β1 integrin, which is one of the counterparts for laminin 511, can induce epithelial cell cytokine responses such as IL-1, -6 and -8.29 Anti-laminin γ1 antibody may induce the activation of integrin signaling pathways, which result in the production of chemoattractants for neutrophils, by disrupting laminin–integrin interaction in vivo and the more elaborate ex vivo system.
Proteolysis of basement membrane or even hemidesmosomal proteins results in fragments that are chemotactic for various cell types.30 Laminins are degraded by neutrophil- and macrophage-derived proteases, such as neutrophil elastase, cathepsin G, proteinase 3 and MMP.23 Laminin-derived peptides have chemotactic activity for neutrophils. Digestion of laminin 111 by human neutrophils, neutrophil elastase, or cathepsin G liberates fragments that were chemotactic for human neutrophils.31 A number of synthetic peptides derived from laminin 111 have also been shown to facilitate either neutrophil chemotaxis or chemokinesis.32 Both laminin 332 fragments generated by neutrophil elastase digestion and neutrophil elastase-digested laminin γ2 peptide were found to be chemotactic for neutrophils.23 Further, a cryptic peptide sequence from the laminin α5 chain, which is composed of laminin 511 with laminin γ1 and exists also in the skin, is chemotactic for both neutrophils and macrophages and induces MMP-9 production in vitro and in vivo, and regulates inflammatory immune responses through TNF-mediated signaling.33–35 These results suggest the existence of possible cross-talk between neutrophils and laminins in anti-laminin γ1 pemphigoid, and psoriasis.
Identification of autoantigen in anti-laminin γ1 pemphigoid provides us prospects in both clinical medicine and basic biology as follows. First, we can directly approach the mechanism of autoimmunity and pathogenicity in this disease. Second, we can develop an accurate and concise diagnostic method, which enables us to treat the patients of this disease more quickly and appropriately. Finally, this new finding would clarify known but very complicated biological phenomena in the epidermal–dermal interface and bring laminin into the limelight, as the hub molecule among matrix biology, immunology and molecular cell biology.
We gratefully appreciate Miss Ayumi Suzuki, Miss Takako Ishikawa and Miss Sachiko Sakaguchi for technical assistance and Miss Akiko Tanaka and Mrs Yasuko Nakayama for secretarial work. We thank the patients for their participation. This work was supported by Grants-in-Aid for Scientific Research and Strategic Research Basis Formation Supporting Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health and Labor Sciences Research Grants and grants for Research on Measures for Intractable Diseases from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by grants form the Uehara Memorial Foundation, the Nakatomi Foundation.