Bullous pemphigoid: interaction of interleukin 5, anti-basement membrane zone antibodies and eosinophils. A preliminary observation.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease characterized by the presence of autoantibodies to normal components of the hemidesmosome (BPAg1 and BPAg2). Histology of skin lesions demonstrate a subepidermal vesicle and a predominantly eosinophilic dermal cellular infiltrate. Interleukin 5 (IL-5) plays a major role in eosinophil recruitment and function. Using an ELISA, we investigated the levels of IL-5 in the sera and blister fluid of BP patients with active disease and those in prolonged clinical remission treated with intravenous immunoglobulin, and compared it to that in normal controls. Significantly increased levels of IL-5 were detected in the serum and particularly the blister fluid of patients with active disease (P=0.0043) when compared to levels in normal controls. There was no significant difference in IL-5 levels in patients in prolonged clinical remission compared to normal control serum. In an immunoblot assay, using bovine gingival lysate as substrate, we determined the presence of IgG and IgE autoantibodies specific to basement membrane zone proteins in blister fluid and serum of BP patients. IgG autoantibodies to BPAg1 and BPAg2 were detected in both blister fluid and serum of patients, whereas IgE autoantibodies, in much lower titers, were detected to only BPAg1. Elevated levels of blood and tissue eosinophilia were observed in these patients. Based on these data, we present a preliminary hypothesis for the initiation, progression and localization of blister formation in BP.     

Basement membrane reactivity of antisera to type IV collagen and sera from patients with bullous pemphigoid and epidermolysis bullosa acquisita

The basement membrane antigenic specificities of antibodies to Type IV collagen were compared to naturally occurring antibodies in sera from patients with bullous pemphigoid and epidermolysis bullosa acquisita (EBA) by indirect immunofluorescence, mixed immunofluorescence and immunoabsorption. Results suggested that the three sera reacted with three different basement membrane antigens. In addition, absorption with Types I, II, III, or IV collagen failed to reduce the basement membrane reactivities of bullous pemphigoid or EBA sera. The antibodies to the basement membrane components should be useful in studying skin and mucous membrane diseases including periodontal diseases.     

Presence of interleukin 10 in the serum and blister fluid of patients with pemphigus vulgaris and pemphigoid

Interleukin 10 (IL-10) is an immunoregulatory cytokine produced by T lymphocytes and macrophages. Recently, it has been suggested that IL-10 may be involved in the pathogenesis of various inflammatory and autoimmune diseases. Using an ELISA we investigated the presence of IL-10 in the serum and blister fluid of pemphigus vulgaris (PV) patients with active disease and those in prolonged clinical remission compared with normal controls. Sera from patients with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), oral pemphigoid (OP) and blister fluid from five patients with BP were also studied. Increased levels of IL-10 were detected in the sera of 87.5% of patients with active PV and were statistically significant (P=0.0003) when compared with levels in normal human serum. Lower levels of IL-10 were detected in 12.5% PV patients in remission and were statistically significant (P=0.0001) when compared with levels in patients with active disease. Levels of IL-10 were detected in sera of 4.6% (1 of 24) of the normal controls. The levels of IL-10 were approximately four times higher in blister fluids than levels in the serum in the same PV patients. This difference was highly statistically significant (P=0.0008). A correlation was observed between serum levels of IL-10 and titres of pemphigus autoantibodies and with disease severity. Elevated level of IL-10 was detected in the blister fluid from five BP patients. Levels of IL-10 in the sera of patients with BP, OCP and OP were not significantly increased. These preliminary data suggest that IL-10 in concert with other cytokines may play an important role in the pathogenesis of PV and BP.     

Bullous pemphigoid‐like skin blistering disease in a rhesus macaque (Macaca mulatta)

Autoimmune bullous disease is very uncommon in non‐human primates. We observed a bullous skin disease in a male rhesus monkey while conducting porcine islet xenotransplantation. Fifty days after the transplantation, multiple bullous skin lesions were observed. There was no mucosal involvement. Skin biopsy results demonstrated a subepidermal blister with no necrotic keratinocytes. Immunofluorescent staining showed linear IgG deposition at the roof of the blister. These skin lesions spontaneously disappeared. Considering these results, this monkey was diagnosed with bullous pemphigoid (BP). As far as we know, this is the first report of BP in non‐human primates.     

Cytokines and bullous pemphigoid.

This report reviews the data presented in the literature concerning the presence and levels of different cytokines in sera, lesional tissue or blister fluids of patients with bullous pemphigoid. The list of cytokines analysed includes 21 molecules: interleukins (IL)-1 => 8, IL-10 => 13, IL-15, granulocyte-monocyte-colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), oncostatin-M (OSM), regulated upon activation normal T cell expressed and presumably secreted (RANTES), transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF). Basic information regarding the functions of these cytokines and their possible involvement in the pathogenetic steps of the disease, such as autoantigen expression, autoantibody induction, complement activation, local cell recruitment and stimulation, resident cell activation, release of various effector molecules and tissue damage are also reported. A specific function for each cytokine in bullous pemphigoid induction cannot be still defined, however, the literature attributes a major role to IL-1, IL-4, IL-5, IL-6, IL-8 and IFN-gamma. On the basis of significant (direct or inverse) correlations found between disease intensity and the blister fluid/serum levels, the following cytokines IL-7, IL-15, RANTES, VEGF and TNF-alpha, besides those previously mentioned, may also be involved in this disease.     

Immunoglobulin light and heavy chain isotypes in skin diseases: restricted distribution in bullous pemphigoid and linear IgA bullous dermatosis

To assess the heterogeneity of immunoglobulins involved in various skin diseases, direct and indirect immunofluorescence studies of skin biopsies and sera, respectively, for kappa and lambda light chains, were performed. The anti-basement membrane zone (anti-BMZ) antibodies of patients with bullous pemphigoid showed a predominance of kappa light chains, and patients with linear IgA bullous dermatosis showed a predominance of one light chain that was sometimes kappa and sometimes lambda. The bullous pemphigoid autoantibodies were then studied for IgG subclass distribution; a predominance of IgG4 was found. Although other explanations are possible, the light chain restriction in bullous pemphigoid most likely reflects heavy chain restriction and preferential association of heavy and light chain isotypes. The basis of the heavy chain restriction is not apparent. The light chain restriction in linear IgA bullous dermatosis may represent a restricted idiotypic repertoire.     

FcR-independent effects of IgE and IgG autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by IgE and IgG class autoantibodies specific for 180-kDa BP Ag 2 (BP180), a protein involved in cell-substrate attachment. Although some direct effects of BP IgG have been observed on keratinocytes, no study to date has examined direct effects of BP IgE. In this study, we use primary cultures of human keratinocytes to demonstrate Ag-specific binding and internalization of BP IgE. Moreover, when BP IgE and BP IgG were compared, both isotypes stimulated FcR- independent production of IL-6 and IL-8, cytokines critical for BP pathology, and elicited changes in culture confluence and viability. We then used a human skin organ culture model to test the direct effects of these Abs on the skin, whereas excluding the immune inflammatory processes that are triggered by these Abs. In these experiments, physiologic concentrations of BP IgE and BP IgG exerted similar effects on human skin by stimulating IL-6 and IL-8 production and decreasing the number of hemidesmosomes localized at the basement membrane zone. We propose that the Ab-mediated loss of hemidesmosomes could weaken attachment of basal keratinocytes to the basement membrane zone of affected skin, thereby contributing to blister formation. In this article, we identify a novel role for IgE class autoantibodies in BP mediated through an interaction with BP180 on the keratinocyte surface. In addition, we provide evidence for an FcR-independent mechanism for both IgE and IgG class autoantibodies that could contribute to BP pathogenesis.     

Human Eosinophils Express the High Affinity IgE Receptor,FcεRI,in Bullous Pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.     

Identification of an epitope within human integrin alpha 6 subunit for the binding of autoantibody and its role in basement membrane separation in oral pemphigoid

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin alpha6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin alpha6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin alpha6. Control sera did not bind to the full-length integrin alpha6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin alpha6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.     

Reconstitution of the epidermal basement membrane after enzymatic dermal-epidermal separation of embryonic mouse skin

Pieces of trypsin-isolated 14-day embryonic mouse epidermis were recombined with various living or non-living dermal or non-dermal substrates, in order to analyse the reconstruction of the dermal-epidermal junction. The constitution and ultrastructure of the epidermal basement membrane were characterized by immunolabelling of laminin, type IV collagen and bullous pemphigoid antigen, and by transmission electron microscopy. Trypsin treatment of dorsal skin followed by dermal-epidermal separation does not visibly damage the epidermal basement membrane, which remains attached to the lower face of epidermis. When freshly isolated epidermis is reassociated with dermis, the basement membrane is first degraded during the first 4 h of culture, then reconstituted within 24 h. When epidermis is cultured in isolation the basement membrane disappears within 4 h and is not reconstructed. Epidermis, precultured for 4 h and thus deprived of its basement membrane prior to reassociation, is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, with muscle tissue, or with a film of fibrous type I collagen. No basement membrane is reconstituted when the epidermis is recombined with heat (100 degrees C) killed dermis. It is concluded that, in the reconstituted epidermal basement membrane, laminin, type IV collagen, bullous pemphigoid antigen, and lamina densa are of exclusive epidermal origin.     

Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE

Nasal lavage fluids from unstimulated individuals contain a histamine-releasing factor (HRF) similar to those which we have previously described from macrophages, platelets, and from blister fluids obtained during the late cutaneous reaction. The nasal HRF was partially purified by ion-exchange chromatography and gel filtration. Although some m.w. heterogeneity was observed, the majority of the HRF eluted at an apparent m.w. range of 15,000 to 30,000. This partially purified HRF induced histamine release from basophils of certain individuals. Histamine release occurred via a mechanism which is IgE-dependent in that: basophils desensitized by exposure to anti-IgE in the absence of calcium no longer respond to HRF, and desensitization with HRF reduces responsiveness to anti-IgE; and removal of IgE from the basophil surface by using lactic acid renders cells unresponsive to HRF. We have further defined this IgE dependence and have shown that the reason that only selected basophil donors respond to HRF is due to a previously unrecognized, functional heterogeneity of IgE. Thus, passive sensitization using sera from responders restored the responsiveness of acid-stripped basophils and conferred responsiveness to basophils of a nonresponder with naturally unoccupied IgE receptors. Sera from nonresponders failed to do this even though similar numbers of IgE molecules were put onto the basophil surface in each case. This property of responder sera was due to IgE because both heating sera at 56 degrees C for 2 hr and passage of sera over anti-IgE-Sepharose (which removes greater than 90% of the IgE) markedly reduced the ability of sera to induce responsiveness, and because an excess of either purified IgE myeloma or purified penicillin-specific IgE antibody from a nonresponder competitively inhibited the ability of IgE from responder sera to induce responsiveness to HRF. We conclude that nasal lavage fluids contain an HRF which induces basophil histamine release in a specific, IgE-dependent fashion but only from individuals with the appropriate type of IgE. Because we have shown that basophils are recruited into the nose during the late-phase reaction, we suggest that nasal HRF may induce these cells to release histamine and other mediators which could contribute to the symptomatology of the late-phase reaction.     

The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo

We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.     

Bullous pemphigoid antigen (BPAG1): cDNA cloning and mapping of the gene to the short arm of human chromosome 6

Bullous pemphigoid antigen (BPAG1), an integral component of the cutaneous basement membrane zone, serves as autoantigen in a blistering disease, bullous pemphigoid. In this study, we have generated cDNAs corresponding to human BPAG1 sequences. Two cDNAs, a 0.45-kb PCR product synthesized with human keratinocyte RNA as template and a 2.2-kb cDNA isolated from human keratinocyte lambda gt11 library, were utilized for chromosomal in situ hybridizations to establish the genomic location of the BPAG1 gene. Metaphase chromosomes of phytohemagglutinin-stimulated human peripheral blood leukocytes were examined by hybridizations with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human BPAG1 gene is at locus 6p11-6p12. This conclusion was supported by hybridizations with a panel of human X rodent hybrid cell DNA, which indicated concordance with human chromosome 6. Because different genes encoding human basement membrane components have been mapped previously to chromosomes other than 6, the results further indicate that human basement membrane zone genes are widely dispersed within the human genome.     

大疱性类天疱疮IgG型抗基底膜带抗体的靶抗原初步分析

目的　研究大疱性类天疱疮 Ｉｇ Ｇ 型抗基底膜带抗体的靶抗原。方法　应用 Ｄ Ｉ Ｆ与 Ｉ Ｉ Ｆ检测的 Ｉｇ Ｇ型抗体阳性 Ｂ Ｐ 血清进行 Ｗ ｅｓｔｅｒｎ 免疫印迹分析。结果　初步研究发现 Ｉｇ Ｇ 阳性的 Ｂ Ｐ 血清能结合抗原１８０ Ｋ Ｄ,１６０ Ｋ Ｄ 以及 １０５ Ｋ Ｄ 与 ７７ Ｋ Ｄ 的多肽。结论　 Ｉｇ Ｇ 型 Ｂ Ｐ自身抗体可能靶向基底膜带不同层出现的不同抗原并提示该病的发生可能涉及不同的免疫机制。     

Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.     

Markers of atopic dermatitis,allergic rhinitis and bronchial asthma in pediatric patients: correlation with filaggrin,eosinophil major basic protein and immunoglobulin E

Background

Allergic reactions have been implicated as contributions in a number of atopic disorders, including atopic dermatitis (AD), allergic rhinitis (AR) and bronchial asthma (BA). However, the potential for filaggrin protein, eosinophil major basic protein (MBP) and immunoglobulin E (IgE) to elicit allergic response or to contribute to atopic disorders remains largely unexplored in pediatric patients. This study was undertaken to investigate the status and contribution of filaggrin protein, eosinophil MBP and total IgE in pediatric patients with AD, AR and BA.

Methods

Sera from 395 pediatric patients of AD, AR or BA with varying levels of disease activity according to the disease activity index and 410 age-matched non-atopic healthy controls were evaluated for serum levels of atopic markers, including filaggrin, eosinophil MBP and IgE.

Results

Serum analysis showed that filaggrin levels were remarkably high in pediatric patients with AD, followed by BA and AR, whereas its levels were low in non-atopic pediatric controls. Eosinophil MBP levels in sera of atopic patients were significantly high as compared with their respective controls, but its levels were highest in AR patients, followed by AD and BA. Total IgE in sera of AD patients was markedly high, followed by AR and BA patients, whereas its levels were low in non-atopic pediatric controls. Interestingly, not only was an increased number of subjects positive for filaggrin protein, eosinophil MBP or total IgE, but also their levels were statistically significantly higher among those atopic patients whose disease activity scores were higher as compared with atopic patients with lower disease activity scores.

Conclusions

These findings strongly support a role of filaggrin protein, eosinophil MBP and IgE in the onset of allergic reactions in pediatric patients with AD, AR and BA. The data suggest that filaggrin, eosinophil MBP or IgE might be useful in evaluating the progression of AD, AR or BA and in elucidating the mechanisms involved in the pathogenesis of these pediatric disorders.

     

Bullous pemphigoid antigen: isolation from normal human skin.

A protein moiety from epidermal PBS-soluble products was isolated by gel filtration (Bio-Gel A-1.5m) and ion exchange chromatography (DEAE-cellulose). This protein (A-1-Epid) was not retarded by DEAE-cellulose in Tris-HCl buffer, 15mM, pH 8.1. By IEP against an antiserum to epidermal antigens, it showed a single cathodal arc. On disc electrophoresis, at low pH (4.3) a single band was apparent. On SDS gels this protein demonstrated two bands, one with a molecular weight of 20,000, and the second with a molecular weight of 9,200. This purified antigen was able to block the staining of the basement membrane zone produced by bullous pemphigoid antibodies on monkey esophagus and normal human skin with the use of indirect immunofluorescence. This study also demonstrates that bullous pemphigoid antigen (A-1-Epid) and a second epidermal protein (A-2-Epid) are present in the PBS-soluble products of human esophageal mucosa, saliva, and urine. These antigens appear to be unrelated with the blood group substances or secretor status of the donors.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

C3d,g is present in normal human epidermal basement membrane

mAb as well as polyclonal anti-human C3d antibodies were found to specifically bind to the epidermal basement membrane zone of normal human adult and neonatal skin in a linear continuous pattern on direct immunofluorescence microscopy. No such binding was found in dermal microvascular basement membranes. Studies of normal adult human skin using a rat mAb specific for C3g revealed the same pattern of epidermal basement membrane staining. Control polyclonal antibodies directed against C3, C3c, C5, IgG, IgA, or IgM showed no evidence of epidermal basement membrane binding or in situ deposits of immune complexes in samples of normal human skin that were all positive for C3d and C3g. Pre-adsorption of monoclonal or polyclonal anti-human C3d with purified human C3d completely blocked these reagents' epidermal basement membrane reactivity. Anti-human C3d epidermal basement membrane binding was not diminished by pre-treatment of substrate with antibodies directed against C3, C3c, C5, laminin, fibronectin, or type IV collagen as well as bullous pemphigoid, KF-1, or epidermolysis bullosa acquisita Ag. Direct immunofluorescence microscopy studies on 1 M NaCl split human skin showed that C3d and C3g were found in the base of the cleavage plane created within the lamina lucida. By immunoelectron microscopy, C3d was found along the base of the lamina densa and in the sublamina densa region of normal human epidermal basement membrane. Although anti-human C3d epidermal basement membrane binding was not altered by treatment of 6 micron skin sections with buffers of varying pH and ionic concentration, binding was abolished by treating dermal portions of salt split skin with 0.1 M dithiothreitol in 8 M urea. Studies of a patient with congenital C3 deficiency revealed that there was no binding of anti-human C3d or anti-human C3g to this subject's epidermal basement membrane. Moreover, treatment of this patient's skin with aged human serum containing C3d,g or purified human C3 did not restore epidermal basement membrane anti-human C3d binding. These studies demonstrate that C3d,g or a closely related C3 fragment is present in the epidermal basement membrane zone of normal human skin.     

14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes

The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin alpha6, integrin beta4, or bullous pemphigoid antigen-1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14-3-3 sigma, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3sigma GST pull-down screening assay and showed that sigma (sigma) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3sigma revealed that only intact 14-3-3sigma molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 sigma is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 sigma interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3sigma isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.