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6ß4 Integrin of Patients Affected by Ocular Cicatricial Pemphigoid Recognize Predominantly Epitopes Within the Large Cytoplasmic Domain of Human ß41
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Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, MA 02115;
Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and
The Massachusetts Eye and Ear Infirmary and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some investigators have reported that like bullous pemphigoid (BP),
mucous membrane pemphigoid or CP patients’ sera recognize 180-kDa
proteins termed as BPAg2 when human epidermal lysate and BPAg2 fusion
proteins were used as substrates (2, 3). This protein
along with BPAg1 (BP230) and
6ß4 integrin are found
in hemidesmosomes, which are structures that link the underlying
basement membranes of stratified as well as other epithelia to the
intermediate filament system. They are composed of an outer plaque,
containing proteins such as BP180 and
6ß4, and an inner
plaque containing BP230 and HD1/plectin, which then associate with the
cytoskeleton. Defects in these proteins can lead to epithelial blisters
in both humans and mouse models (4)
We have observed that after preabsorption of epidermal or conjunctival or tumor cell lysates with BP sera, OCP sera recognize a 205-kDa protein (5, 6), identified as the ß4 integrin subunit (7). These findings raised the possibility that a putative target Ag for OCP may be the ß4 integrin subunit (7). In fact, we have recently reported that OCP sera, immunoaffinity-purified OCP Abs eluted from the 205-kDa band on nitrocellulose, and anti-ß4 Abs cause blistering in an in vitro human conjunctival model (8). This result suggests that Abs from OCP sera against ß4 could be pathogenic in vivo.
A subset of CP patients has been reported to be characterized by the
presence of autoantibodies against epiligrin, which is now identified
as the 3 subunit of laminin-5 (9, 10). Interestingly, many of the OCP sera used in the present
study and in our earlier reported studies (5, 6) did not
bind to epiligrin (K. Yancey, unpublished observation).
Since CP is a heterogeneous disease, it is possible that clinical subsets may correlate with a variety of anti-BMZ autoantibodies with different specificities that recognize different target molecules within the complex of cell surface proteins and extracellular matrix proteins in the BMZ.
Given the possible pathogenic role of anti-ß4 Abs in OCP sera, the purpose of this study was to determine the region of ß4 recognized by these Abs. Surprisingly, we have found that the anti-ß4 Abs of OCP bind to the intracellular region of human ß4 integrin and we were unable to detect, using immunoblotting, immunoprecipitation, and FACS analysis, any binding to the extracellular domain of ß4 integrin subunit.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sera were obtained from 15 patients with active OCP in the acute phase of the disease before the institution of therapy. The diagnosis of OCP was confirmed in each patient by clinical presentation, routine histology, and immunopathological analysis of biopsied conjunctiva. When tested on salt split skin, the anti-BMZ autoantibody bound to the epidermal side of the split.
Total IgG of OCP autoantibodies was prepared from 10 active OCP patients by EZ-SEP kit obtained from Middlesex Sciences (Foxborough, MA). Immunoaffinity-purified OCP autoantibodies were eluted from the nitrocellulose blots, as described previously (11). In another Western blot, it was confirmed that the immunoaffinity-purified OCP autoantibodies bound to a 205-kDa protein in normal human conjunctiva and epidermis in exactly the same manner as OCP sera. mAb to human ß4 (3E1) and polyclonal Ab to extracellular and cytoplasmic domains of human ß4 integrin were described elsewhere (12, 13). The 9E10 anti-myc mAb was prepared by the hybridoma core facility at Memorial Sloan-Kettering Cancer Center (New York, NY). Control sera were obtained from 10 healthy individuals and from 1 patient each with active disease with pemphigus vulgaris (PV), BP, oral pemphigoid (OP), and dermatitis herpetiformis (DH). All the control and patient sera were stored at -80°C until used. The study has been reviewed by our Institutional Review Board. Sera were obtained after appropriate informed consent procedures.
Preparation of lysate from human skin and conjunctiva
The epidermis of the human skin was separated from underlying dermis, and clear lysates of Ags were prepared as reported earlier (14). Briefly, the human epidermis and conjunctiva were homogenized on ice with 1.5% SDS, 62.5 mM Tris-HCl buffer, pH 6.8, supplemented with 5% mercaptoethanol, 2 mM PMSF, and 10 µg/ml of pepstatin A, antipain, leupeptin, and chymostatin (Sigma, St. Louis, MO). The lysates were then boiled for 5 min, vortexed, and centrifuged at 15,000 x g for 30 min. The clear supernatant of human epidermal lysate and human conjunctival lysate was centrifuged again at high speed in a microfuge centrifuge for 30 min, and supernatant was concentrated. Protein content was measured at 280 nm, and aliquots were stored at -80°C until used.
Cells and transfection
The 804G rat bladder carcinoma cells and 293T human embryonic
kidney were grown in DMEM containing 10% bovine calf serum. The 804G
cells stably expressing recombinant human full-length
ß4 (clone A), extracellular domain of
ß4 (clone F), and intracellular domain of
ß4 integrin (clone L) were described elsewhere
(13, 15). The 293T cells, which do not express endogenous
ß4, were cotransfected by calcium phosphate
precipitation method with cDNAs of human
6 and versions of
ß4 (13, 15, 16) cloned into the
EcoRI site of the PRK5 expression vectors. Typically,
subconfluent cells were extracted for 30 min on ice in Triton X-100
lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 4 mM EDTA,
1% Triton X-100, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and
10 µg/ml each pepstatin A, aprotinin, and leupeptin). Lysates were
clarified by centrifugation for 10 min at 14,000 rpm in a refrigerated
centrifuge.
Western blotting
SDS-PAGE and Western blot were performed as previously described (17, 18). A sensitive Western blotting assay was used in this study, as described previously (7). In brief, blotted nitrocellulose membrane was blocked with 3% skimmed milk. Blotted proteins (nitrocellulose strips) after extensive washing with TBS-containing 0.05% Tween 20 were incubated with diluted test sera or Abs. After 4x wash, nitrocellulose strips were incubated with HRP-conjugated secondary Abs (anti-human, anti-rabbit, and anti-mouse), and then the final step was detected by using the ECL Western blotting kit (Amersham Life Sciences, Arlington Heights, IL), according to the manufacturer’s protocol.
Immunoprecipitation of OCP-Ag/ß4 integrin
Immunoprecipitation was performed as described earlier (16). Briefly, 50 µl of packed protein G-agarose washed three times with lysis buffer, resuspended in 500 µl of lysis buffer, then 10–50 µl of OCP sera or Abs to ß4 integrin was added to it and incubated for 2 h at 4°C. The protein G was washed three times with lysis buffer. The cell lysates (500 µg/500 µl) suspended in lysis buffer were added to the OCP sera or ß4 Ab-coated protein G and incubated overnight at 4°C. The immunoprecipitates were washed five times in cold lysis buffer and then resuspended in 50 µl of sample buffer boiled for 5 min and analyzed by immunoblotting.
In these experiments, we used 804G (rat bladder) and 293T fibroblast cell lysates (untransfected and transfected with full-length, extracellular, and intracellular domains of ß4 integrin) and immunoprecipitated with OCP autoantibodies.
FACS
The 293T cells expressing full-length ß4 were detached briefly with 0.02% trypsin and incubated for 20 min in ice with either 10 µg/ml of 3E1 mAb, or diluted OCP Abs, or normal human IgG, followed by FITC-conjugated goat anti-mouse or anti-human secondary Abs. After fixation in 3.7% formaldehyde, cells were analyzed by flow cytometry using a Becton Dickinson FACS machine and Cell Quest Ware.
Cytoplasmic localization of OCP autoantibody in cultured cells by direct immunofluorescence
This study was done according to the methods described by other investigators (19, 20). Briefly, ß4-expressing cell lines (MDA-435) were grown on chamber slides (Nunc, Naperville, IL) and allowed to adhere and proliferate. After 48 h, the cells were nearly confluent and washed with RPMI 1640 medium, then incubated with OCP sera, Abs to cytoplasmic, and extracellular domains of ß4 integrin and normal human serum for 24 h. After incubation, the slides were washed three times with PBS, then fixed in methanol acetone at -20OC for 15 min. After fixation, the cells were washed in PBS and blocked with 3% BSA in PBS for 30 min. Then the cells were washed and incubated with fluorescein-labeled goat anti-human or anti-rabbit IgG (Sigma) for 45 min, then washed, mounted, and observed by fluorescent microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 804G cells are derived from a rat bladder carcinoma and are widely used for studies on hemidesmosomes. These cells were transfected with cDNA constructs encoding wild-type ß4, a ß4 subunit lacking the ectodomain (headless), or one lacking the cytoplasmic domain (tailless). Stable clones expressing comparable surface levels of each recombinant protein were analyzed by immunoblot and immunoprecipitation experiments. Untransfected 804G cells were used as controls.
Immunoblot experiments
Extracts from various cell lines were separated by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with either OCP sera
or control Abs against either the cytoplasmic or extracellular domains
of ß4 (Fig. 1
).
OCP sera reacted with wild-type (205 kDa) and headless
ß4 (140 kDa), but did not react with tailless
ß4 (100 kDa). Immunoblotting with rabbit
polyclonal Abs against the cytoplasmic and extracellular domains of
ß4 confirmed the expression of the transfected
constructs. Wild-type ß4 (205-kDa band) is
observed in all lanes because it is endogenously expressed by 804G
cells.
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Lysates from the various 804G cell lines were immunoprecipitated
with OCP sera. These immunoprecipitates were then subjected to
SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted
with polyclonal Ab to extracellular and cytoplasmic domains of
ß4 integrin (Fig. 2). OCP sera was able to
immunoprecipitate the wild-type and headless versions of
ß4, but did not immunoprecipitate with the
tailless ß4. Wild-type
ß4 is observed in all lanes because 804G cells
express endogenous ß4. Similar results were
seen using IgG fractions of OCP sera and immunoaffinity-purified OCP
Abs (data not shown). Sera from patients with PV, BP, OP, DH, or normal
human serum did not immunoprecipitate with ß4
(data not shown). These experiments demonstrate that autoantibodies
from OCP sera react predominantly with the cytoplasmic, and not
extracellular domain of ß4 integrin.
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Reactivity of OCP autoantibodies with wild-type and mutant human ß4 integrin subunits expressed in 293T cells
Immunoblot experiments.
Human embryonic kidney 293T cells were transiently transfected with
cDNA constructs encoding the wild-type ß4, the
ß4 subunit lacking the ectodomain (headless),
or the ß4 subunit lacking the cytoplasmic
domain (tailless). Extracts from these cells were separated by
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with IgG
fractions from OCP sera or immunoaffinity-purified OCP autoantibodies,
as well as normal human IgG as a negative control or rabbit polyclonal
Abs against the ß4 cytoplasmic and
extracellular domains as positive controls (Fig. 3). Immunoaffinity-purified OCP
autoantibodies and IgG fractions of OCP sera recognized wild-type
ß4 and headless ß4, but
not tailless ß4. Similar results were observed
using whole sera from OCP patients (data not shown). Normal human IgG
did not react with ß4. Expression of wild-type
ß4 and ß4 mutants was
confirmed by immunoblotting with rabbit polyclonal Abs to the
cytoplasmic or extracellular domains of ß4.
Lysates from untransfected 293T cells did not show any reactivity with
OCP sera or control anti-ß4 Abs (data not
shown).
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). The results demonstrate that OCP sera
can immunoprecipitate the wild-type and headless
ß4, but not tailless
ß4. When the same lysates were
immunoprecipitated with sera from PV, BP, OP, DH, or normal human
serum, no reactivity with ß4 was observed (data
not shown).
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FACS analysis
Although the OCP autoantibodies do not bind to the
ß4 extracellular domain by immunoblot or
immunoprecipitation analysis, it is possible that a small, but
potentially pathogenic pool of OCP autoantibodies can also recognize
the ß4 extracellular domain in its native
conformation at the cell surface. Therefore, we tested the ability of
the OCP autoantibodies to bind the extracellular domain of
ß4 in intact cells by flow cytometry with
nonpermeabilized cells. Under these conditions, we could not detect any
binding of OCP autoantibodies to the cell surface, although the control
Ab 3E1 against ß4 extracellular domain bound
strongly (Fig. 5).
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When MDA-435 cells, which endogenously produce full-length
ß4, were incubated with OCP sera, Abs to
cytoplasmic and extracellular domains of ß4
integrin, and normal human serum for 24 h, the following
observations were made. OCP sera bind only to the cytoplasm of the
cultured cells (Fig. 6C). Ab
to cytoplasmic domain of ß4 integrin binds in a
similar pattern to only the cytoplasm of the cultured cells (Fig. 6B). No extracellular binding was seen in cells cultured
with OCP sera or Ab to cytoplasmic domain of ß4
integrin. Ab to extracellular domain of ß4
integrin demonstrated binding to the cell surface only (Fig. 6A). No cytoplasmic binding was seen. No binding to either
the cell surface or cytoplasm was seen when the cells were cultured
with normal human serum (Fig. 6D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of the OCP sera and Ab to cytoplasmic domain of ß4 to bind the cytoplasmic tail of ß4 was studied in vitro using cells in culture. We showed that OCP sera and Ab to cytoplasmic domain of ß4 demonstrate intracellular binding only.
No extracellular binding was observed. The control Ab to the extracellular domain of ß4 bound only to the cell membrane and showed no binding to the cytoplasm.
We preferred to use MDA-435 cell lines instead of the 293T-transfected cells because it produced full-length ß4, and any cross-reactivity between these Abs to the extracellular and cytoplasmic domains or OCP sera would have been detected. Although we have used all means available to detect the presence of Abs against extracellular domain of ß4 integrin, it is important to note that they may exist as a small and undetectable, yet potentially pathogenic pool of such autoantibody. Advances in technology or evaluation of patients in preclinical states may detect them.
The intracellular portion of ß4 integrin is large and consists of 1000 aa, and contains four type III fibronectin-like modules (4, 21). The cytoplasmic portion of ß4 has been shown to interact with two other hemidesmosomal proteins, BP180 and HD1/plectin (4), and is required for hemidesmosome formation and stable adhesion of stratified epithelia, such as the skin and pyloris (22).
The basic pathology in OCP is a subepithelial vesicle or blister formation. In recent studies, we have demonstrated that when normal human conjunctiva is placed in organ culture with OCP sera, immunoaffinity-purified OCP autoantibodeis, or Abs to human ß4 integrin, but not normal human sera, a separation between the conjunctival epithelium and underlying submucosa is observed. This separation is remarkably similar to that observed in human conjunctival pathology in vivo in OCP patients (8). These observations suggest that the autoantibodies to ß4 in OCP can contribute to the initiation or progression of the disease.
The experiments in this study strongly suggest that autoantibodies to ß4 contribute to the pathogenesis of OCP. The observation in this study presents the dilemma of the role of the extracellular domain of ß4 in the pathogenesis of OCP and access of OCP Ab to its intracellular target Ag. Three hypothetical but plausible explanations deserve consideration. First, the disease could be initiated by Abs directed to the ectodomain of ß4. However, we did not detect such Abs in the sera of the patients we studied with active disease. Hence, it is likely that such Abs could be transient and are present only in the preclinical stage of the disease, and therefore not detected.
Second, the inciting Ag initially involved may be irrelevant, but epitope spreading may involve epitopes that have homology to ß4. Alternatively, autoantibodies could enter the cell to initiate injury that is subsequently propagated by Abs to exposed Ag on the cell surface. Nonetheless, Abs to the cytoplasmic epitopes of ß4, BP180, and BP230 are generated and cause disruption of hemidesmosomal assembly-resultant blister formation due to BMZ separation and clinical disease.
Our direct immunofluorescence studies using cultured cells demonstrate that OCP autoantibodies and Ab to cytoplasmic domain of ß4 integrin bind intracellularly to cells. A number of previous observations demonstrate that autoantibodies can penetrate into living cells, bind to their intracellular targets, and influence cellular function (23). For example, autoantibodies to ubiquitous intracellular Ags are commonly found in the sera of patients with systemic lupus erythematosus, poly and dermatomyositis, progressive systemic sclerosis, and Sjögren’s syndrome (24). Investigators have demonstrated that specific ant-DNA Abs can penetrate cells in culture (19, 20).
Furthermore, when these anti-DNA antibodies are injected into normal mice, they produce glomerular damage and proteinuria, indicating the functional capability to the autoantibodies (19). Anti-nuclear and anti-cytoplasmic Abs have been detected in vivo in human skin biopsies from the patients with connective tissue diseases (25, 26).
There are several mechanisms by which autoantibodies could penetrate cells and gain access to the intracytoplasmic target Ags. The mechanisms are probably different in different diseases and may be different in different subsets of the same disease. Based on the work of several investigators, one hypothesis has emerged and has gained some acceptance. Intracellular molecules that contain sites targeted by autoantibodies may be presented on cell membranes by surrogate molecules (27). The surrogate membrane molecule mimics the structure of the intracellular Ag (28). The complex containing the autoantibody and surrogate Ag is internalized within cell. The autoantibody is then released and binds to the pathogenic intracellular Ag and initiates the events that may eventually lead to autoimmunity (29, 30).
The observations made in this study may help in providing an explanation for the two divergent observations made by different investigators studying immunoelectron microscopy (IEM) of CP. In the first group of studies, authors report deposition of immunoreactants on the lower lamina lucida and lamina densa (31, 32, 33). The sera used by two investigators bound to the dermal side of salt split skin on indirect immunofluorescence assay and lamina densa on IEM contained Abs to laminin-5 (34). In the second group, investigators demonstrate that immunodeposits occur on hemidesmosomes and basal keratinocyte cytoplasm or the junction between hemidesmosomes and the inner plasma membrane of keratinocytes (33, 34, 35). These investigators observed that sera that produced such immune deposition did not contain Abs to laminin-5 or BP (33). In this study, using transfected cell lines, we demonstrated that patients with CP whose autoantibody binds to the epidermal side of salt split skin have Abs that bind to the cytoplasmic domain of ß4 integrin. Hence, we propose that the sera studied by the second group contained Abs to cytoplasmic domain of ß4 integrin, which accounts for their IEM pattern.
A definitive role for Abs to human ß4 integrin in the pathogenesis of OCP can come from in vivo animal model studies. Further studies on the role of ß4 in the generation of autoimmunity are important since ß4 is the first integrin to be implicated in the pathogenesis of an autoimmune epithelial blistering disease. In addition, OCP may provide a model to study the possible role of integrins in initiation of progression or disease process.
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Footnotes |
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2 K.C.B. and M.J.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. A. R. Ahmed, Department of Oral Medicine, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115.
4 Abbreviations used in this paper: OCP, ocular cicatricial pemphigoid; BMZ, basement membrane zone; BP, bullous pemphigoid; CP, cicatricial pemphigoid; DH, dermatitis herpetiformis; IEM, immunoelectron microscopy; OP, oral pemphigoid; PV, pemphigus vulgaris.
Received for publication January 6, 2000. Accepted for publication June 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6 ß4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J. 16:2365.[Medline]
6ß4 integrin with the cytoskeleton of hemidesmosomes. Mol. Biol. Cell 4:871.[Abstract]
6ß4-mediated cell adhesion to laminins. J. Cell Biol. 129:473.6ß4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell basement membrane adhesion. J. Cell Biol. 113:907.This article has been cited by other articles:
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  K.C. Bhol, L. Goss, S. Kumari, J.E. Colon, and A.R. Ahmed Autoantibodies to Human {alpha}6 Integrin in Patients with Oral Pemphigoid Journal of Dental Research, August 1, 2001; 80(8): 1711 - 1715. [Abstract] [PDF]
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 S. Kumari, K. C. Bhol, R. K. Simmons, M. S. Razzaque, E. Letko, C. S. Foster, and A. R. Ahmed Identification of Ocular Cicatricial Pemphigoid Antibody Binding Site(s) in Human {beta}4 Integrin Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 379 - 385. [Abstract] [Full Text]
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