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1 From the Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts; and the 2 Immunology and Uveitis Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Clone the fragments representing the extracellular and intracellular domain of ß4 molecule from normal human conjunctival mRNA into an expression vector; map the region to which sera from OCP patients bind by Western blot analysis. Determine the role of the immunodominant region in pathogenesis by demonstrating the ability of the rabbit antibody to the immunodominant region to produce separation of basement membrane zone (BMZ) from the basal epithelial layer when incubated with normal human conjunctiva in an in vitro organ culture model.
RESULTS. Majority of the OCP sera tested bound to the C-terminal end of the intracellular domain (IC3.0) of the human ß4 integrin. Further subcloning of IC3.0 demonstrated that a smaller fragment extending from 1489 aa to 1572 aa (IC3.4) was responsible for this binding. This region may have multiple antibody binding sites. Antibody to human IC3.0 and IC3.4 produced in rabbit, resulted in BMZ separation, histologically identical with that observed when normal human conjunctiva was cultured with OCP sera in an human conjunctival organ culture model.
CONCLUSIONS. These observations identify IC3.4 as the antibody binding site for sera of OCP patients and suggest a possible role for it in blister formation. Indirectly it highlights certain important aspects of the structural and functional dynamics of the biology of the hemidesmosomes and basement membranes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our recent observations suggest that human ß4 integrin may play an important role in the pathogenesis of OCP.4 5 Integrins comprise of a large family of heterodimeric receptors that mediate cell adhesion to extracellular matrices and other cells.6 In addition, they are involved in extracellular signal transduction and hemidesmosomal assembly.7 8 9 Integrins probably play an important role in adherence of basal epithelial cells to underlying basement membranes.10
There are several human autoimmune diseases in which the target autoantigens are identified as being intracellular in location.11 12 13 The exact mechanism by which the autoantibodies enter the cell and bind to these target antigens is not clearly known or understood. The purpose of the present study is to identify specific site(s) on human ß4 molecule to which sera from OCP patients bind and to determine its role in the process of blister formation. In the present study, we demonstrate that OCP sera binds to intracellular regions (IC3.0 and IC3.4) of the human ß4 molecule. Rabbit antibodies to these fragments produces BMZ separation in an in vitro human conjunctiva organ culture model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA Preparation and RT-PCR
Total RNA was extracted from normal human conjunctiva (NHC),
using Trizol followed by ethanol precipitation.14
Approximately 3 µg of total RNA was used to make cDNA using Reverse
Transcriptase Superscript II (Gibco, BRL, Gaithersburg, MD) and
gene-specific primer (5'-GGGGCAGGGTGCGGTCAAGTGTTTGGAAGAAC-3') was
designed based on the C-terminal sequence of the ß4
molecule.4
The resulting cDNA was used as the template for
all the RT-PCR reactions. RT-PCR conditions were as follows: 94°C
melting temperature; 60°C annealing temperature for 1 minute; 72°C
extension temperature for 1 minute; and this entire procedure was
repeated for 25 cycles. The reaction was performed with a hot
start. Products obtained after 25 cycles of amplification in a DNA
thermal cycler (Perkin–Elmer Corp., Norwalk, CT) were extracted using
magic PCR purification resin.
Cloning and Characterization of the Different Fragments
Representing the Extracellular and Intracellular Domain of the ß4
Molecule from Normal Human Conjunctival RNA
The resulting cDNA from the conjunctival RNA was used for PCR
amplification. The different fragments representing the extracellular
(EC) and intracellular (IC) domain of human ß4 integrin molecule were
amplified and sequenced by the di-deoxy chain termination method at the
Molecular Biology Core Facility of the Dana Farber Cancer Institute,
Boston, MA. For accuracy purposes, both the strands were sequenced. The
sequence of the fragments were compared to the sequence of the human
ß4 integrin molecule, Accession No. 53587.15
The fragments representing the extracellular domain are designated as
EC1.0 (28 aa–366 aa), and EC2.0 (367 aa–710 aa); and those
representing the intracellular domain are designated as IC1.0 (734
aa–1074 aa), IC2.0 (1075 aa–1488 aa), and IC3.0 (1489 aa–1822 aa).
The fragments used in this study are schematically represented in
Figure 1
. The PCR products were resolved on 1.2% agarose gel, purified using
Promega PCR prep resin, digested with the respective restriction enzyme
(EcoRI and XhoI), and ligated into pET28a
(Novagen Inc., Madison WI). The restriction sites EcoRI and
XhoI were created in the 5' and 3' primers, respectively, to
facilitate the subcloning procedure. The ligated products were
transformed into Escherichia coli DH5
, and the positive
clones were identified by restriction endonucleases
analysis.14
The selected recombinants were expressed in
E. coli BL21
DE3plysS. IC3.0 (1489 aa–1822 aa)
was further subcloned as IC3.1 (1489 aa–1654 aa), and IC3.2 (1655
aa–1822 aa). Based on a recent report, which determined that the very
end of the C-terminal portion of the intracellular tail of ß4 was an
important site for interaction between ß4 and plectin.10
Hence we produced a construct designated IC3.3 (1489 aa–1803 aa),
which eliminates the last 19 aa of ß4 to determine its capacity to
bind to OCP sera. For the purpose of epitope-mapping, IC3.1 was
subcloned into two fragments as IC3.4 (1489 aa–1572 aa) and IC3.5
(1573 aa– 1654 aa). Finally, another construct, IC3.6, extending from
1573 aa–1822 aa (representing a combination of IC3.2 and IC3.5) was
made. This construct was made to verify the binding of the OCP antibody
to a cDNA clone selected while screening of a keratinocyte cDNA library
in gt11 as reported in our earlier study.4
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DE3plysS. The pET-28a
(Novagen Inc.) allows expression, by means of the bacteriophage T7
10 promoter, of a His-Tag sequence fused to the N-terminus
of the different fragments.16
Cells of the resultant
transformants were grown to an OD610 of 
0.4 at
37°C in LB containing kanamycin (40 µg/ml), induced by the addition
of 0.4 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). After a 3-hour
incubation under induction conditions, the cells were harvested by
centrifugation at 3000g for 20 minutes at 4°C. The cell
pellets were resuspended and solubilized in bug-buster buffer as per
manufacturer’s instructions (Novagen, Inc.).
Identification and Characterization of Fusion Proteins by Western
Blot Analysis
Characterization of the fusion proteins was done by standard
Western blot analysis.4
The primary antibodies consisted
of the panel of patient’s sera, control sera, and antibodies to
extracellular and intracellular domain of human ß4 molecule raised in
rabbit (provided by F. Giancotti, Memorial Sloan Kettering Cancer
Center, New York, NY). The binding was detected using ECL reagents
(Amersham–Pharmacia Biotech, Inc., Piscataway, NJ).
Immunization and Production of Rabbit IgG
New Zealand rabbits were immunized SC as described
earlier17
with 100 µg of purified EC1.0, EC2.0, IC1.0,
IC2.0, IC3.0, and IC3.4 fragments of ß4 molecule. Pre- and
postimmunization sera were collected from the respective rabbits.
Indirect Immunofluorescence
Indirect immunofluorescence was performed using normal human
conjunctiva as substrate as described earlier5
to test the
presence of anti-BMZ antibodies. Test reagents included OCP sera
(n = 20), and rabbit antibodies to EC1.0, EC2.0, IC1.0,
IC2.0, IC3.0, and IC3.4. Sera from normal human individuals and
pre-immune rabbit sera served as negative control, whereas sera from
patients with PV (n = 5) served as a positive control
in this assay.
In Vitro Culture of Normal Human Conjunctiva
Normal human conjunctiva was obtained during cataract surgery.
Then, 4 to 5 mm2 size pieces of normal human
conjunctiva were incubated in a 24-well tissue culture plate in
complete RPMI-1640 medium supplemented with different test reagents.
Next, 30% v/v of sera from patients with OCP (n = 5),
PV (n = 5), normal human individuals (n = 5), pre-immune rabbit sera, and rabbit antibodies to EC1.0, EC2.0,
IC1.0, IC2.0, IC3.0, and IC3.4 were added to the wells and incubated
with 5% CO2 for 24, 36, and 48 hours. The
optimal concentrations and timings were obtained from our earlier
studies.5
After incubation, tissue samples were examined
by routine histology. The experiment was repeated three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gt11. The sequence of the putative clone was identical with the
C-terminal end of the ß4 molecule.15
Cloning of the
different fragments representing the extracellular and intracellular
domain of ß4 molecule from cDNA of normal human conjunctiva was done
primarily to determine whether there were any additional binding sites
on the ß4 integrin molecule for OCP autoantibody, besides the one
described earlier.4
Molecular Characterization of the Fragments of Human ß4 Molecule
The various fragments representing the extracellular and
intracellular domains are schematically represented in Figure 1
and
were PCR amplified and resolved in 1.2% agarose gel. All the amplified
fragments were approximately 1 kb in length. These were: EC1.0, 1.0 kb;
EC2.0, 1.06 kb; IC1.0, 1.0 kb; IC2.0, 1.2 kb; and IC3.0, 1.0 kb. These
PCR products representing the extracellular and intracellular domain of
the putative OCP gene are shown in Figure 2
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39 kDa; EC2, 40 kDa; IC1,
38 kDA; IC2, 42 kDa; IC3, 37 kDa (data not shown).
Binding of OCP Sera to the Fragments of ß4 Molecule
Binding pattern of OCP sera to various fragments is presented in
Figure 3
. All the OCP sera (n = 20) demonstrated binding to
IC3.0. However, five OCP sera samples bound to both IC2.0 and IC3.0,
and two OCP sera reacted to all three fragments IC1.0, IC2.0, and IC3.0
(data not shown). The reactivity to IC1.0 and IC2.0 was significantly
less than IC3.0. OCP sera did not demonstrate binding to the fragments
(EC1.0 and EC2.0) representing the extracellular domain of ß4
molecule. Polyclonal antibody to the extracellular domain of ß4
demonstrated binding to EC1.0 and EC2.0. Antibody to intracellular
domain of ß4 demonstrated binding only to IC3.0. This demonstrates
that the binding pattern of antibody to intracellular domain of ß4 is
identical with the binding pattern of OCP sera. The polyclonal antibody
to the intracellular domain of ß4 used in this study was raised
against the terminal 33 aa of ß4.18
19
The
anti-intracellular antibody did not bind to either EC1.0 or EC2.0.
Normal human sera and sera from patients with bullous pemphigoid (BP)
and pemphigus vulgaris (PV) did not exhibit any detectable level of
binding to any of the cloned fragments of ß4. This demonstrates the
specificity of the binding of the OCP sera to the intracellular domain
of ß4 molecule.
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Indirect Immunofluorescence
Normal human conjunctiva sections incubated with OCP sera and
rabbit antibodies to IC3.0 and IC3.4 demonstrated a smooth homogenous
linear BMZ staining (Fig. 5)
. PV sera exhibited binding to intercellular cement or epithelial cell
surface of the normal human conjunctiva. No binding was observed in the
sections of normal human conjunctiva incubated with normal human serum
and pre-immune rabbit serum. Rabbit antibodies to EC1.0, EC2.0, IC1.0
and IC2.0 bound to BMZ on normal human conjunctiva.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrated that the intracellular domain of human ß4 molecule contains peptides to which OCP sera bind. All the sera tested bound to IC3.0, 25% bound to both IC2.0 and IC3.0 and only 10% bound to the entire intracellular fragment IC1.0, IC2.0 and IC3.0, suggesting that the important epitope(s) lie within IC3.0, with the presence of minor or secondary epitope(s). However, our in vitro organ culture studies indicated that antibodies to these minor epitopes are probably not as important in BMZ separation.
OCP sera demonstrate strong binding to IC3.1. Further subcloning of this region, demonstrated that a smaller peptide IC3.4 may account for most of this binding. A 19 aa-stretch, extending from 1801 aa to 1822 aa or IC3.3, may be important for the binding of the anti-intracellular antibody, indicating that multiple epitopes may be possibly involved. In our indirect immunofluorescence assay we demonstrated that rabbit antibodies to all these fragments specially IC3.0 and IC3.4 binds to the conjunctival BMZ in a pattern similar to OCP sera, confirming our binding in the immunoblot assay.
In our in vitro conjunctival organ culture model, rabbit antibody to IC3.0 and IC3.4 were capable of producing BMZ separation, identical with that produced by OCP sera. The demonstration of the production of acantholysis of epithelial cells by PV sera is our positive control. The production of in vitro acantholysis in skin organ culture eventually lead to the demonstration of the ability of PV sera to produce clinical disease in vivo in neonatal mice.20 21
Model of the algorithm for antigenic determinants proposed by Jameson and Wolfe22 suggests that regions containing IC3.4 and IC3.5 have a high probability of being antigenic. IC3.4 contains three stretches of highly antigenic region (residues 1497–1510, 1518–1531, and 1546–1558), whereas IC3.5 contains only one stretch of highly antigenic region extending from 1605 to 1616. Furthermore, the regions extending from 1497–1510, 1546–1558 (IC3.4), and 1605–1616 (IC3.5) are hydrophilic as predicted by the hydrophilicity plot.23
The binding to IC3.4 is stronger than IC3.5 or IC3.6. Hence it may indicate that there is epitope hierarchy and possible phenomenon of epitope spreading. There is significant evidence for the presence of multiple epitopes for binding of autoantibodies in human autoimmune diseases such as SLE,24 myasthenia gravis,25 and in animal models for autoimmune diseases such as nonobese diabetic (NOD)26 27 and murine experimental autoimmune encephalomyelitis.28
A group of patients with MMP-like disease, now called anti-epiligrin MMP involving multiple mucous membranes, have anti-epiligrin antibodies that bind to multiple epitopes within laminin 5.29 30 The sera of these patients bind to the dermal side of salt split skin. In immuno-electron microscopy (IEM) this sera demonstrates the deposition of immuno-reactants on the lower lamina lucida and lamina densa.3 31 32 The sera of patients tested in the present study do not contain autoantibodies to laminin 5 (unpublished data) and bind to the epidermal side of salt split skin. Such sera bind to the hemidesmosome and the cytoplasm of the basal keratinocyte between hemidesmosomes and the inner plasma membrane of keratinocyte or their junction.3 33 34 Therefore, the subset of MMP patients in this report are distinct and different from anti-epiligrin MMP patients.
MMP is a heterogeneous disease with a wide spectrum of phenotypic presentation.2 We realize that there may be several subsets of MMP and that each subset may correlate with one or more autoantibody systems. Hence it is likely that sera of some patients with MMP may bind to the molecules other than ß4. Investigators have demonstrated the binding of MMP sera to BP180 (BPAg2).35 The importance of such binding and its role in the pathogenesis of MMP is not clear. Neonatal Balb/C mice injected with antibodies to BP180 do not demonstrate mucosal disease.17 BP sera containing antibodies to BP180 do not produce any subepithelial BMZ separation in mucous membrane in organ culture models.5 When BP patients containing antibody to BP180 are examined, the majority of them produce subepidermal blisters only on the skin.36 These observations would suggest that BP and MMP are clinically very distinct,37 and different diseases though the antigens recognized or targeted by their autoantibodies may lie in the BMZ.
The large cytoplasmic domain of ß4 displays a high level of structural and functional complexity.38 39 The observation that OCP sera bind to a 165 aa-stretch (IC3.1) in the cytoplasmic portion of the C-terminus of the ß4 molecule may provide insight into the molecular pathogenesis of BMZ separation. Adjacent on either side of the region of ß4 (IC3.4) where the OCP sera bind, are the sites where BP180, BP230, and ß4 interact with each other.10 40 On one side, BP230 and BP180 bind to each other; and on the other side, BP230 and ß4 bind to BP180.41 42 43 Therefore, it is possible that binding of OCP sera to IC3.4 results in instability of the hemidesmosomes, and its interaction with the basement membrane proteins and may ultimately influence the process of BMZ separation.
In this and earlier studies,44 we have demonstrated that the OCP sera binds preferentially to the intracellular region of ß4. There was no detectable binding to the extracellular domain. We recognize that such binding is possible and that we are unable to detect it due to the limitations of the methodology and technology used. In these studies, the fusion proteins were expressed in E. coli, and protein expressed in mammalian systems could provide different results.
These observations would indicate that BMZ autoantibodies can penetrate cell membrane of basal epithelial cells and reach their intracellular binding site (IC3.4). The ability of autoantibodies to penetrate living cells has been well established.45 Investigators have demonstrated that anti-RNP and anti-DNA antibodies,11 46 anti-La/SSB, anti-Ro/SSA, and anti-Scl7012 13 can penetrate cells, bind to target antigens, and modify cell functions. The mechanism by which the autoantibody enters the cell may be different for each autoantibody, and the resultant effects on the functions of the cell may be different for each cell and each disease process.
The significance of the observations made in this study are threefold. First, they identify human ß4 integrin as a possible autoantigen. The congenital absence of ß4 has been associated with development of blister in epidermolysis bullosa and confirmed in ß4 knockout mice.47 48 However, this study would implicate a direct role for human ß4 in an autoimmune disease with a clinical distribution limited to mucous membranes and occasionally the skin. Second, it provides some meaningful insights into the important sites of interactions, connections and interdependency of cytoplasmic structures that maintain hemidesmosomal stability and integrity. Third, it identifies a specific molecular event during the pathogenesis of OCP and concomitant BMZ separation. Several events must be present between the systemic production of the anti-BMZ antibody and its binding to BMZ antigens in mucosal tissue eventually producing subepithelial blisters. Once these multiple events are identified and characterized, it will be possible to devise strategies and create models that would facilitate development of disease and site specific therapeutic intervention.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 22, 2000; accepted October 16, 2000.
Commercial relationships policy: N.
Corresponding author: A. Razzaque Ahmed, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115. razzaque_ahmed{at}hms.harvard.edu
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References |
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6ß4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human ß4 J Immunol 165,2824-2829This article has been cited by other articles:
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  K. A. Rashid, J. N. H. Stern, and A. R. Ahmed 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 J. Immunol., February 1, 2006; 176(3): 1968 - 1977. [Abstract] [Full Text] [PDF]
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 M Zierhut, N Stubiger, K Siepmann, and C M. Deuter MMF and eye disease Lupus, March 1, 2005; 14(3_suppl): s50 - s54. [Abstract] [PDF]
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M. Zierhut, N. Stubiger, K. Siepmann, and C. Deuter MMF and eye disease Lupus, January 1, 2005; 14(1_suppl): s50 - s54. [Abstract] [PDF]
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 J. J. Rowsey, Y. Macias-Rodriguez, and C. Cukrowski A New Method for Measuring Progression in Patients With Ocular Cicatricial Pemphigoid Arch Ophthalmol, February 1, 2004; 122(2): 179 - 184. [Abstract] [Full Text] [PDF]
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