The Second Study of Clinical and Immunological Findings in Anti-laminin 332-Type Mucous Membrane Pemphigoid Examined at Kurume University—Diagnosis Criteria Suggested by Summary of 133 Cases

BackgroundRecently, we published an article retrospectively summarizing the results in 55 anti-laminin 332 (LM332)-type mucous membrane pemphigoid (MMP) cases examined at Kurume University, which were diagnosed by strict inclusion criteria, including positive reactivity in direct immunofluorescence and absence of antibodies to non-LM332 autoantigens. However, indirect immunofluorescence using 1M-NaCl-split normal human skin (ssIIF) is also valuable for diagnosis of anti-LM332-type MMP.MethodsIn this second study, we selected 133 anti-LM332-type MMP cases, which were diagnosed by our different inclusion criteria: (i) immunoglobulin G (IgG) deposition to basement membrane zone (BMZ) by direct immunofluorescence or IgG reactivity with dermal side of split skin by ssIIF, (ii) positivity for at least one of the three subunits of LM332 by immunoblotting of purified human LM332, and (iii) the presence of mucosal lesions. Clinical, histopathological, and immunological findings were summarized and analyzed statistically. Although these cases included the 55 previous cases, the more detailed study for larger scale of patients was conducted for further characterization.ResultsClinically, among the 133 patients, 89% and 43% patients had oral and ocular mucosal lesions, respectively, 71% had cutaneous lesions, and 17% had associated malignancies. Histopathologically, 93% patients showed subepidermal blisters. The sensitivities of ssIIF and direct immunofluorescence are similar but are significantly higher than indirect immunofluorescence using non-split human skin (both p < 0.001). In immunoblotting of purified LM332, patient IgG antibodies most frequently reacted with LMγ2 subunit (58%), followed by LMα3 (49%) and LMβ3 (36%). Thirty-four percent patients recognized additional non-LM332 autoantigens. Statistical analysis revealed that autoantibodies against non-LM332 autoantigens might stimulate the production of anti-LMγ2 antibodies.ConclusionsThis retrospective study further characterized in more detail the clinical and immunological features of 133 cases of anti-LM332-type MMP, in which the new diagnostic criteria without positive direct immunofluorescence reactivity were useful for the diagnosis. Higher frequency with anti-LMγ2 antibodies suggested more significant pathogenic role of this subunit. Additional autoantibodies to non-LM332 autoantigens detected in one-third of the patients may contribute to complexity in anti-LM332-type MMP, including the induction of anti-LMγ2 antibodies.

Subunit-Specific Reactivity of Autoantibodies Against Laminin-332 Reveals Direct Inflammatory Mechanisms on Keratinocytes

Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology.

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid

Anti-laminin 332 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by predominant mucosal lesions and autoantibodies against laminin 332. The exact diagnosis of anti-laminin 332 MMP is important since nearly 30% of patients develop solid cancers. This study compared two independently developed diagnostic indirect immunofluorescence (IF) tests based on recombinant laminin 332 expressed in HEK239 cells (biochip mosaic assay) and the migration trails of cultured keratinocytes rich in laminin 332 (footprint assay). The sera of 54 anti-laminin 332 MMP, 35 non-anti-laminin 332 MMP, and 30 pemphigus vulgaris patients as well as 20 healthy blood donors were analyzed blindly and independently. Fifty-two of 54 and 54/54 anti-laminin 332 MMP sera were positive in the biochip mosaic and the footprint assay, respectively. In the 35 non-anti-laminin 332 MMP sera, 3 were positive in both tests and 4 others showed weak reactivity in the footprint assay. In conclusion, both assays are easy to perform, highly sensitive, and specific, which will further facilitate the diagnosis of anti-laminin 332 MMP.

Gene Expression in Amnion-Derived Cells Cultured on Recombinant Laminin 332—A Preliminary Study

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

Anti-P 200 pemphigoid – The most common floor binding subepidermal autoimmune bullous disease in a tertiary care center in south India

Background: The pemphigoid group of diseases may present clinically and immunologically in a very similar fashion. Indirect immunofluorescence microscopy with readily available salt-split human skin in a BIOCHIP™ helps to classify these conditions as those with either with roof binding or floor binding of immunoreactants. Epidermolysis bullosa acquisita, anti-laminin 332 pemphigoid and anti-p200 pemphigoid show floor binding, while in the most frequent type of pemphigoid disease, bullous pemphigoid, epidermal side staining pattern is seen on salt-split skin Aims: The aim of the study was to detect the target antigens in sub-epidermal bullous diseases. Methods: Forty patients with bullous pemphigoid diagnosed by lesional histopathology and direct immunofluorescence microscopy were re-evaluated by a BIOCHIP™ mosaic containing both tissue substrates and recombinant target antigens. Sera with floor pattern staining on salt-split skin were further evaluated by immunoblotting with dermal extract. Results: Five patients with floor staining had anti-p200 pemphigoid. Limitations: We could not perform serration pattern analysis of direct immunofluorescence in our patients. Conclusion: Histopathology and direct immunofluorescence microscopy cannot differentiate between various entities of pemphigoid diseases. A multivariant approach using a BIOCHIP™ mosaic including salt-split skin followed by immunoblotting with dermal extract helps to identify the target antigen.

A Novel Phenotype of Junctional Epidermolysis Bullosa with Transient Skin Fragility and Predominant Ocular Involvement Responsive to Human Amniotic Membrane Eyedrops

Junctional epidermolysis bullosa (JEB) is a clinically and genetically heterogeneous skin fragility disorder frequently caused by mutations in genes encoding the epithelial laminin isoform, laminin-332. JEB patients also present mucosal involvement, including painful corneal lesions. Recurrent corneal abrasions may lead to corneal opacities and visual impairment. Current treatments are merely supportive. We report a novel JEB phenotype distinguished by the complete resolution of skin fragility in infancy and persistent ocular involvement with unremitting and painful corneal abrasions. Biallelic LAMB3 mutations c.3052-5C>G and c.3492_3493delCG were identified as the molecular basis for this phenotype, with one mutation being a hypomorphic splice variant that allows residual wild-type laminin-332 production. The reduced laminin-332 level was associated with impaired keratinocyte adhesion. Then, we also investigated the therapeutic power of a human amniotic membrane (AM) eyedrop preparation for corneal lesions. AM were isolated from placenta donors, according to a procedure preserving the AM biological characteristics as a tissue, and confirmed to contain laminin-332. We found that AM eyedrop preparation could restore keratinocyte adhesion in an in vitro assay. Of note, AM eyedrop administration to the patient resulted in long-lasting remission of her ocular manifestations. Our findings suggest that AM eyedrops could represent an effective, non-invasive, simple-to-handle treatment for corneal lesions in patients with JEB and possibly other EB forms.