Granzyme B inhibition reduces disease severity in autoimmune blistering diseases.

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.

A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.

Mediators of mast cells in bullous pemphigoid and dermatitis herpetiformis.

Bullous pemphigoid (BP) and dermatitis herpetiformis (DH) are skin diseases associated with inflammation. However, few findings exist concerning the role of mast cells in autoimmune blistering disease. Skin biopsies were taken from 27 BP and 14 DH patients, as well as 20 healthy individuals. Immunohistochemistry was used to identify the localization and mast cell expression of TNFα and MMP9 in skin lesions and perilesional skin. The serum concentrations of TNFα, MMP9, chymase, tryptase, PAF, and IL-4 were measured by immunoassay. TNFα and MMP9 expression in the epidermis and in inflammatory influxed cells in the dermis was detected in skin biopsies from patients. Although these mediators were found to be expressed in the perilesional skin of all patients, the level was much lower than that in lesional skin. Increased serum PAF levels were observed in BP patients. Mast cells may play an essential role in activating inflammation, which ultimately contributes to the tissue damage observed in BP and DH. Our findings suggest that differences in the pattern of cytokine expression directly contribute to variations in cellular infiltration in DH and BP.

Autoimmunity-driven enzymatic remodeling of the dermal-epidermal junction in bullous pemphigoid and dermatitis herpetiformis.

Pathogenesis of blister formation in bullous pemphigoid (BP) and dermatitis herpetiformis (DH) is associated with destruction of numerous components of the dermal--epidermal junction. Proteolytic enzymes (PE) are involved in a multitude of physiological reactions and may have impact on the epidermal--dermal integrity. Involvement of various PE in inflammation and blister formation in BP and DH is intensively investigated using both morphologic and functional approaches, particularly in BP. The development into the full-blown stage in BP and DH may be caused by an impairment of the human Fc receptor regulatory system that may cause the inefficiently controlled activation of inflammatory cells and subsequent secretion of various proteases.

Hepatotoxicity and liver enzyme alteration in patients with immunobullous diseases receiving immunosuppressive therapy.

To avoid complications of high dose corticosteroid, pemphigus patients are usually co-treated with other immunosuppressive agents. Liver enzyme abnormality occurs commonly during treatment and occasionally causes discontinuation of drugs. To assess the rate of therapy-induced hepatotoxicity in patients with immunobullous diseases, we conducted a study of 250 pemphigus patients under immunosuppressive therapy prospectively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) plasma levels were recorded before the start of treatment and every week under treatment (up to 3 weeks). Hepatotoxicity was defined as the rise in the ALT plasma levels to greater than twice the upper normal limit. Approximately 81% of patients received prednisolone and azathioprine. Approximately 12% received only prednisolone. Hepatotoxicity occurred in 2.9% (n = 8) of patients after 1 week, in 7.8% (n = 20) after 2 weeks and in 11.5% (n = 29) after 3 weeks. No patient had jaundice or other clinical manifestations of hepatitis. The mean values of ALT and AST before the start of treatment were 20.7 ± 13.7 and 17.6 ± 10.8 U/L, respectively that grew to 47.5 ± 28.5 and 26.8 ± 14.5 U/L, 3 weeks after the initiation of treatment. Distribution of changes was not significantly different among groups of age, sex, immunosuppressive drugs and isoniazid consumption. Under usual treatment of pemphigus, hepatotoxicity occurs in 10% of patients during the first 3 weeks of therapy that does not seem to be associated with azathioprine or mycophenolate mofetil exclusively. High doses of prednisolone may play a role.

Dual targets for mouse mast cell protease-4 in mediating tissue damage in experimental bullous pemphigoid.

Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.

NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.

The contribution of phagocyte-derived reactive oxygen species to tissue injury in autoimmune inflammatory diseases is unclear. Here we report that granulocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase crucially contributes to tissue injury in experimental models of the antibody-mediated autoimmune disease epidermolysis bullosa acquisita. Neutrophil cytosolic factor 1-deficient mice lacking functional NADPH oxidase were resistant to skin blistering by the passive transfer of antibodies against type VII collagen. Pharmacological inhibition or deficiency of human NADPH oxidase abolished dermal-epidermal separation caused by autoantibodies and granulocytes ex vivo. In addition, recruitment of granulocytes into the skin was required for tissue injury, as demonstrated by the resistance to experimental blistering of wild-type mice depleted of neutrophils and of CD18-deficient mice. Transfer of neutrophil cytosolic factor 1-sufficient granulocytes into neutrophil cytosolic factor 1-deficient mice demonstrated that granulocytes provide the NADPH oxidase required for tissue damage. Our findings identify granulocyte-derived NADPH oxidase as a key molecular effector engaged by pathogenic autoantibodies and provide relevant targets for prevention of tissue damage in granulocyte-mediated autoimmune diseases.

Glutathione-S-transferase pi expression in toxic epidermal necrolysis: a marker of putative oxidative stress in keratinocytes.

BACKGROUND: Toxic epidermal necrolysis (TEN) is a dramatic drug-induced emergency related to extensive destruction of the epidermis. There is evidence that its pathomechanism involves impaired detoxication of xenobiotics. Glutathione-S-transferase pi (GST-pi) is a phase II detoxifying enzyme involved in drug metabolization by human keratinocytes. METHOD: Immunohistochemistry was performed in order to assess the expression of GST-pi in keratinocytes of TEN, other cutaneous adverse drug reactions and bullous pemphigoid. RESULTS: GST-pi was disclosed in the involved epidermis of 16/16 TEN patients. It was present in the cytoplasm of suprabasal keratinocytes. GST-pi was also expressed in the clinically uninvolved skin in a majority (8/12) of TEN patients. By contrast, it was rarely and poorly expressed in the other tested dermatoses. CONCLUSION: The pathomechanism of TEN is not related to an impaired quantitative expression of GST-pi. GST-pi expression is an early event in TEN. As oxidative stress is a major inducer of GST-pi, this mechanism might be involved in TEN. Its GST-pi expression mainly restricted to the suprabasal keratinocytes suggests that the pathomechanisms leading to keratinocyte death in TEN are distinct at different levels of the epidermis.

Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid.

BACKGROUND: Matrix metalloproteinase (MMP)-2, MMP-9 and MMP-13 can degrade type IV collagen which is the major component of the basement membrane zone (BMZ). In bullous pemphigoid (BP), the separation occurs within the BMZ. OBJECTIVE: To evaluate the involvement of MMPs in the pathogenesis of BP, we examined the expression of MMP-2, MMP-9 and MMP-13 in the lesional skin of BP patients. METHODS: The expression of MMP-2, MMP-9, MMP-13, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 was analyzed by immunohistochemistry in the lesional skin of BP patients in comparison with that in normal human skin. Next, the cellular sources of MMP-2, MMP-9 and MMP-13 were analyzed by double immunohistochemistry. Finally, the levels of these MMPs in the serum and blister fluid of BP patients were measured by ELISA. RESULTS: The number of cells expressing MMP-2, MMP-9 and MMP-13 were significantly increased in the lesional skin of BP patients as compared to that in normal skin. Although the number of cells expressing TIMP-1 and TIMP-2 were also increased in the lesional skin of BP patients as compared to that in normal skin, the ratio of MMPs to TIMPs in the lesional skin of BP patients was high (2.4:1). T cells comprised the major source of MMP-2, MMP-9 and MMP-13, while a proportion of mast cells and eosinophils also expressed these MMPs. Furthermore, marked expression of MMP-2 was detected in the epidermal keratinocytes. The levels of these MMPs in the blister fluid were significantly greater than those in the serum. CONCLUSION: These results suggest that MMP-2, MMP-9 and MMP-13 may be involved in the mechanism of blister formation in BP and that besides infiltrating inflammatory cells, structural cells like epidermal keratinocytes may also participate in the induction of blister formation in BP.

Synergy between a plasminogen cascade and MMP-9 in autoimmune disease.

Extracellular proteolysis by the plasminogen/plasmin (Plg/plasmin) system and MMPs is required for tissue injury in autoimmune and inflammatory diseases. We demonstrate that a Plg cascade synergizes with MMP-9/gelatinase B in vivo during dermal-epidermal separation in an experimental model of bullous pemphigoid (BP), an autoimmune disease. BP was induced in mice by antibodies to the hemidesmosomal antigen BP180. Mice deficient in MMP-9 were resistant to experimental BP, while mice deficient in Plg and both tissue Plg activator (tPA) and urokinase Plg activator (uPA) showed delayed and less intense blister formation induced by antibodies to BP180. Plg-deficient mice reconstituted locally with Plg or the active form of MMP-9 (actMMP-9), but not the proenzyme form of MMP-9 (proMMP-9), developed BP. In contrast, proMMP-9 or actMMP-9, but not Plg, reconstituted susceptibility of MMP-9-deficient mice to the skin disease. In addition, MMP-3-deficient mice injected with pathogenic IgG developed the same degree of BP and expressed levels of actMMP-9 in the skin similar to those of WT controls. Thus, the Plg/plasmin system is epistatic to MMP-9 activation and subsequent dermal-epidermal separation in BP.

Elevated expression and release of tissue-type, but not urokinase-type, plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes.

In bullous pemphigoid (BP), the binding of BP180-specific antibodies to their hemidesmosomal target antigen is not sufficient for blister formation, but must be accompanied by the release of proteases. Using plasminogen activator (PA) knock-out mice, the PA system has previously been shown to be a prerequisite for blister formation in experimental murine BP. Here, we found elevated levels of plasmin and tPA, but not of uPA, in blister fluid from BP patients (n = 7) compared to blisters from patients with toxic epidermal necrolysis (n = 4) and suction blisters in healthy controls (n = 7). Subsequently, we addressed the question whether keratinocytes release PA in response to the binding of anti-BP180 antibodies. Treatment of cultured normal human keratinocytes with BP IgG, but not with control IgG, led to both increased protein and mRNA levels of tPA, but not of uPA, as determined by ELISA and RT-PCR, respectively. The specificity of this finding was confirmed using BP180-deficient keratinocytes from a patient with generalized atrophic benign epidermolysis bullosa, where no tPA release was observed after stimulation with BP IgG. Our results show the elevated expression and release of tPA from normal human keratinocytes upon stimulation with antibodies to human BP180. Keratinocytes, by secreting tPA, may thus play an active role in blister formation of BP.

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.

Increased tryptase and myeloperoxidase levels in blister fluids of patients with bullous pemphigoid: correlations with cytokines, adhesion molecules and anti-basement membrane zone antibodies.

Tryptase and myeloperoxidase respectively represent 2 specific markers of activated mast cells or neutrophils. Therefore, establishing the levels of these enzymes may be useful to quantify the cell involvement in the tissues or fluids of different origins and in different pathologies. The aim of this study was to analyse the levels of these 2 markers in both the sera and blister fluids of patients affected with bullous pemphigoid. These levels were then correlated to the concentrations of 19 cytokines and 2 soluble adhesion molecules determined in the same samples and also with the log (anti-basement membrane zone antibody) titres, evaluated in the patients' sera. For these purposes, 15 patients with bullous pemphigoid (10 males and 5 females; median age: 84 years, range 66-87; median disease duration: 0 years, range 0-3: median number of skin lesions: 17, range 14-30; median anti-basement membrane zone antibody titre: 1:320, range 0.0-1:2560) and 15 normal subjects (11 males and 4 females, median age: 81 years, range 59-86) were analysed by means of commercially available kits. Results showed that blister fluid myeloperoxidase and tryptase levels were increased as compared with the respective sera (P<0.01) and several correlations were observed with cytokines and adhesion molecules. In fact, significant correlations of blister fluid tryptase levels were observed with IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, VEGF, RANTES and sICAM-1, while myeloperoxidase was correlated with IL-1beta, IL-13 and IL-15. The blister fluid tryptase levels were also significantly correlated with the anti-basement membrane zone antibody titres (R=0.53, P=0.05). In conclusion, these findings are in accord with an involvement of both mast cells and neutrophils in bullous pemphigoid and their recruitment may be mediated by different biological modulators. Our findings seem to indicate that the cytokine (IL-3, IFN-gamma and OSM) or adhesion molecule (sICAM-1) concentrations in blister fluid are logarithmically related to the anti-basement membrane zone antibody titers.

A critical role for neutrophil elastase in experimental bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by subepidermal blisters and autoantibodies against 2 hemidesmosome-associated proteins, BP180 and BP230. The immunopathologic features of BP can be reproduced in mice by passive transfer of anti-BP180 antibodies. Lesion formation in this animal model depends upon complement activation and neutrophil recruitment. In the present study, we investigated the role of neutrophil elastase (NE) in antibody-induced blister formation in experimental BP. Abnormally high levels of caseinolytic activity, consistent with NE, were detected in extracts of lesional skin and blister fluid of mice injected with anti-BP180 IgG. The pathogenic anti-BP180 IgG failed to induce subepidermal blistering in NE-null (NE(-/-)) mutant mice. NE(-/-) mice reconstituted with neutrophils from wild-type mice became susceptible to experimental BP. Wild-type mice given NE inhibitors (alpha1-proteinase inhibitor and Me-O-Suc-Ala-Ala-Pro-Val-CH(2)Cl), but not mice given cathepsin G/chymase inhibitors (alpha1-antichymotrypsin or Z-Gly-Leu-Phe-CH(2)Cl), were resistant to the pathogenic activity of anti-BP180 antibodies. Incubation of murine skin with NE induced BP-like epidermal-dermal detachment. Finally, NE cleaved BP180 in vitro and in vivo. These results implicate NE directly in the dermal-epidermal cleavage induced by anti-BP180 antibodies in the experimental BP model.

Evaluation of an avidin-biotin-peroxidase method with a monoclonal antibody to type IV collagen in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita.

There are reports in which an immunohistochemical technique with a monoclonal antibody to type IV collagen has been employed for differentiating between bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). The aim of this study was to determine whether this method could be used routinely. Biopsies (paraffin-embedded lesional skin containing a blister) from currently diagnosed patients with clinical features suggesting BP or EBA were examined by an avidin-biotin-peroxidase (ABC) technique. Sera were tested by indirect immunofluorescence on salt-split skin (IF) and immunoblotting (IB). In all cases which exhibited clear type IV collagen staining, the results of the ABC technique agreed with results of both IF and IB. In one confirmed EBA case, it was impossible to unequivocally localize type IV collagen, because it stained very faintly. Taking into consideration the results of our study, data indicating that the level of blistering might not coincide with the localization of immunoreactants in EBA cases and the possibility of an enzymatic destruction of lamina densa, we conclude that the ABC method is unsuitable for differentiation between BP and EBA.

Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis.

Keratinocytes synthesize urokinase-type plasminogen activator (uPA) and a specific cell surface receptor for uPA (uPA-R, CD 87). Plasminogen is present in plasma and interstitial fluids from where it is bound to cell surfaces via plasmin(ogen) binding sites. uPA binds to the uPA-R in an autocrine manner and activates cell-bound plasminogen: a mechanism, which provides plasmin for pericellular proteolysis. Cell-bound uPA is regulated by plasminogen activator inhibitor type-1 (PAI-1) or type-2 (PAI-2). Bullous pemphigoid is an autoimmune inflammatory skin disease characterized by subepidermal blisters. Although circumstantial evidence suggested plasminogen activation in lesional epidermis of bullous pemphigoid, immunohistological data on the type of plasminogen activators, on the uPA-receptor or the type of plasminogen activator inhibitors in the lesions of bullous pemphigoid are lacking so far. To obtain this information we have performed the present immunohistological study. The presence of uPA and its receptor as well as PAI-2 was disclosed in epidermal keratinocytes in the roof of the subepidermal blisters. Moreover, keratinocytes at the bottom of the blister, which most likely represent keratinocytes during reepithelialization were stained. Co-localization was found for uPA and its receptor, uPA and plasmin(ogen) as well as for uPA and PAI-2. In non-lesional epidermis of bullous pemphigoid only PAI-2 was found. We propose that the expression of uPA and uPA-R, as well as the upregulation of PAI-2 in keratinocytes of lesional epidermis is part of the repair and reepithelialization process following lesion formation, i.e. epidermo-dermal dyshesion, in bullous pemphigoid.

92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.

Eosinophils are prominent in bullous pemphigoid (BP), and proteases secreted from these and other inflammatory cells may induce disruption of the basement membrane. We used in situ hybridization and immunohistochemistry to localize the sites of 92-kD gelatinase expression in BP lesions. In all samples (20/20), a strong signal for gelatinase mRNA was detected only in eosinophils and was most pronounced where these cells accumulated at the floor of forming blisters. No other cells were positive for enzyme mRNA. Both eosinophils and neutrophils, however, contained immunoreactive 92-kD gelatinase indicating that active expression occurred only in eosinophils. Degranulated eosinophils were also seen near blisters, and as demonstrated by gelatin zymography, immunoblotting, and ELISA, 92-kD gelatinase protein was prominent in BP blister fluid. No other gelatinolytic activity was specifically detected in BP fluid, and only small amounts of 92-kD gelatinase were present in suction blister fluids. As demonstrated in vitro, 92-kD gelatinase cleaved the extracellular, collagenous domain of recombinant 180-kD BP autoantigen (BP180, BPAG2, HD4, type XVII collagen), a transmembrane molecule of the epidermal hemidesmosome. Our results suggest that production and release 92-kD gelatinase by eosinophils contributes significantly to tissue damage in BP.

The autoimmune blistering skin disease bullous pemphigoid. The presence of plasmin/alpha 2-antiplasmin complexes in skin blister fluid indicates plasmin generation in lesional skin.

Plasminogen activators produced locally in the skin have been implicated in blistering skin diseases. To explore whether plasminogen activators convert their substrate plasminogen into plasmin locally in the lesional skin we have analyzed the autoimmune blistering skin disease bullous pemphigoid. Enzyme activity was detected in bullous pemphigoid skin blister fluid by using a low molecular weight chromogenic substrate for plasmin. Enzyme activity was detected neither in suction blister fluid raised on normal skin nor in normal plasma. Immunoprecipitation or fractionation by molecular sieve chromatography of bullous pemphigoid skin blister fluid followed by testing in immunoassays disclosed putative plasmin/alpha 2-macroglobulin complexes and plasmin/alpha 2-antiplasmin complexes. Enzyme activity detected in bullous pemphigoid skin blister fluid by the low molecular weight chromogenic peptide assay was ascribed to the putative plasmin pha 2-macroglobulin complexes. Because formation of plasmin-inhibitor complexes requires the active plasmin, our findings indicate previous activation of plasminogen to plasmin in skin lesions. There was no evidence for free plasmin (i.e., plasmin not complexed to inhibitors) in bullous pemphigoid blister fluid, suction blister fluid, or plasma.