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Ocena zjawisk immunologicznych i enzymatycznych w patogenezie wybranych podnaskórkowych chorób pęcherzowych

100%

Żebrowska A.

Folia Medica Lodziensia

2011

vol. 38

issue suplement 2

5-260

The pathogenesis of subepidermal autoimmune blistering diseases, such as pemphigoid (BP, ang. bullous pemphigoid) and dermatitis herpetiformis (DH, ang. dermatitis herpetiformis, morbus Duhring) is connected with the destruction of basement membrane components and blister formation. Ultrastructural studies confirmed the basement membrane zone as a structure responsible for the integrity of dermo-epidermal junction. The binding of autoantibodies in BMZ zone leads to the activation of immunological and enzymatic mechanisms leading to the destruction of hemidesmosomes and anchoring fibres. Despite a similar initial stage: the connection of autoantibodies with autoantigens ,which are the proteins of the basement membrane structures, the further development of the disease mechanisms is different. The Differences of antigens and the activation of other cytokines, chemokines and enzymes imply the characteristic histological immunological picture, and require a different therapy. In BP and DH the impact of the disorders of certain cytokines expression , especially proinflammatory and complement components, on the development of inflammatory infiltrates and the formation of subepidermal blisters was shown. These phenomena are the cause of the activation of adhesion molecules and subsequent inflammatory mediators such as chemokines, neuropeptides and proteolitic enzymes. Recent experimental studies have also shown that these processes can activate procoagulant factors causing the development of blood thrombus, which is the most common cause of complications in patients with BP. The goal of the study was: 1. The assessment of the role of cytokine network components, such as adhesion molecules, chemokines, neuropeptides, and markers of activation of eosinophils and mast cells in the formation of skin lesions in pemphigoid and dermatitis herpetiformis. 2. The assessment of the role of selected proteolytic enzymes: metalloproteinases, adamalysins and their tissue inhibitors in the development of subepidermal blisters. 3. The assessment of procoagulation factors activity and basement membrane destruction caused by them in pemphigoid and dermatitis herpetiformis. 4. The indication of coagulation parameters useful in the prevention of complications in therapy of BP patients. The study included 61 persons: 27 untreated patients with BP (range: 58 to 84 years, average - 68,5) and 14 with DH (range: 18 to 70 years, average – 49,8) in an active stage of the disease. A Control group consisted of 20 healthy individuals (range:50 to 80 years, average – 71,6 for BP patients and ranged:19 to 49, average – 42 for DH patients). All the study subjects gave their consent before entering the study and the study protocol was approved by The Local Ethical Committee of Medical University of Lodz. Paraffin-embedded sections were used for routine staining and for immunohistochemistry to assess the expression of adhesion molecules, chemokines and their receptors, neuropeptides, metalloproteinases and their inhibitors and adamalysines. The following primary monoclonal antibodies were used: 1. adhesive molecules: anty- CD29 (β1 family), CD61 (GPIII) (β3 family), CD104 (β4 family) CD 62E (E-selectin), CD62L (L-selectin) from Novocastra 2. chemokines and their receptors: anty- eotaksyna, TARC, MCP-1, CCR- 1, CXCR-1, CXCR-2 from R&D. 3. neuropeptides: anty- Neurokinin B antibody, Endothelin B Receptor, CGRP, Corticotropin Releasing Factor and Substance P, from Abcam. 4. metalloproteinases: anty- MMP1, MMP2, MMP9, MMP1, TIMP1, TIMP2, TIMP 3 from Novocastra. 5. adamalisyns: anty- ADAM8, ADAM10, ADAM15, ADAM17 from R&D. 6. anty- TNF α from R&D. 7. tissue factors: anty- TF from American Diagnostica Inc. The Significantly increased expression of adamalysines: ADAM8, ADAM10, ADAM15, ADAM17 in the tissues (immunohistochemistry technique assessed) was confirmed by in situ hybridization and immunoblotting. In order to determine the concentrations of the examined protein in the serum the enzyme-linked immunoassay method was used using ELISA kits: TARC, MCP-1, eotaxin from R&D ECP – ELISA Kit for Human Ribonuclease A3 from Uscn Life Science; PAF ELISA Kit for Human Platelet Activating Factor (PAF from Uscn Life Science, Tryptaza, ELISA Kit for Human Tryptase) from Uscn Life Science, Chymaza ELISA Kit for Human Chymase 1 Mast Cell from Uscn Life Science, TNFα Human TNFα -ELISA Kit from Gen-Probe Diaclone, IL-4 Human IL-4 ELISA Kit from Gen-Probe Diaclone, Neurokinin B, Endotelin B, CGRP, Substance P ELISA kit from R&D, MMP1, MMP2, MMP9 i TIMP2 ELISA kit from Quantikine, R&D Systems, IMUBIND Tissue Factor ELISA Kit from American Diagnostica Inc. The Indications of d-dimers were performed using the method of optical test apparatus Turbidity STA Compact (Roche Diagnostics). The Determination of the concentration of fibrinogen was performed using chronometric Clauss and Turbidity method (during the determination coagulation parameters). The results were statistically analyzed. The Results were considered statistically significant with p < 0.05. The localization and expression of adhesive molecules: E- and L-selectins and β1, β3, β4 integrins was studied by immunohistochemistry.The Expression of selectins was detected in the epithelium and skin leukocytes in all DH and BP samples. The expression of β1, β3 integrins was detected mainly in basal keratinocytes as well as focally in the other layers of the epidermis. The expression of β4 integrin was irregular and detected mainly in the blister. In the control group only weak expression of the examined molecules was detected. The expression of one of the chemokines: eotaxin was found in the epidermis and in the cells of inflammatory infiltrate in the epidermis. The expression of eotaxin was significantly higher in the patients with BP compared to the healthy subjects, as well as in the patients with DH compared to the healthy subjects (infiltration - p < 0.02, keratinocytes - p < 0.03). In the patients with BP TARC (thymus and activation regulated chemokine) expression was present in keratinocytes of the lower layers of the epidermis around lesions in individual cells and the inflammatory infiltrate located in the epidermis. TARC expression was significantly higher in the patients with BP compared to the healthy subjects (infiltration - p < 0.001, keratinocytes – p < 0.006) and skin lesions in relation to the perilesional skin in BP (infiltration p < 0.009 and keratinocytes, p < 0.001). In the biopsies from the skin lesions in DH the average intensity of expression TARC was observed in most cases. In keratinocytes within the lesions it differed significantly between the patients with expression of BP and DH (p < 0.05), as well as in keratinocytes of the perilesional skin (p < 0.05). In the control group there was very weak, focal expression of TARC of single keratinocytes located in the lower layer of the epidermis. The expression of chemokin MCP-1 (monocyte chemoattractant protein-1) was present in skin lesions and the perilesional skin in the patients with BP. There was no expression of MCP-1 in DH patients and the control group. The expression of chemokine receptor: CCR-1 in skin lesions and the perilesional skin of the patients with BP was similarly weak. There was no statistically significant difference between the expression of CCR-1 in skin lesions and the perilesional skin in BP and DH. The expression of receptor for chemokines CXCR-1 was present in the biopsies of skin lesions in the patients with BP. CXCR-1 expression was significantly higher in the skin lesions in relation to the perilesional skin in BP (p < 0.03). In all the cases of patients with DH cells expressing CXCR-1were found in lesions. CXCR-1 expression was significantly higher in the patients with DH compared to the healthy subjects (p < 0.001). The Patients with DH showed higher expression of CXCR-1 in the biopsies of lesions compared to the patients with BP (0.67±0.18 vs. 0.15±0.24, p < 0.001). In the skin lesions in BP patients, receptor for chemokines CXCR-2 expression was demonstrated in all specimens and was significantly higher compared to the perilesional skin in BP (p < 0.02) as well as the expression in the healthy subjects (p < 0.04). In the biopsies taken from the blisters in DH patients in all cases the expression of CXCR-2 was present. The Patients with DH showed significantly higher expression of CXCR-2 in skin lesions compared to the patients with BP (1.1±0.87 vs. 0.31±0.24, p < 0.001). There was no expression of CCR-1, CXCR-1, CXCR-2 in the control group. The concentrations of MCP-1 levels were statistically higher in the patients with BP (374.8±30.8 pg/mL) compared to the patients with DH (282.2±21 pg/mL, p < 0.05), although there was no statistically significant difference in comparison to the control group. The concentration of ECP (283.9±66.92 pg/mL) and PAF (136.9±29.87 pg/mL) levels was significantly higher in BP than in the healthy subjects and the patients with DH (p < 0.05). The Concentration of chymase (0.21±0.98 pg/mL) was significantly higher in the patients with BP compared to the healthy subjects and the patients with DH. There was no difference in the concentrations of tryptase, IL-4 and TNF in the treatment groups. The expression of TNFα was demonstrated in inflammatory infiltrates and keratinocytes and in blister liquid of BP and DH patients. The Expression of TNFα was also confirmed in the perilesional skin. Morphometric analysis showed a higher expression of TNFα in the skin lesions as amended in relation to the perilesional skin (1.59±0.58 BP vs. 1.01±0.43, DH 1.39±0.84 vs. 0.69±0.59) as well as in the control groups vs. the control group (p < 0.01). The expression of neither the examined chemokines nor their receptors was observed in the biopsies from the healthy people. The expression of the neuropeptide: corticoliberin CRF (corticotrophin releasing factor) was present in keratinocytes, cells of the sebaceous glands and sweat glands, and the infiltration of inflammatory cells. The results had significantly higher expression of CRF in the biopsies derived from the skin lesions than in the skin biopsies taken from the perilesional skin, and this applied both to the individuals with BP (1.58±0.64 vs. 1.04±0.48, p < 0.001) and with the DH (1.36±0.89 vs. 0.71±0.61, p < 0.05). The lowest expression of CRF was demonstrated in the healthy subjects (0.36±0.15). The expression of endothelin receptor B was found in the endothelial cells, vascular muscle and keratinocytes. This phenomenon was observed for the increased expression of endothelin receptor B in the individuals with DH. This value was significantly higher in the skin lesions than in the perilesional skin (1.24±0.81 vs. 0.66±0.43, p < 0.04). There was no difference in the expression of endothelin B receptor in the perilesional skin in DH patients and the control group (0.66±0.43 vs. 0.58±0.32). The Expression of Substance P, CGRP and neurokinin B was demonstrated in the nerve endings in the epidermis and the skin. The expression of these neuropeptides in the groups was not statistically significant. The expression of metalloproteinases - MMP1, 2, 9 and 10 (MMP, matrix metalloproteinase) in patients with BP was found both in the basal layer and the whole epidermis and in the fluid blister and inflammatory infiltrates. In biopsies from the skin lesions with the blister the linear expression of MMPs has been shown in the bottom of the blister. In patients with DH MMPs expression was found in the basal layer of keratinocytes in all the biopsies from the lesion, in most biopsies it was very intense. The Expression of MMPs is not limited to the basal layer of keratinocytes, but was also present in medium intensity in the other layers of epidermal keratinocytes. The expression of these MMPs was also found in neutrophils, blister fluid and microabscesses. In contrast to the MMPs, the expression of their inhibitors - TIMPs (tissue inhibitor of martix metelloproteinases) was moderately or poorly pronounced. The expression of TIMP1, 2, 3 in the patients with BP was found throughout the epidermis. In the presence of the blister it was ascertained at its bottom. Moderate activity of these enzymes was demonstrated in inflammatory infiltrates and blister fluid. The expression of TIMP1, 2, 3 of medium intensity in all specimens was demonstrated throughout the epidermis of the patients with DH, as well as in neutrophilic microabscesses. In the studied serum of the patients with DH and BP the concentrations of MMP1, MMP2 and TIMP2 are within normal limits. Individual values of MMP9 concentrations were elevated in the patients with BP, in the remaining members of the studied groups they were normal. In the comparison group they were not elevated in the serum concentrations of these enzymes. The expression of adamalysins: ADAM8, 10, 15 and 17 (ADAM, a disintegrin and metalloproteinases domain) was found in the cells of the basal layer and other layers of the epidermis and dermis in BP and DH. Eosinophilic and neutrophilic infiltration in the skin lesions of patients with BP showed a mean intensity expression. The Expression of ADAMs was also shown in perivascular influx and microabscesses in DH. The Expression of ADAM8, 10, 15 and 17 in the control group in all the biopsies was detected only in single keratinocytes. Morphometric analysis showed a higher expression of ADAM8, 10, 15 and 17 in the skin lesions compared to the perilesional skin in keratinocytes, inflammatory infiltration in both the group of patients with BP and DH. Morphometric analysis showed a significantly higher expression of ADAM15 in the skin lesions in BP and in the perilesional skin compared to the patients with DH (p < 0.001). In a study by immunoblotting a significantly higher expression of ADAM17 was detected in the patients with BP compared to the patients with DH (p = 0.007 and 0.007, respectively) and the control group (p = 0.007 and p = 0.006 respectively). The Immunohistochemical study was confirmed by in situ hybridization. The expression of tissue factor (TF, tissue factor) was demonstrated in the neutrophils in inflammatory infiltrates and keratinocytes and blisters fluid of the patients with BP. TF expression was also shown in the perilesional skin. Morphometric analysis showed a higher expression of TF in the skin lesions in comparsion to the perilesional skin in the patients with BP (p < 0.02). The studies of the patients with DH showed no significant expression of the tissue factor in pathologically changed, and normal appearing skin. TF was also detected in the specimens from the healthy individuals. The Expression, however, in this group was significantly lower than in the lesions in BP and comparable to the lesions in DH. In the group of the patients with BP serum TF concentration was 847.2±402.7 values, in the patients with DH 285.7±187.7 pg/mL, and the comparison group for BP - 252.6±169.3 pg/mL, and for DH 221.9±153.3 pg/mL. These results indicate a statistically significant difference in the TF values between the group of patients with BP and DH and BP and the control group (p <0.001). The values of d-dimers in the group of patients with BP before the treatment, most patients were in the upper limits of normal and averaged: 0.49±0.40 mg/mL (0.33-2.9 mg/mL). In 7 patients the values were elevated and had a range between 1.2 and 2.9 μg/mL. These values were higher than in the group of patients with DH and the comparison group. In all patients with DH values of d-dimers correspond to the standard dimers and their average value was 0.31±0.19 mg/mL (0.26-0.36 g/mL). In the control group for patients with BP in an individual d-dimer values exceeded the normal in the other corresponded to the standard 0.29±0.3 mg/mL (0.24-0.67 g/mL), the comparison group for all patients with DH values were normal - mean: 0.34±0.6 mg/mL. There is a statistically significant difference between the group of patients with BP and DH and BP and a group comparison of the value of d-dimers (p < 0.05). In the patients with BP the value of fibrinogen is an average 4.50±0.18 g/L at normal 2.0-5.0 g/L. The Patients with DH had a mean fibrinogen value 2.80±0.14 g/L in the control group for BP - 2.60±0.3 g/L and for DH - 2.45±0.26 g/L. There is a statistically significant difference between the group of patients with BP and DH and BP and the comparison group in the value of fibrinogen (p < 0.05). The obtained results allowed to formulate the following conclusions: 1. Differences in the expression of adhesion molecules and chemokines and their receptors are the cause of a different composition of the inflammatory infiltrate in dermatitis herpetiformis and pemphigoid, in which a special role is played by tissue factor activating eosinophiles. 2. Increase in expression of neuropeptides is one of the factors of development neuroimmunological inflammation and itching in the DH and BP. 3. The increased activity of metalloproteinases and adamalysines and their tissue inhibitors and their balance disorder is the cause of the activation of cytokines and tissue destruction in the course of BP and DH. These data offer new prospects for therapeutic BP and DH. 4. The increase in tissue factor expression in skin lesions and the serum of patients with BP indicates the activation of the coagulation cascade and the coexistence of elevated levels of d-dimer and fibrinogen, points to the particular predisposition to thromboembolic complications of glicocorticosteroids in therapy. 5. The determination of coagulation parameters should be the standard before the planned treatment with glicocorticosteroids in patients with pemphigoid.

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1 Folia Medica Lodziensia

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1 Żebrowska A.

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Ocena zjawisk immunologicznych i enzymatycznych w patogenezie wybranych podnaskórkowych chorób pęcherzowych