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 TNF
Dermatitis herpetiformis (DH) is one of the subepidermal autoimmune bullous diseases, which is characterized by skin and intestinal lesions. Skin lesions include polymorphic eruption accompanied by severe pruritus. Intestinal lesions are characterized by atrophy of intestinal villi resulting from immunological process [
Bullous pemphigoid (BP) is a blistering disease characterized by inflammatory infiltrate in the dermis and the presence of IgG and C3 deposits along the BMZ and circulating IgG autoantibodies. Autoantibodies bound to autoantigens, the glycoproteins BPAG1 (230 kD) and BPAG2 (180 kD), localized in the basement membrane of the epidermis activate a series of immunological and enzymatic phenomena leading to the destruction of basement membrane components and the formation of blisters, as observed in DH [
In the dermis, inflammatory infiltrates formed by eosinophils and neutrophils and bound
Infiltrate formation is preceded by early accumulation of leukocytes, depending on the activity of adhesion molecules. The binding of autoantibodies leads to the activation of keratinocytes, which release cytokines, as well as the activation of metalloproteinases and the C5 component of the complement [
Mast cells are a source of many mediators, cytokines, and enzymes which may affect the course of inflammation in the skin in different ways. One mediator which plays a very important role in the development skin lesions in autoimmune skin diseases is tumor necrosis factor
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. The control group comprised 20 healthy individuals in total. The mean age of control group number 1 (10 patients), for BP patients, was 71.6 years (range 50 to 80 years) while the mean age of control group number 2 (10 patients), DH patients, was 42 years (range 19 to 49 years). The control groups consisted of unrelated volunteers matched for sex and age.
In all DH cases, histological examination revealed perivascular neutrophilic infiltrates, the presence of Pierrard’s abscesses, and small subepidermal blisters. In most samples, large unilocular blisters displaying multiple neutrophilic papillary microabscesses were found. All histopathological findings according to Ackerman were fully developed [
Twenty-seven patients (16 women, 11 men; mean age 68.5 years; range: 58–84) with BP were included in the study. Pemphigoid was diagnosed based on clinical picture and histological and immunological findings. The patients were at an active stage of the disease; 22 of the 27 patients presented with skin blisters, vesicles, and itching papules, whereas the rest had only small vesicles and urticarial papules. In all cases, the histopathology findings were fully developed according to Ackerman et al. [
All the patients gave their informed written consent before entering the study. The study protocol (RNN/132/07/KB) was approved by the Local Ethical Committee of the Medical University of Lodz.
Paraffin-embedded sections, 3-4
For immunohistochemistry, the paraffin-embedded sections were placed on adhesive plates and dried at 56°C for 24 hours and were later deparaffinated in a series of xylenes and alcohols with decreasing concentrations. The activity of endogenous peroxidase was inhibited with 3% hydrogen peroxide solution in methanol for 5 minutes.
In order to retrieve the antigenicity of tissues and allow them to react with antibodies, specific procedures were used for each tested antibody, according to the manufacturer’s instructions. After incubation with diluted antibodies for 60 minutes at room temperature, they were washed with Tris buffer twice. DAKO EnVision double-step visualization system was then applied in order to visualize the antigen-antibody reaction. In the case of a positive immunohistochemical reaction, cellular nuclei were stained with Meyer haematoxylin for 2 minutes. After dehydration and processing through a series of acetones and xylenes, the sections were fixed in Canadian balm.
In each specimen, the staining intensity of MMP9 and TNF
Histological morphometry of MMP9 and TNF
The percentage of immunopositive inflammatory cells was estimated by counting 100–120 inflammatory cells in 7–10 adjacent high power fields on each slide using the semiautomatic function. The staining in the epithelial cells was measured using the point-counting method, based on Weibel [
Median levels of TNF and MMP9 for patients with BP and DH in lesions (L) and surrounding (S) were compared using the nonparametric Mann-Whitney
In order to determine the concentrations of the examined protein in the serum, the enzyme-linked immunoassay method was used: chymase: ELISA Kit for Human Chymase 1 Mast Cell (Uscn Life Science), tryptase: ELISA Kit for Human Tryptase (Uscn Life Science), TNF
Chymase, tryptase, TNF
All data was presented as median and range. Nonparametric Kruskal-Wallis analysis was performed followed by a median test. Significance at
PAF levels were statistically higher in BP patients (218.19 +/− 29.874) than healthy subjects 29.87 +/− 2.973 (
Serum levels of examined mediators in BP, DH, and control groups.
MMP9 levels were significantly higher in BP patients (364.79 +/− 41.383) and patients with dermatitis herpetiformis (243.10 +/− 77.110) compared to healthy subjects (52.61 +/− 0.779):
The chymase (34.05 +/− 0.209 versus 24.30 +/− 3.438 versus 32.10 +/− 0.823), tryptase (19.79 +/− 1.983 versus 20.74 +/− 6.083 versus 19.33 +/− 3.231), IL-4 (68.66 +/− 6.653 versus 71.50 +/− 20.219 versus 66.98 +/− 10.850), and TNF
Moderate expression of TNF
Immunoexpression of TNF
Morphometric analysis revealed that TNF
The quantitative data of TNF
TNF |
DH | BP | Control |
---|---|---|---|
L | |||
Mean | 1.39 | 1.59 | 0.16 |
SEM | 0.069 | 0.100 | 0.017 |
Median | 1.38 | 1.48 | 0.15 |
Range | 0.50 | 1.52 | 0.20 |
|
|||
S | |||
Mean | 0.62 | 1.01 | 0.16 |
SEM | 0.050 | 0.045 | 0.017 |
Median | 0.64 | 0.94 | 0.15 |
Range | 0.35 | 0.87 | 0.20 |
|
|||
MMP9 | DH | BP | Control |
|
|||
L | |||
Mean | 2.34 | 7.41 | 2.15 |
SEM | 0.356 | 0.814 | 0.276 |
Median | 2.51 | 7.66 | 2.68 |
Range | 2.52 | 14.57 | 2.63 |
|
|||
S | |||
Mean | 1.69 | 2.01 | 2.15 |
SEM | 0.511 | 0.183 | 0.276 |
Median | 1.09 | 1.81 | 2.68 |
Range | 3.64 | 3.76 | 2.63 |
Morphometry of the immunoexpression of MMP9 and TNF
Morphometry of the immunoexpression of MMP9 and TNF
Moderate expression of MMP9 was found in inflammation cells in BP patients, as well as in the epidermal basal cells in DH patients (Figure
Morphometric analysis revealed that MMP9 expression was significantly higher in lesions than the surrounding skin in patients with BP (
D’Auria et al. [
It seems important to determine which of the mediators secreted from mast cells are involved in the development of skin lesions in blistering diseases. Mast cell markers include tryptase, chymase, IL-4, PAF, TNF
As demonstrated by Heimbach et al. [
Using an animal BP model, Chen et al. [
The mechanisms leading to mast cell activation and release of proteases in these diseases are poorly understood, but C3a and C5a anaphylatoxins, neuropeptides, and cytokines could be involved as well as numerous other proteins or peptides. The involvement of mast cells in inflammation and blister formation in subepidermal bullous diseases has been suggested, and since early skin lesions have an urticarial appearance, the mast cells are hypogranulated, and their granules are spread extracellularly and blister fluid contains increased histamine and tryptase levels. In addition, injection of the mast cell degranulator, compound 48/80, into the skin of patients with dermatitis herpetiformis causes a DH-like bullous lesion in some cases [
The expression of TNF
Our findings confirm the role of TNF
The destruction of tissue in the disorders associated with the increased activity of matrix metalloproteinases is often the result of an imbalance between the activity of these enzymes and their inhibitors. Recently, MMPs have been reported to play a significant role in the pathogenesis of skin diseases, including blistering diseases [
Inflammatory cells are the main source of MMPs. Neutrophils forming influxes in the skin release the matrix and immunoglobulin-degrading enzymes [
Recent literature data shows that IgA-Ag complexes presented in the tissue in a course of different IgA-mediated diseases, also DH, are responsible for tissue damage due to migration induction as well as granulocyte activation. This is why granulocytes are unable to destruct the complexes which lead to attract new multinuclear cells, sustain inflammation, and, as a consequence, cause tissue damage [
Our findings confirming the presence of TNF
The autocrine and paracrine effects of cytokines on the inflammatory cells and keratinocytes appear to cause an imbalance between MMPs and their inhibitors. This phenomenon leads to changes in the architecture of the extracellular matrix [
Previous reports confirm the increased activity of stromelysin 1 in skin lesions in patients with DH. It is an enzyme which degrades the proteins of the basement membrane and activates procolagenasis. No increased expression of other MMPs such as matrilysin has been observed, although they may potentially cause proteolysis of basement membrane proteins such as entactin and type IV collagen [
Our experiences confirm those reports. The high expression of MMP9 was observed in basal keratinocytes, in areas of intense neutrophilic infiltration, and in blister fluid. However, the results show the possibility of stimulating the production of metalloproteinases by other layers of the epidermis. MMP9 is probably the primary element responsible for the formation of lesions in the course of BP [
Recent reports suggest that collagen XVII and collagen VII, which are the basis of anchoring fibers, undergo proteolysis under the influence of a specific group of metalloproteinases. In murine models of bullous pemphigoid, Verraes et al. [
Based on the obtained results, it can be concluded that the increased activity of matrix metalloproteinase 9 and the consequent imbalance between the groups of enzymes are responsible for tissue destruction in the course of BP. Considering the increasing number of studies concerning the participation of MMPs in the pathology of skin diseases, research regarding the therapeutic use of MMP inhibitors can be expected in the near future.
The findings of the present study demonstrate increased expression of TNF
The authors declare that there is no conflict of interests regarding the publication of this paper.
The study was funded by Medical University of Lodz research projects no. 503/1-152-01/503-01 and The Polish Science Committee Grant no. 4746/B/PO1/2009/37.