Aberrant expression of complement regulatory proteins, membrane cofactor protein and decay accelerating factor, in the involved epidermis of patients with vitiligo.

BACKGROUND: Vitiligo is a pigmentary disorder of the skin characterized by the complete absence of melanocytes from the lesion. Complement-activating antimelanocyte antibodies have been implicated in vitiligo pathogenesis. As membrane regulators of complement activation, membrane cofactor protein, decay accelerating factor and CD59 protect cells from elimination by autologous complement, their absence or downregulation on melanocytes may be associated with autoantibody and complement-mediated melanocyte destruction in vitiligo. OBJECTIVES: We studied the expression of these regulatory proteins in non-lesional, perilesional and lesional vitiligo skin compared with those of control specimens. METHODS: We used immunohistochemistry to study the expression of the regulatory proteins, and flow cytometric analysis of cultured melanocytes to investigate possible constitutive changes in the expression levels of these molecules. We also investigated whether melanocytes can influence keratinocyte susceptibility to autologous complement by regulating keratinocytic decay accelerating factor and membrane cofactor protein expression levels. RESULTS: Immunohistochemical data showed that expression of membrane cofactor protein and decay accelerating factor in whole epidermis was lower in lesional and perilesional skin in comparison with non-lesional skin. The reduced in situ expression appeared to be specific to vitiligo. However, coculture experiments indicated that melanocytes do not influence keratinocyte susceptibility to autologous complement. Further, flow cytometric analysis of cultured melanocytes convincingly demonstrated that non-lesional vitiligo and control melanocytes have comparable decay accelerating factor, membrane cofactor protein and CD59 expression levels. CONCLUSIONS: It is therefore concluded that there is no constitutive melanocyte defect per se that could be related to the in vivo expression of these molecules in vitiligo. Nevertheless, the present data suggest that both keratinocytes and melanocytes in the involved vitiliginous whole epidermis express lower levels of decay accelerating factor and membrane cofactor protein compared with controls that could render them more vulnerable to autologous complement attack.

Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo.

Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte-expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl-2 and Bax revealed no differences in in vitro expression levels between normal control and non-lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non-lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte-expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non-lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB-induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.

Altered expression of interferon-gamma and interleukin-4 in inflammatory bowel disease.

Experimental data indicate that mucosal CD4+ T cells play an important role in the pathogenesis of inflammatory bowel disease (IBD). Based on the pattern of cytokine production, CD4+ T cells may be distinguished into two different phenotypes. Th1 responses are characterized by secretion of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, lymphotoxin, and interferon (IFN)-gamma and are associated with delayed-type hypersensitivity reactions, whereas Th2 responses, which are characterized by secretion of IL-4, IL-5, and IL-10, have been associated with humoral immune responses and allergy. To assess the number of IFN-alpha and IL-4 positive cells in IBD and normal intestinal specimens, frozen sections from intestinal specimens from 10 Crohn's disease (CD), 8 ulcerative colitis (UC), and 8 healthy controls were examined by immunohistochemistry. Monoclonal antibodies for CD3, CD8, IFN-gamma, and IL-4 were used. T-lymphocyte infiltration and cytokine expression by epithelial, lamina propria, and submucosal cells were scored on a four-point scale by two independent observers who were blinded for the clinical data. One-way analysis of variance (ANOVA) testing was used for statistical analysis. In intestinal specimens from IBD patients, the number of CD3+ cells was found increased in the lamina propria and, within the submucosa, this increase was significant (p < 0.001). In CD the number of lamina propria IFN-gamma positive cells was significantly increased as compared with controls (p < 0.002). In UC the number of both IFN-gamma and IL-4 producing cells in the lamina propria was not significantly increased as compared with controls. The present results confirm the existence of a Th1-biased pattern production in CD but not in UC.

Morphoea lesions are associated with aberrant expression of membrane cofactor protein and decay accelerating factor in vascular endothelium.

One of the main features of systemic and localized forms of scleroderma is vascular damage, the mechanism of which is not yet understood. Recently, we have shown undetectable or decreased expression of complement regulatory molecules, membrane cofactor protein (MCP) and decay-accelerating factor (DAF), in cutaneous endothelium of patients with systemic sclerosis (SSc). In some patients, CD59 expression in endothelium was also altered. As these molecules protect endothelial cells from damage by autologous complement, their decreased expression could be part of the mechanism of vascular damage in SSc. In the present study, we investigated the expression of MCP, DAF and CD59 in the endothelium of lesional and non-lesional skin of patients with localized morphoea. Normal skin and lesional skin from patients with systemic lupus erythematosus, and three inflammatory diseases, were included as relevant controls. The results showed that the extent of expression of the three molecules in non-lesional skin of patients with morphoea, on all the skin cells and structures, was identical to that of normal skin. In lesional skin, however, the expression of MCP and DAF in endothelium was either undetectable or only present to a very slight degree. CD59 expression in endothelium in lesional skin was normal. No such aberrant expression was observed in the lesions of any of the control diseases. These results indicate a decreased ability of endothelial cells in lesional areas to protect themselves from autologous complement, and this could contribute to vascular damage in morphoea lesions.

Fibrous and lipid-rich atherosclerotic plaques are part of interchangeable morphologies related to inflammation: a concept.

BACKGROUND: The morphology of advanced atherosclerotic plaques varies from solid fibrous lesions, considered essentially stable, to lipid-rich lesions with large atheromas prone to rupture. The latter situation is often associated with large amounts of foam cells. An in-situ inflammatory process influenced by activated T cells and macrophages can be demonstrated in atherosclerotic plaques; however, the relationship between the inflammation and clinically relevant morphological types has not yet been investigated. A study of plaque morphology, focusing on the relationship between inflammatory cells, smooth muscle cells, and the collagen matrix, on the one hand, and the 'classic' plaque morphologies (fibrous versus lipid-rich), on the other, may shed light on this concept. MATERIALS AND METHODS: Immunocytochemical techniques were used in combination with connective tissue stains to study the topographic distribution of smooth muscle cells, collagen, and inflammatory cells in different morphologic types of advanced atherosclerotic plaques in aortic and carotid arteries obtained at autopsy. RESULTS: Lesions with an inconspicuous lipid core were defined as fibrous (n = 7). They contained a dense collagen matrix and the dominant cell type was the smooth muscle cell; lymphocytes and macrophages were sparse. Lesions with a large lipid core were defined as lipid-rich (n = 13). They contained a thin fibrous cap with a loosely arranged matrix dominated by macrophages and T cells. Most lesions (n = 21), however, had a morphology that ranged between fibrous and lipid-rich. The cellular components consisted either of mixed smooth muscle and inflammatory cells or of local distinct zones of inflammatory cells within a fibrous cap otherwise dominated by smooth muscle cells and collagen. Thus, zones of severe inflammation were invariably associated with dissolution of the connective tissue matrix and abundant human leukocyte antigen-DR expression on inflammatory cells and the remaining smooth muscle cells. CONCLUSION: Our observations support the concept that inflammatory mechanisms modulate plaque morphology, by promoting either synthesis or lysis of the fibrous cap. Our hypothesis is that fibrous and atheromatous lesions are essentially interchangeable.

Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions.

Atherosclerotic lesions show features of a cell-mediated immune inflammatory process. From this viewpoint, the potential role of arterial endothelium in the recruitment of mononuclear cells (T lymphocytes and macrophages) was studied. The endothelium of diffuse intimal thickening (DIT) and atheromatous plaques (AP) in human coronary arteries and abdominal aortas was characterized for the expression of adhesion molecules ELAM-1, ICAM-1, and the major histocompatibility complex (MHC) class II antigens HLA-DR/DP. A marked increase in expression of ICAM-1 and ELAM-1, and to a lesser extent HLA-DR/DP was observed on endothelial cells that were adjacent to subendothelial infiltrates of T lymphocytes (CD3+, CD11a+, HLA-DR/DP+) and macrophages (CD14+, CD11a+, CD11c+, HLA-DR/DP+). This contrasted with a lower or absent expression of these activation markers at sites without prominent inflammatory cell infiltrates. These findings could be demonstrated in DIT as well as in AP. The observations suggest that cytokines produced by the subintimal infiltrates may activate the endothelium in a similar way as is observed in the microvasculature at sites of immune inflammation. The expression of these activation markers in the microvasculature is associated with enhanced leukocyte adhesion, permeability for macromolecules, and procoagulant activity, features known to occur also in early experimental atherosclerosis. The findings therefore support the concept that arterial endothelium plays an active role in the recruitment of mononuclear cells in atherosclerotic lesions.

Macrophage differentiation in atherosclerosis. An in situ immunohistochemical analysis in humans.

The differentiation of macrophages present in diffuse intimal thickening, fatty streaks, and atheromatous plaques, was analyzed with immunohistochemical methods, using segments of aorta, coronary, and carotid arteries obtained at autopsy. Various differentiation antigens were studied with the monoclonal antibodies anti-HLA-DR, EBM-11, Leu M3, OKM1, and OKM5. Adjacent sections were stained for lipids (oil red O) and lysosomal activity (acid phosphatase). Almost all macrophages identified with the pan-macrophage antibody EBM-11, also stained with the anti-HLA-DR antibody. Diffuse intimal thickening showed a predominance of Leu M3+ cells; fatty streaks also showed OKM1+ and OKM5+ macrophages. Classical atheromatous plaques showed a gradual shift in phenotypic expression towards the center of the lesion. Cells in the superficial layers were positive only with Leu M3, deeper localized cells showed double expression of Leu M3 and OKM1 or double expression of OKM1 and OKM5. Cells that were localized adjacent to the atheromatous debris stained only with OKM5. The phenotypic changes occurred in parallel with an increase in both fat uptake and lysosomal activity of the macrophages. This shift in phenotypic expression suggests a process of differentiation and maturation of the macrophages involved. The results indicate that macrophages within the arterial intima are activated and mature towards cells that express receptors for adhesion proteins and complement during the development of atherosclerotic plaques. This may imply that the macrophages involved in lipid metabolism also have a potential to act as effector cells in a chronic inflammatory process, and thus, may contribute to the progression of an atherosclerotic plaque. Functional studies of macrophage subpopulations are needed to verify this hypothesis.

Potential antigen-presenting cells in normal extraocular muscles demonstrated with double immunoenzyme staining.

Antigen-presenting cells are of crucial importance for the initiation and regulation of regional immune responses. In a previous study, indirect morphological evidence that morphologically normal human orbital tissues contain HLA-DR-positive macrophages, which may represent antigen-presenting cells, has ben obtained. In the present study, these cells were characterized in detail using double immunoenzyme staining techniques with monoclonal antibodies directed against several well-characterized monocyte/macrophage markers and against HLA-DR gene products. The orbital muscular tissues appear to contain numerous HLA-DR, monocyte/macrophage marker double-stained cells, which are considered to be potential antigen-presenting cells. The cells are widely distributed in the connective tissue of all the orbital muscular tissues studied and consist of several subsets with different phenotypes. Furthermore, site-specific differences were shown between recti muscles and the levator/Müller's muscles with respect to the distribution of HLA-DR and one monocyte/macrophage marker (OKM5). Many of the orbital antigen-presenting cells appear to be of the dendritic type and are considered to be of major importance in regulating local orbital immunity.

Expression of the nuclear oncogene p53 in colon tumours.

The nuclear tumour antigen p53 is expressed by a gene localized on the p-arm of human chromosome 17, a region frequently deleted in colon carcinomas. Using a monoclonal antibody to p53 antigen, immunohistochemical analysis of carcinomas and dysplastic tubular adenomas of the colon has been performed to study the relation between p53 expression and dysplasia or malignancy. With this methods p53 was detectable in 55 per cent of colon carcinomas (n = 29). In 8 per cent of adenomas (n = 74), focal nuclear p53 expression was found in dysplastic epithelial cells. In general, these p53-positive regions of the polyps were histologically indistinguishable from the neighbouring tubuli. Sometimes the p53-positive nuclei were found in a focus of more highly dysplastic epithelium. The results suggest that expression of the p53 gene may be part of the process of malignant transformation of dysplastic colon polyps.

Enzyme histochemical studies on the conducting system of the human heart.

In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres. As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, beta-glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.

X-linked ichthyosis and X-linked placental sulfatase deficiency: a disease entity. Histochemical observations.

The combined occurrence of X-linked steroid sulfatase deficiency of the placenta and X-linked ichthyosis is reported in 6 unrelated boys. Placental steroid sulfatase deficiency was diagnosed on the basis of a very low total estrogen excretion (6 cases), verified prenatally by the dehydroepiandrosterone sulfate (DHEAS) loading test in 4 cases and postnatally by clinical investigations (6 cases) and by biochemical investigations (5 cases). In addition, microsomal arylsulfatase C (MAS) could not be detected in the placental homogenate of the five cases investigated. Lysosomal arylsulfatases were within the normal range. All boys developed well except for X-linked ichthyosis. In the 5 cases investigated the skin biopsy showed the same MAS deficiency histochemically in the granular layer of the epidermis as in the trophoblast cells. The same holds true for the skin of carriers. Steroid sulfatase activity of cultured skin fibroblasts from the boys was almost nil (3 cases). The histochemical technique offers a practical approach in the scientific investigation of keratotic conditions.

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.