Pranab K. Das

Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site.

In situ immune infiltrates in lesional, perilesional, and nonlesional skin biopsies from patients with vitiligo were analyzed by immunohistochemistry and compared with immune infiltrates found in the skin of normal healthy donors and relevant disease controls. An increased influx of activated skin-homing T cells and macrophages were seen in the perilesional biopsies. The overall percentages of cutaneous leukocyte-associated antigen-positive (CLA+) T cells were similar to those found in normal healthy donors. This is compatible with the similar expression of E-selectin. Most strikingly, however, the CLA+ T cells in perilesional skin were mainly clustered in the vicinity of disappearing melanocytes, and 60% to 66% of these interacting T cells expressed perforin and granzyme-B. The perforin+/granzyme-B+ cells were not seen in locations different from that of disappearing melanocytes. Interestingly, the majority of the infiltrating T cells were HLA-DR/CD8+. Another hallmark of the present study is the focal expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR in the epidermis at the site of interaction between the immune infiltrates and the disappearing melanocytes. The data presented in this study are consistent with a major role for skin-homing T cells in the death of melanocytes seen in vitiligo.

Predominance of «memory» T cells (CD4+, CDw29+) over «naive» T cells (CD4+, CD45R+) in both normal and diseased human skin

SummaryAbsolute numbers of CD3+ T lymphocytes and their subpopulations were determined and statistically evaluated in the lesional skin of psoriasis, atopic dermatitis, nummular dermatitis, pityriasis rosea, and lichen planus. Skin sections were divided into horizontal layers and the numbers of CD3+ T cells as well as CD4+ inducer and CD8+ suppressor-cytotoxic T-cell subsets were counted. In addition, absolute numbers of the two subpopulations of inducer T cells, i.e., “memory” (4B4+ 2H4-) and “naive” (4B4- 2H4+) were evaluated. Unexpectedly, epidermal infiltration by T cells was highest in psoriasis and lowest in atopic dermatitis. In most cases, this exocytosis was dominated by CD8+ suppressor/cytotoxic T lymphocytes, with a minimal epidermal mean CD4/mean CD8 ratio of 0.04 in pityriasis rosea and a maximum of 0.48 in psoriasis. Inducer T cells within the epidermis were almost exclusively of the 4B4+ 2H4- “memory” T-cell subpopulation, whereas 4B4- 2H4+ “naive” T cells were extremely uncommon in lesional epidermis. Similar results were obtained for dermal T cells in all diseases studied, i.e., 4B4- 2H4+ “naive” T cells were relatively rare. Papillary dermis infiltration by T cells was highest in lichen planus where a mean CD4/mean CD8 ratio of 1.10, the minimum in this comparative study, was obtained. The mean CD4/mean CD8 ratio of the papillary infiltrate was highest in atopic dermatitis (4.12). Our results indicate disease-specific and significantly different infiltration patterns of T-lymphocyte subsets in the chronic inflammatory dermatoses investigated. The predominant presence of the CD4+ 2H4- “memory” subpopulation of CD4+ T cells in all diseases studied as well as in normal human skin (reported previously) seems to indicate that the skin immune system is rather unidirectional in its increase in this subpopulation of the inducer T-cell subset. This predominance of the “memory” subpopulation thus indicates that most T cells of normal and diseased human skin are already primed, i.e., have already met their specific ligand in a MHC II context.

Review of the etiopathomechanism of vitiligo: A convergence theory

Abstract Vitiligo is an acquired melanin pigmentary disorder manifesting itself by expanding depigmented lesions of the skin. To date, the etiopathomechanism of vitiligo has not been convincingly elucidated and a number of seemingly mutually opposed hypotheses with equal likelihood still coexist. Concurrent theories on vitiligo etiology, together with supportive evidence, are reviewed here. Due to the observed variation in clinical manifestations of the disease, it seems likely that the etiology of vitiligo may differ among patients. Therefore several theories on vitiligo etiopathogenesis have been combined to formulate a convergence theory for vitiligo. also presented in this article. This theory stales that stress, accumulation of toxic compounds, infection, autoimmunity. mutations, altered cellular environment and impaired melanocyte migration and or proliferation can all contribute to vitiligo etiopathogenesis in varying proportions.

Immunopolarization of CD4 + and CD8 + T Cells to Type-1–Like is Associated with Melanocyte Loss in Human Vitiligo

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. High frequencies of melanocyte-reactive cytotoxic T cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional T-cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. We isolated T cells from both perilesional and nonlesional skin biopsies obtained from five vitiligo patients, then cloned and analyzed their profile of cytokine production after short-term, nonspecific expansion in vitro. Perilesional T-cell clones (TCC) derived from patients with vitiligo exhibited a predominant Type-1–like cytokine secretion profile, whereas the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Detailed analysis of broad spectrum of cytokines produced by perilesional- and nonlesional-derived CD4+ and CD8+ TCC confirmed polarization toward Type-1–like in both CD4 and CD8 compartments, which paralleled depigmentation process observed locally in the skin. Furthermore, CD8+ TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8+ TCC isolated from perilesional biopsies of two patients with vitiligo. Finally, in two of five patients, tetramer analysis revealed presence of high frequencies of Mart-1–specific CD8 T cells in T-cell lines derived from perilesional skin. Altogether our data support the role of cellular mechanisms playing a significant part in the destruction of melanocytes in human autoimmune vitiligo.

A symbiotic concept of autoimmunity and tumour immunity: lessons from vitiligo

Vitiligo is a skin disease in which melanocytes (MCs) are eradicated from lesional epidermis, resulting in disfiguring loss of pigment. MCs are destroyed by MC-reactive T cells, as well as other non-immune and immune components. Similarities exist between the autoimmunity observed in vitiligo and the tumour immunity observed in melanoma immuno-surveillance. An analysis of these mechanisms might lead to the development of new therapies for both vitiligo and melanoma.

Production of catecholamines in the human epidermis.

Cell-free extracts from human full thickness skin (i.e., epidermis and dermis), suction blister roofs (i.e., epidermis) and from human keratinocytes express biopterin-dependent tyrosine hydroxylase a well as phenylethanolamine-N-methyl transferase, both representing key enzymes for the biosynthesis of epinephrine. These enzyme activities could not be detected in cell extracts from human melanocytes and human fibroblasts. Since keratinocytes in the human epidermis, and in cell cultures, express a high density of beta-2-adrenoceptors, and this signal transduction system regulates intracellular calcium homeostasis, it can be concluded that epinephrine production in the epidermis activates calcium transport via the beta-2-adrenoceptor system. Our results show for the first time that the human epidermis has the capacity to independently produce epinephrine.

Multiple immunoenzyme staining techniques use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).

Costimulatory molecules in human atherosclerotic plaques: an indication of antigen specific T lymphocyte activation

Atherosclerotic plaques contain inflammation, composed largely of macrophages and lymphocytes. A proportion of lymphocytes shows signs of activation, but the question arises whether they are activated in an antigen specific way. The expression of costimulatory molecules-receptors that provide accessory signals during antigen-specific activation is a prerequisite for such a condition. This aspect of inflammation in atherosclerotic lesions has not been investigated. Human arterial segments with diffuse intimal thickening, fatty streaks and atherosclerotic plaques were studied with immuno-single and double staining methods. Macrophages and T lymphocytes were stained with CD68 and CD3, respectively, and pan-B cell markers CD19 and CD22 were also used. Costimulatory molecules B7-1 and B7-2, together with their common ligand CD28, and CD27 with its ligand CD70, were stained with specific monoclonal antibodies. The results show that most T lymphocytes were CD27 positive and that only a subpopulation of these (5-15%) was positive also for B7-1, CD28 and CD70. Macrophages expressed B7-1, B7-2, CD28 and CD70, while macrophages positive for CD28 and CD70 have not been reported yet. The expression of costimulatory molecules was most pronounced in the superficial layers at the fibrous cap, but decreased towards the lipid core. This study shows, therefore, that atherosclerotic plaques provide costimulatory signals generally accepted as a prerequisite for adequate T cell stimulation. In addition, this study reveals that only approximately 5-15% of the lymphocytes appears actively involved in the inflammatory reaction.

Increased expression of adhesion receptors in both lesional and non-lesional psoriatic skin.

Adhesion receptors and their ligands play a vital role in the immune system. We studied the expression of different adhesion receptors, using single- and double-staining immunohistochemical techniques, in both lesional and non-lesional skin specimens from seven psoriasis patients and in skin biopsy specimens from eight normal healthy controls. Our results showed an overall increased expression of several adhesion receptors in both lesional and non-lesional psoriatic skin. We consistently found an increased expression in particular of ICAM-1 and E-selectin on endothelial cells, and ICAM-1 on T cells and Langerhans cells. In contrast, a weak expression of VCAM-1 was found on endothelial cells and mononuclear cells in lesional psoriatic skin specimens alone. Interestingly, LFA-1 was also expressed on Langerhans cells, with a greater frequency in skin from lesional than from non-lesional sites, but was never expressed in skin from normal healthy individuals. Furthermore, significantly increased numbers of Langerhans cells and T cells with a positive reactivity for MAb HECA-452 were found in both lesional and non-lesional psoriatic skin. We hypothesize that the enhanced expression of adhesion receptors on migrating immunocompetent cells and endothelial cells of psoriatic skin in general facilitates the increased influx of activated T lymphocytes and other immunocomponent cells into the skin, and thus underscores the generalized character of the disease.

Skin-homing T lymphocytes : detection of cutaneous lymphocyteassociated antigen (CLA) by HECA-452 in normal human skin

The immigration of circulating T cells into specific tissues is directed by the interaction between adhesion molecules on lymphocyte subpopulations and their ligands on vascular endothelium. Of these, endothelial leucocyte adhesion molecule (ELAM-1), weakly expressed in normal human skin (NHS), seems to be the counter-structure for cutaneous lymphocyte-associated antigen (CLA). CLA is a 200 kDa cell-surface glycoprotein of which the sugar moieties sialyl Le(a) and sialyl Le(x) are the possible epitopes recognized by the monoclonal antibody HECA-452. HECA-452 was originally described as a marker for lymphoid organ high endothelial cells, but 16% of peripheral-blood-derived T cells react with this antibody. We studied the expression of CLA on the cellular constituents of the skin immune system (SIS). By applying immunohistochemical double staining, 41% of CD3+ T cells, 44% of CD4+ T cells and 31% of CD8+ T cells were found to express CLA. Keratinocytes, CD1a+ Langerhans cells (LC) and endothelial cells did not express HECA-452 in significant numbers in NHS. Monocytes were found to express HECA-452 in 14% of CD68+ cells. CLA expression was present on a relatively low percentage of T cells and subsets localized distant from NHS vessels, suggesting loss of the molecule during further migration after transendothelial passage. However, intraepidermal T cells expressed CLA in similar percentages to T cells localized directly perivascularly. Our findings support the notion that CLA expression by T cells is associated with their homing into cutaneous structures.