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Apoptosis resistance in peripheral blood lymphocytes of alopecia areata patients Studies involving peripheral blood mononuclear cells have revealed significant changes in patients with alopecia areata in comparison with normal individuals. Reports indicate that there is involvement of T-cell activation and the blood levels of CD16 positive cells and cytokine signaling chemical Tumor necrosis factor-alpha are at higher levels in patients with alopecia areata. Although not life threatening, the condition is cosmetically distressing. In many instances those afflicted with the disease have associated autoimmune disorders of which hypothyroidism is the most common along with nail dystrophy and eye defects. Typically, restoration of hair loss is encouraged with the use of corticosteroid injections or the use of contact sensitizing agents. Since the mechanism underlying the onset and progression of alopecia areata is still elusive, peripheral blood mononuclear cells have been studied for various parameters to assess the reasons behind the changes that occur in alopecic patients, especially the higher percentage of activated T cells. In this study, conducted on Caucasian patients and appropriate controls, the number of CD4+/CD25+ regulatory T cells in alopecic patients was considered. This study is an extension of studies carried out on mouse models which indicated that there was deficiency in CD4+/CD25+ cells in alopecic mice. When this cell population was increased by cell injection in the mice, there was marked improvement in the hair loss condition. Since this finding had a potential base for drug molecular interactions, the study was extended to the human system. Regulatory T-cells have already been studied in some detail in thymectomized mice. These mice developed autoimmune diseases after thymectomy, but showed reversal of disease symptoms when they were injected with CD4+/CD25+ regulatory cells. These cells are produced naturally by the thymus and to a certain extent by the peripheral T cells too, and they express a molecular cell surface structure called CD152. They also help in arresting the multiplication of CD4+ and CD8+ cells which play important roles in alopecia areata. They secrete Interleukin-4 and 10 and tumor growth factor beta. It was observed in mouse models that secretion of Interleukin-10 has a profound effect on CD4+/CD25- cells bringing about a non responsiveness to stimulation. When CD40L is deficient in individuals CD4+/CD25+ are also seen to decrease. Likewise CD44, which is an accessory molecule required by leukocytes to penetrate the walls of blood vessels and enter into the surrounding tissues, has many isoforms, some of which have definite roles in autoimmune diseases. CD40-CD40L induces the expression of the CD44v7 isoform. A high level of CD44v7 has been observed in several autoimmune diseases. An interesting observation is that autoimmune diseases can set in when CD40-CD40L is absent. Hence the mechanism of interaction and control of these cells is intriguing and very complex. How do the CD4+/CD25+ cells in peripheral blood get regulated or activated and does reduced apoptosis [programmed cell death] susceptibility help in activation of T cells? To find answers to the above question, blood samples from 31 healthy volunteers and 43 alopecic patients in different stages of alopecia areata were collected and the peripheral blood mononuclear cells were isolated using a Ficoll-Hypaque centrifugation technique. CD4+, CD8+, CD4+/CD25- and CD4+/CD25+ cells were separated from the peripheral blood mononuclear cells. Flow cytometry, intracellular staining of CD152 and cytokines, double fluorescence analysis of cells and double staining with Annexin V-FITC and propidium chloride for studying apoptotic cell death, and a peripheral blood mononuclear cell proliferation assay were carried out. All the results were analyzed statistically using two-tailed student-t test and multifactorial ANOVA for any significance of difference. The results revealed the following findings:- 1. The percentage of activated T-cells was high in patients with progressive alopecia areata. This was evident from the observation of increase in Interleukin-10 regulated by certain accessory molecules in patients with the progressive condition. There was however no significant increase in activated regulatory T cells in alopecic patients. But this does not give any concrete evidence of the non involvement of the regulatory T cells in alopecia areata. 2. Unlike the mouse models, the human system showed increased levels of CD4+/CD25+ cells even in patients with progressive alopecia areata. 3. Expression of CD95L by CD4+/CD25- was seen in healthy individuals while the progressive alopecic patients showed no CD95L cells giving an impression that deficiency of apoptosis promoting molecules may have some role in progression of the condition. 4. CD44v7 which prevents activation induced cell death was seen to be higher in unstimulated alopecic patients and was expressed in CD4+/CD25- cells in greater levels than CD4+/CD25+ cells. So CD44v7 in association with CD4/CD25- cells did not express CD95L or CD152 while that associated with CD4+/CD25+ showed expression for both CD95L and CD152. Interestingly in the progressive alopecic patients, CD44v7 with CD4+/CD25+ cells also expressed CD154+ and they were totally apoptosis resistant. 5. What was consistent with the mouse model study was the loss of regulatory T cells in the initial period of onset of alopecia areata. Probably the presence of regulatory T cells during the onset of alopecia areata may actually prevent/inhibit the condition to be expressed by controlling T cell activation. The study highlights the differences seen between mouse models and the human system behavior in different stages of alopecia areata. Although a lot more needs to be done to formulate effective drugs to alleviate the condition, the results obtained from this study give a more dependable view of what can be expected in alopecia areata affecting humans.
Apoptosis resistance in peripheral blood lymphocytes of alopecia areata patients references
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