After confirming the clinical diagnosis by hematoxylin and eosin staining of all sections, immunohistochemical staining using HSP60 antibodies was done.
By immunohistochemistry two patterns of subcellular localization were observed, cytoplasmic in most of the cases where the staining was in the cytoplasmic perinuclear area with a granular pattern, as well as nuclear localization in some cases (Figs. 1–6).
Calculation of the mean total IRIDI score for HSP60 expression in LP cases ranged from 2 to 24 with a mean±SD of 14.2±5.17, while that of the controls was 9.0 with a mean±SD of 9.0±0.0. The correlation between them was statistically highly significant with a P value of less than 0.001 (Table 2).
The mean IRIDI score for HSP60 expression in the basal layer of LP patients ranged from 1 to 9 with a mean±SD of 7.9±2.37 (Table 2 and Figs 1–6), while that of the controls was 3.0 with a mean±SD of 3.0±0.0 (Fig. 1). The difference between them was statistically highly significant with a P value of less than 0.001 (Table 2).
The mean IRIDI score for HSP60 expression in the suprabasal layer in LP cases ranged from 1 to 9 with a mean±SD of 5.13±2.56 (Table 1), which was also statistically significantly higher than the in controls, which was 3.0 with a mean±SD of 3.0±0.0, and with a P value of less than 0.05 (Table 2).
In contrast, the mean IRIDI score for HSP60 expression in the superficial layer of LP cases ranged from 0 to 6 with a mean of 1.18±1.81, and was statistically significantly lower than in the normal skin, which was 3.0 with a mean±SD of 3.0±0.0, and with a P value of less than 0.001 (Table 2).
The mean IRIDI score of HSP60 in dermal inflammatory infiltrate ranged from 1 to 9 with a mean±SD of 2.95±2.98. There is a high statistical significant positive correlation between IRIDI scores for dermal infiltrate and total epidermal IRIDI score (r=0.45 and P<0.001).
Correlation of age of LP patients with total IRIDI scores, basal IRIDI scores, or suprabasal IRIDI scores for HSP60 expression revealed no statistically significant correlation. However, a statistically significant negative correlation was found between age of LP patients and superficial IRIDI scores (Table 3).
The mean total and superficial IRIDI scores for HSP60 showed no statistically significant difference among the different types of cutaneous LP. However, the mean basal IRIDI score was significantly lowest in follicular type and the mean suprabasal IRIDI score was significantly highest in atrophic type and significantly lowest in follicular type (Table 4).
There was no statistically significant difference in the mean total IRIDI score for HSP60 expression, in different sites of LP (Table 5).
With regard to the histopathological features, there was statistically significant increase in the mean total IRIDI score in the presence of apoptotic bodies than in their absence. However, there was no statistically significant relationship with other histopathological features (Table 6).
LP is a relatively common papulosquamous dermatosis for which the exact etiology remains obscure despite considerable studies. However, significant evidence exist for an immunologically-mediated pathogenesis in which basal keratinocytes are destroyed by infiltrating T cells .
Either an autoimmune response to basal cell antigens or a perimmune response to antigens shared by microbial agents and basal cells, is believed to cause this T lymphocyte-mediated condition . Both CD4+ and CD8+ T cells are found in lesional skin of LP. T cell lines from donor skin also contain a distinctive population of γδ T cells that are not found in normal skin . This subtype of T lymphocytes is known to respond to antigenic portions of certain HSP molecules [4,13].
HSPs are classified into several families according to molecular weight, homology, and function. Human HSP60 has more than 50% homology with mycobacterial HSP65. HSP60 is recognized by γδ T cells that are distributed in epithelial linings and recognize foreign pathogens directly without the assistance of professional antigen-processing cells in the immune response .
This study found two patterns of cellular localization of HSP60 within the LP cases in positive cells of epidermis; the first was cytoplasmic perinuclear in most of LP cases. This pattern is in accordance with earlier studies [6,14]. Besides, another pattern was observed where HSP60 was present in the nucleus as well as in the cytoplasm. This nuclear pattern was elicited by Shah et al.  where they found that HSP60 was localized primarily in the nucleus. The nuclear localization could be involved in the premessenger RNA splicing within the nucleus. However, the significance of nuclear trafficking remains unclear. Thus, further studies investigating the role of nuclear HSP60 expression in the epidermis of LP cases are needed.
In this study, expression of HSP60 in LP patients and controls was done by a semiquantitative way of immunohistochemistry evaluating the IRIDI. It was found that the mean total, basal, and suprabasal IRIDI scores for HSP60 expression in LP cases were higher than those of controls, while mean IRIDI score was significantly lower in the superficial layer than that of the controls. In accordance with these results, Bayramgürler et al.  had shown upregulated expression of HSP60 in cutaneous LP when compared with normal skin in the total IRIDI score. However, they found that there was upregulation even in IRIDI scores of the three layers of the epidermis and not only in the basal and suprabasal layers, as shown in this study.
Chaiyarit et al.  have shown that the increased expression of HSP60 in oral LP, like the results of this study, was confined only to basal epithelial cells and not all epidermal layers. Similar results were reported by Bramanti et al.  and Sugerman et al.  in oral LP, who showed increased HSP60 expression in oral LP cases when compared with normal oral mucosa.
In this study, there was no statistically significant correlation of total, basal, and suprabasal IRIDI scores of HSP60 with respect to age of LP patients. Gandour-Edwards et al.  stated that the age did not influence HSP60 expression in the skin. However, in this study, statistically significant negative correlation was found between the mean superficial IRIDI score of HSP60 and age. This can be explained as with aging the expression of HSPs in tissues decreases as the capability of aged cells to express HSPs upon stress exposure is reduced .
In agreement with Bayramgürler et al. , in this study there was no statistically significant difference among the mean total IRIDI score of HSP60 in different clinical types of LP. The mean basal IRIDI score of HSP60 was lowest in follicular type. In addition, the mean suprabasal IRIDI score was lowest in follicular type and highest in atrophic type. No statistically significant difference in the mean total IRIDI score for HSP60 expression was found among the different sites of the lesions in LP patients. To the best of our knowledge, these correlations were not studied before. Relating the mean total IRIDI for HSP60 with histopathological features revealed that the only statistically significant association was found between total IRIDI score and apoptotic bodies where in absence of apoptotic bodies IRIDI score was very low (2.0±0.0). This may be explained by the fact that apoptotic bodies increase due to increase in basal cell destruction which results from increase in the inflammatory process in which HSP60 plays an important role .
In agreement with Bayramgürler et al.  it was found that the mean IRIDI score for HSP60 in the inflammatory cells (mainly lymphocytes) in the dermis (dermal infiltrate) correlated positively with the total epidermal IRIDI score.
Although, the significance of this finding to be specific for LP is questionable, because Bayramgürler et al.  found no significant alteration in levels of HSP60 protein between the cases of LP and psoriasis vulgaris. Whether these findings are primary in the pathogenesis of LP or only reflect secondary reactive changes induced by inflammation could not be determined.
Results of this study prove the relation between HSP60 and cutaneous LP. This relation may primarily be pathogenic or reflect secondary reactive changes . The primary role of HSP60 is explained by the molecular mimicry (epitope homology) between microbial and human HSPs . An immunologic cross reactivity between self-HSP and microbial epitopes may cause a direct upregulation of HSP expression by keratinocytes . An alternate speculation that can be made is that microbial agent could induce a cell-mediated immunity with T lymphocytes infiltration in the epidermis. Cytokines generated by these cells may upregulate HSPs expression in the adjacent basal keratinocytes. If a patient is predisposed to react to HSPs by certain human leucocytic antigens, a cytotoxic T lymphocyte response targeting basal keratinocytes resulting in tissue destruction develops [6,20]. Altered HSP60 in LP is probably a consequence of inflammatory stresses placed on the cell. Increased HSP60 protein in both epidermal cells and infiltrating inflammatory cells of LP might be partly explained by physical stress to skin, which induces HSP60 expression. However, precise determination whether HSP60 expression is primary or secondary is not possible.
In conclusion, according to these results a possible role for this protein exist in the pathogenesis of LP.
There is no conflict of interest to declare.
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Keywords:© 2011 Egyptian Women's Dermatologic Society
heat shock protein 60; immunoreactivity intensity distribution index score; lichen planus