Secondary Logo

Journal Logo

Expression of heat shock protein 60 in cutaneous lichen planus: an immunohistochemical study

Attia, Abdallah M.a; Hammam, Mostafa A.a; Abd El-Wahed, Moshira M.b; Abd El-Wahed, Mohammeda; Anter, Azza G.a; Fawzy, Dalia M.a

Journal of the Egyptian Women's Dermatologic Society: January 2011 - Volume 8 - Issue 1 - p 43–49
doi: 10.1097/01.EWX.0000392816.83337.a4
Original Articles
Free

Background Lichen planus (LP) is a chronic inflammatory papulosquamous skin disease recognized as a T cell-mediated disease. Heat shock proteins (HSPs) are expressed by most living cells, and they play fundamental role in many biological processes. The expression of certain HSPs in oral LP was reported in many studies. However, the expression of certain HSPs in cutaneous LP was reported in only in few studies.

Objective To investigate the expression of HSP60 in lesions of cutaneous LP and compare it with normal skin to know if it has a role in LP pathogenesis or not.

Method Forty LP patients and 10 controls of apparently normal skin were studied. Skin biopsies were taken for hematoxylin and eosin examination and for the immunohistochemistry using mouse anti-HSP60 monoclonal antibody. An immunoreactivity intensity distribution index (IRIDI) was calculated for each case and control.

Results The mean total IRIDI scores for HSP60 expression in epidermal layers and cells of the inflammatory infiltrate (mostly lymphocytes) of cutaneous LP showed statistically significantly increase than those of normal skin (P=0.001). The mean IRIDI score for HSP60 expression in basal and suprabasal layers of LP patients also showed statistically significantly increase than those of normal skin (P=0.001 and P=0.05, respectively). The mean IRIDI score for HSP60 expression in the superficial epidermal layer of LP patients was statistically lower than that of normal skin (P=0.001). A positive statistically significant correlation was found in LP patients between the mean IRIDI score for the dermal infiltrate and the mean total epidermal IRIDI score.

Conclusion HSP60 could have a role in cutaneous LP pathogenesis.

aDepartments of Pathology

bDermatology and Andrology S.T.Ds, Menoufiya University, Egypt

Correspondence to Mohamed Abd El-Wahed, Assistant Professor, Department of Dermatology and Andrology S.T.Ds, Menoufiya University, Egypt Tel: +2 012 220 7617; fax: +2 048 2326810; e-mail: drmohamedgaber2000@yahoo.com

Received January 4, 2009

Accepted April 30, 2009

Back to Top | Article Outline

Introduction

Lichen planus (LP) is a mucocutaneous disease of inflammatory nature. Many clinical and immunohistochemical studies suggest the immunologic basis for LP [1]. The underlying mechanism can be a T cell-mediated immune response against foreign or autogenous antigens in which presentation of antigens by Langerhan's cells to T helper cells, elaboration of cytokines to activated T cells, and induction of cytokine production by keratinocytes occurs [2].

Heat shock proteins (HSPs) are certain proteins expressed in all cells known for the maintenance of cellular homeostasis and are inducible under variety of stresses, including oxidative injury and infection with intracellular antigens, to enable cellular survival. They are mainly involved in the proper folding of other proteins and hence referred to as molecular chaperons. The accumulation of HSPs is seen in pathological conditions and tumors [3]. Although, they are induced by any form of stress, it is known that HSPs are expressed in normal (unstressed) cells playing a fundamental role in many biological processes such as control of apoptosis, protection against ultraviolet damage, wound healing, regulation of steroid aporeceptors, kinases, and other protein remodeling processes [4].

Heat shock proteins 60 (HSP60) belongs to multigene families that range in molecular size from 10 to 150 kDa and are found in all major cellular compartments and are subdivided into groups based on their molecular weight (e.g. HSP60 is a 60 kDa protein) [5]. Although it is known that they play an important role in immune and inflammatory responses of the skin, the role of HSPs in the pathogenesis of skin diseases has been studied in only limited skin diseases such as psoriasis, atopic dermatitis, systemic lupus erythematosus, graft versus host disease, Behçet's disease, and palmoplantar pustulosis [6].

Heat Shock proteins 60 has primarily been known as a mitochondrial protein that is important for folding key proteins after being imported into the mitochondria, it is now clear that a significant amount of it is also present in the extra-mitochondrial cytosol of many cells [7]. HSP60 can be recognized by γδ T cells, which are distributed in the epithelial linings and recognizes foreign pathogens directly without the assistance of professional antigen-processing cells in the immune response [8]. Although an altered expression of certain HSPs was frequently reported in oral LP lesion, its expression in cutaneous LP was reported only in one study [6]. Therefore, the aim of this study was to investigate the expression of HSP60 in cutaneous LP lesion and compare its expression in normal skin trying to know if HSP60 has a role in LP pathogenesis or not.

Back to Top | Article Outline

Patients and methods

This study was carried out on 40 patients with LP and 10 healthy individuals as a control group. Patients were selected from the Dermatological Outpatient Clinic, Faculty of Medicine, Menoufiya University.

Back to Top | Article Outline

Methods

The patients were subjected to the following

  • (1) Complete history with special stress to age, sex, occupation, family history, complaint, duration of disease, predisposing factors such as prolonged exposure to sun, trauma, radiation, and presence of complications, and earlier treatment;
  • (2) Clinical examination: complete general and local examination of the patients; the local examination includes: site, size, shape and configuration of the lesions, types (clinical variants);
  • (3) Skin biopsy: the lesional area was biopsed after taking a written informed consent from the patients. In addition, biopsies of normal skin from the control group were taken for histopathological examination.

One section was stained with hematoxylin and eosin for histopathological examination and another two sections were used for immunohistochemical examination (HSP60).

Immunohistochemical staining: paraffin embedded 4 μm tissue sections were deparaffinized with xylene, dehydrated in graded series of ethanol and then incubated with 3% hydrogen peroxide. Slides were rinsed in phosphate buffered saline and then exposed to heat induced epitope retrieval in citrate buffer solution for 20 min. After cooling, the slides were incubated with an epitope specific purified monoclonal mouse anti-HSP60 antibody clone LK-1 (1–50) (SPA-806, Biomol, Stressgen Bioreagent, USA) overnight. Detection of HSP60 was achieved using ultravision detection system ready to use anti-polyvalent horseradish peroxidase-diaminodbenzidine (Labvision Corporation, cat no: TM-015-HA, Neomarker, USA). Finally, the reaction was visualized by appropriate substrate(3,3′-diaminodbenzidine) reagent. Counter stain was done using Mayer's hematoxylin (Bio Genex, Cat No.: 94583, UK).

Back to Top | Article Outline

Interpretation of immunohistochemistry

The method used in this study for immunohistochemical analysis was a semiquantitative way of immunohistochemical evaluation.

All the epidermal cells were counted and included into the immunohistochemical scoring. On microscopic examination, the epidermis was divided into three layers as basal, suprabasal, and superficial. Each layer was evaluated for the proportion of immunoreactive cells and their staining intensity.

The proportion of the immunoreactive cells in each layer was assessed as: 0=no immunoreactive cells; 1=less than 25% of the cells are immunoreactive; 2=25–50% of the cells are immunoreactive; 3=more than 50% of the cells are immunoreactive.

The staining intensity of the cells was graded as: 0=no staining; 1=light staining; 2=moderate staining; 3=intense staining.

An immunoreactivity intensity distribution index (IRIDI) was calculated for each case and controls as follows: the score of the staining intensity for each layer was multiplied by the score of the proportion of the immunoreactive cells in that layer to express the IRIDI for that layer. The resulting scores of the three layers were then added to provide the total IRIDI for each case and control [6].

Back to Top | Article Outline

Statistical analysis

Data were collected, tabulated, and statistically analyzed using the statistical package for the social sciences (SPSS) version 12 (Cherry Hill, New Jersey, USA) on IBM compatible computer, Kruskal–Wallis tests, Student's t-test, and Pearson's correlation analysis. Differences were considered statistically significant with a P value of less than 0.05 and highly significant with a P value of less than 0.001 [9].

Back to Top | Article Outline

Results

This study included 50 individuals categorized into two groups

  • (1) Group I (LP): 40 patients clinically diagnosed and histopathologically confirmed as cutaneous LP. They were 21 males (52.5%) and 19 females (47.5%). Their age ranged from 10 to 9 years, with a mean±standard deviation (SD) of 33.68±15.07 years. Demographical and histopathological features are shown in Tables 1 and 2, respectively;
  • (2) Group II (control): 10 individuals with apparently healthy skin serving as controls. They were five females (50%) and five males (50%). Their age ranged from 10 to 68 years, with a mean±SD of 37.5±18.92 years.
Table 1

Table 1

Table 2

Table 2

Back to Top | Article Outline

Immunohistochemical results

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).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 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).

Table 3

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).

Table 4

Table 4

There was no statistically significant difference in the mean total IRIDI score for HSP60 expression, in different sites of LP (Table 5).

Table 5

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).

Table 6

Table 6

Back to Top | Article Outline

Discussion

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 [10].

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 [11]. 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 [12]. 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 [8].

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. [15] 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. [6] 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. [16] 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. [11] and Sugerman et al. [17] 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. [18] 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 [19].

In agreement with Bayramgürler et al. [6], 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 [6].

In agreement with Bayramgürler et al. [6] 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. [6] 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 [6]. The primary role of HSP60 is explained by the molecular mimicry (epitope homology) between microbial and human HSPs [4]. An immunologic cross reactivity between self-HSP and microbial epitopes may cause a direct upregulation of HSP expression by keratinocytes [16]. 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.

Back to Top | Article Outline

References

1. El-Zefzaf E, Abd El-Razzak M, Ghoneim S, Abu-Azma N. Demonstration of T-cell subsets in oral lichen planus. Egypt Dent J. 1994;40:633–638
2. Santoro A, Majorana A, Bardellini E, Festa S, Sapelli P, Facchetti F. NF-kappa β expression in oral and cutaneous lichen planus. J Pathol. 2003;201:466–472
3. Paul MK, Mukhopadhyay AK. Tyrosine kinase-role and significance in cancer. Int J Med Sci. 2004;1:101–115
4. Morris SD. Heat shock proteins and the skin. Clin Exp Dermatol. 2002;27:220–224
5. Xu Q. Infections, heat shock proteins, and atherosclerosis. Curr Opin Cardiol. 2003;18:245–252
6. Bayramgürler D, Özkara SK, Apaydin R, Ercin C, Bilen N. Heat shock proteins 60 and 70 expression of cutaneous lichen planus: comparison with normal skin and psoriasis vulgaris. J Cutan Pathol. 2004;31:586–594
7. Gupta S, Knowlton AA. HSP 60, Bax, apoptoris and the heart. J Cell Mol Med. 2005;9:51–58
8. Nishioka K, Groreishi M, Yokozeki H. Heat shock proteins and skin diseases. Curr Opin Infect Dis. 1999;12:171
9. Petrie A, Sabin C Medical statistics at a glance. 20001st ed Oxford Blackwell:52–69
10. Daoud MS, Pittellkow MRFreedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI. Lichen planus. Fitzpatrick's dermatology in general medicine. 20036th ed New York: McGraw-Hill Professional:464–477
11. Bramanti TE, Dekker NP, Lozada-Nur F, Sauk JJ, Regezi JA. Heat shock(stress) proteins and gamma delta T lymphocytes in oral lichenplanus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995;80:698–704
12. Gadenne AS, Strucke R, Dunn D, Wagner M, Bleicher P, Bigby M. T-cell lines derived from lesional skin of lichen planus patients contain a distinctive population of T-cell receptor gamma delta-bearing cells. J Invest Dermatol. 1994;103:347–351
13. Hirsh MI, Junger WG. Roles of heat shock proteins and gamma delta T cells in inflammation. Am J Respir Cell Mol Biol. 2008;39:509–513
14. Soltys BJ, Gupta RS. Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp Cell Res. 1996;222:16–27
15. Shah M, Stanek J, Handwerger S. Differential localization of heat shock proteins 90, 70, 60 and 27 in human decidua and placenta during pregnancy. Histochem J. 1998;30:509–518
16. Chaiyarit P, Kafrawy AH, Miles DA, Zunt SL, Van Dis ML, Gregory RL. Oral lichen planus: an immunohistochemical study of heat shock proteins (HSPs) and cytokeratins (CKs) and a unifying hypothesis of pathogenesis. J Oral Pathol Med. 1999;28:210–215
17. Sugerman PB, Savage NW, Xu LJ, Walsh LJ, Seymour GJ. Heat shock protein expression in oral lichen planus. J Oral Pathol Med. 1995;24:1–8
18. Gandour-Edwards R, McClaren M, Isseroff RR. Immunolocalization of low-molecular-weight stress protein HSP 27 in normal skin and common cutaneous lesions. Am J Dermatopathol. 1994;16:504–509
19. Soti C, Csermely P. Chaperones and aging: role in neurodegeneration and in other civilizational diseases. Neurochem Int. 2002;41:383–389
20. Shiohara T, Mizukawa Y, Takahashi R, Kano Y. Pathomechanisms of lichen planus autoimmunity elicited by cross-reactive T cells. Curr Dir Autoimmun. 2008;10:206–226
Keywords:

heat shock protein 60; immunoreactivity intensity distribution index score; lichen planus

© 2011 Egyptian Women's Dermatologic Society