In situ localization of IFN-gamma-positive cells in psoriatic lesional epidermis

ARTICLE

Skin lesions of psoriasis are thought to be a result of immune response that is mediated by various cytokines [1]. Accumulated evidence from both in vivo and in vitro studies show that interferon-gamma (IFN-gamma) is a critical element in the induction of keratinocyte hyperproliferation in psoriasis [2-4]. Psoriasis is accompanied by an infiltration of activated T cells in the papillary dermis and the epidermis. The presence and potential functional importance of T cells in the epidermis was emphasized in the pathogenesis of psoriasis. By using flow cytometry with intracellular staining, T cells freshly isolated directly from psoriatic lesional epidermis were found to be capable of producing IFN-gamma after stimulation with ionomycin/phorbol myristate acetate [5, 6]. This finding indicates IFN-gamma-producing potential rather than ongoing production of IFN-gamma by individual epidermal T cells in psoriasis. However, it is not known whether psoriatic epidermal T cells actually produce and secrete IFN-gamma within the lesion.

A recently developed immunohistochemical technique was used to analyze cytokine expression in situ [7-11]. Pretreatment by several methods enabled the cellular ultrastructure and protein antigenicity to be preserved, and immunohistochemical identification of IFN-gamma producing cells in formalin-fixed, paraffin-embedded sections has been reported previously [7, 8].

In this study, we detected IFN-gamma-positive cells immunohistochemically in situ at the protein level to obtain histological evidence of epidermal IFN-gamma-producing T cells in psoriasis.

Materials and methods

Specimens and reagents

The subjects were five patients with psoriasis vulgaris (chronic plaque-type) and six patients with pustular psoriasis (three patients with generalized-type and three patients with localized-type), all of whom had been referred to Kyushu University Hospital. All patients were untreated for at least two weeks. The lesional skins were biopsied after obtaining informed consent from the patients. Formalin-fixed, paraffin-embedded sections were used for examination in this study.

Mouse monoclonal antibodies against CD8 (clone: C8/144B, IgG1, Nichirei, Tokyo, Japan), CD4 (clone: 1F6, IgG1, Nichirei) and IFN-gamma (clone: 25718.11, IgG2a, Genzyme/Techne, USA) were used as primary antibodies. Control mouse IgG1 and IgG2a were purchased from Pharmingen, San Diego, CA, USA.

Immunohistochemistry

The sections were stained using a Histofine SAB-AP kit (Nichirei) according to the manufacturer's instruction. Antigen retrieval was achieved by several methods. The skin sections were pretreated with 0.1% trypsin for 60 min at 37°C for immunostaining for IFN-gamma [8]. Heat pretreatment was performed by incubating the sections in 10 mM citrate buffer (pH 6.0) in a pressure cooker at 110° C for 5 min for immunostaining for CD4 and CD8 [12]. The sections were incubated with each primary antibody overnight at 37° C. New fuchsin was used as a substrate for the Histofine SAB-AP kit, and the sections were then counterstained with hematoxylin. The isotype-matched mouse antibodies always showed negative staining as a negative control. Blocking experiments were performed to test the specificity of IFN-gamma staining. The anti-IFN-gamma antibody was incubated with a 10-fold excess of recombinant IFN-gamma (Genzyme/Techne) at 4° C overnight and was used for immunohistochemical staining.

Results

Immunophenotypical characterization of inflammatory cells in the psoriatic lesional epidermis

To identify T cells present in the lesional epidermis of psoriasis, we performed immunohistochemistry using anti-CD4 and anti-CD8 antibodies. Mixtures of CD4⁺ T cells and CD8⁺ T cells were found to be present in the papillary dermis and the epidermis of psoriasis. CD8⁺ T cells seemed to be dominant in the epidermis (Fig. 1).

IFN-gamma-positive cells in the psoriatic lesional epidermis

Next, the possible presence of IFN-gamma-positive cells in the lesions of psoriasis was investigated in situ using immunohistochemical techniques. Considerable amounts of IFN-gamma-positive cells were detected in the papillary dermis of the lesions (Fig. 2). The pattern of IFN-gamma staining in the dermis appeared to be intracellular in mononuclear lymphoid cells. The cytoplasm of IFN-gamma-positive cells occasionally exhibited a fine granular appearance.

IFN-gamma-positive cells were also detected in the epidermis of the lesions. The pattern of IFN-gamma staining in the epidermis appeared to be a combination of intracellular staining in mononuclear lymphoid cells (Fig. 3) and extracellular deposition in the surrounding areas (Fig. 4). Although there was some variation between patients, IFN-gamma-positive cells in the epidermis were obvious in the lesions of pustular psoriasis.

The staining was considered to be highly specific because it could be completely blocked by preabsorption with recombinant IFN-gamma.

Discussion

IFN-gamma is believed to be an important mediator in psoriasis. Accumulated evidence from both in vivo and in vitro studies show that IFN-gamma is a critical element in the induction of keratinocyte hyperproliferation in psoriasis [2-4]. IFN-gamma has been detected in psoriatic lesions by immunohistochemical methods [13, 14] and polymerase chain reaction [15, 16]. Cloned T cells from psoriatic lesions showed IFN-gamma production [16, 17]. IFN-gamma-producing mast cells are significantly increased in the psoriatic skin [18], and expression of interleukine-12 (IL-12) on mononuclear cells is increased in the psoriatic skin [19]. IL-12 promotes IFN-gamma secretion by T cells.

Epidermal T cells are currently considered to be important in the pathogenesis of psoriasis. Successful treatment of psoriasis is accompanied by selective depletion of intraepidermal T cells [20]. Recent progress in the identification of individual cells and their functions by intracellular cytokine staining has enabled characterization of T cells isolated from the psoriatic lesional epidermis. By using flow cytometry with intracellular staining, T cells in the psoriatic epidermis have been analyzed at the single cell level, and IFN-gamma-producing capability after stimulation with ionomycin/phorbol myristate acetate has been shown [5, 6]. However, these findings do not show whether there is actual ongoing production of IFN-gamma by individual epidermal T cells in psoriasis. Therefore, the aim of the present study was to determine in situ localization of IFN-gamma-producing epidermal T cells from psoriasis.

It was previously reported that IFN-gamma was detected immunohistochemically in the mononuclear cell infiltrate in some endothelial cells of the papillary dermis and in the stratum corneum and in some keratinocytes around microabscesses of psoriatic lesions by using frozen samples [12]. However, it could not be determined morphologically whether epidermal T cells were actually positive by IFN-gamma staining. In our study, IFN-gamma was detected immunohistochemically in epidermal T cells in psoriatic lesions by using formalin-fixed, paraffin-embedded skin biopsy sections. A series of preliminary experiments was done to optimize the antigen retrieval system for IFN-gamma staining, and pretreatment with trypsin was found to be effective in our study. Thus, it was found that stained cells are more morphologically identifiable in paraffin-embedded samples than in frozen samples.

IFN-gamma staining could be identified at the single-cell level by the immunohistochemical procedures used in this study. Since it is recognized that IFN-gamma is produced mainly by T cells, T cells seem to be the dominant phenotype of IFN-gamma staining cells detected in our study. A double staining method would be helpful to clarify the IFN-gamma positive cells. The pattern of IFN-gamma staining in the epidermis appears to be a combination of intracellular staining in mononuclear lymphoid cells (Fig. 3) and extracellular deposition in the surrounding areas (Fig. 4). These staining patterns resembled those of cytokine-producing cells in human tissues reported previously [9, 10]. Intracellular staining of IFN-gamma seen in the mononuclear lymphoid cells reflects intracellular IFN-gamma production rather than IFN-gamma uptake [9]. Probably, extracellular staining of IFN-gamma observed in our study may be produced by supposed T cells.

Locally produced IFN-gamma from epidermal T cells may have profound effects on keratinocytes in psoriasis. Class II major histocompatibility complex proteins and intercellular adhesion molecule-1 may be induced on keratinocytes by IFN-gamma produced by epidermal T cells. The presence of IFN-gamma-producing T cells within the hyperplastic epidermis is likely to stimulate the growth of keratinocyte in vivo.

There is little information available regarding IFN-gamma-producing epidermal T cells in other skin diseases, and we cannot say whether the observed frequency of IFN-gamma-producing epidermal T cells in psoriasis patients represents a high or a low level. Our preliminary data showed that very few IFN-gamma-positive cells were found in the epidermis of chronic eczema and lichen planus. Further study is needed to clarify the functional implications of these cells in the pathogenesis of psoriasis.

Article accepted on 31/7/01

CONCLUSION

Acknowledgements

We thank Mr. T. Kanemaru at Morphology Core, Faculty of Medicine, Kyushu University for his help. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, and also from the Ministry of Health and Welfare of Japan. Dr. H. Duan is kindly supported by a postdoctoral fellowship program for foreign researchers of Japan Society for the Promotion of Science.

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