Immunohistochemical detection of interferon-gamma-producing cells in dermatophytosis

ARTICLE

Although various components of the host-dermatophyte relationship have been explored, the immunological mechanisms involved in the prevention and control of dermatophytosis have not yet been investigated in detail. A delayed-type hypersensitivity (DTH) response to a dermatophyte antigen is one of the host defense mechanisms [1]. Th1 cytokines are involved in the elicitation of the DTH response, and interferon-gamma (IFN-gamma) is regarded as a major factor in the effector phase of the DTH reaction [2]. In recent studies, the production pattern of cytokines has been investigated in the DTH response in dermatophytosis [3-6]. IFN-gamma may be involved in the immune response of dermatophytosis, but the precise localization of IFN-gamma-producing cells in the skin lesion is not known.

A recently developed immunohistochemical technique was used to analyze cytokine expression in situ [7-10]. Pretreatment by several methods enabled the cellular ultrastrucure and protein antigenicity to be preserved. Immunohistochemical identification of IFN-gamma-producing cells in formalin-fixed, paraffin-embedded sections has been reported previously [7].

In this study, we detected, immunohistochemically, IFN-gamma-positive cells in situ at the protein level to obtain the histological evidence of lesional Th1 response in superficial dermatophyte infection.

Material and methods

Specimens and reagents

The subjects of this study were four patients with acute inflammatory tinea lesions in which the scales were KOH-positive. Their lesions were located on the neck, forearm, groin and thigh respectively. Trichophyton rubrum was cultured in one patient, Microsporum canis in one patient, and a causative dermatophyte was not identified in two patients. After informed consent had been obtained, skin biopsies were taken from the margins of scaly erythematous lesions. Formalin-fixed, paraffin-embedded sections were examined in this study.

Mouse monoclonal antibodies against CD1a (clone: O10, IgG1, Immunotech, Marseille, France), CD68 (clone: KP-1, IgG1, DAKO, Glostrup, Denmark), 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 recommendation. 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. 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, CD8, CD68 and CD1a. The sections were incubated with each primary antibody overnight at 37° C. We used new fuchsin as a substrate for 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. 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 IFN-gamma staining.

Results

Immunophenotypical characterization of inflammatory cells in the skin lesions of dermatophytosis

Histological examination of the skin lesions revealed acute inflammation with moderate cellular infiltration of the upper dermis. PAS-positive fungal hyphae were observed in the stratum corneum.

To identify T cells present in lesional skin of dermatophytosis, we performed immunohistochemistry using anti-CD4 and anti-CD8 antibodies. Mixtures of CD4-positive T cells and CD8-positive T cells were present in the dermal infiltrates of the lesions. CD8-positive T cells seemed to be dominant. Considerable amounts of CD1a-positive cells were detected in the upper dermis and epidermis (Fig. 1). A marked accumulation of CD68-positive cells was found in the upper dermis.

IFN-gamma-positive cells in the skin lesions of dermatophytosis

In the next experiment, we examined whether IFN-gamma-positive cells were present in the skin lesions of dermatophytosis in situ by immunohistochemical techniques. IFN-gamma-positive cells were found in the upper dermis of the lesions (Fig. 2). In all samples studied, anti-IFN-gamma antibody stained approximately 2 to 3% of mononuclear lymphoid cells. The pattern of IFN-gamma staining in the skin lesion appeared to be intracellular in mononuclear lymphoid cells (Fig. 3). IFN-gamma-positive cells were fairly uniform in size and often spherical with smooth contours. The cytoplasma of IFN-gamma-positive cells occasionally exhibited a fine granular appearance. The staining was considered to be highly specific because it could be completely blocked by preabsorption with recombinant IFN-gamma.

Discussion

The histological findings in our study indicated that the inflammatory dermal infiltrates of dermatophytosis mainly consisted of CD4-positive T cells and CD8-positive T cells, complemented by CD68-positive macrophages and CD1a-positive Langerhans cells. Similar findings have been obtained in previous studies on characterization of the phenotypes of infiltrating cell components in dermatophytosis [11, 12].

Skin lesions of dermatophytosis are thought to be a result of a T cell-dependent inflammatory response that is mediated by various cytokines. A recently developed immunohistochemical technique was used to analyze cytokine expression in situ [7-10]. IFN-gamma staining was detected in mononuclear lymphoid cells mainly present in the upper dermis of the lesions. IFN-gamma-positive cells were fairly uniform in size and often spherical with smooth contours resembling lymphocytes. Since it is now recognized that IFN-gamma is produced by CD4-positive T cells, CD4-positive T cells seem to be the dominant phenotype of cells detected in our study. A double staining method would be helpful to clarify the IFN-gamma positive cells. IFN-gamma staining can be identified at the single cell level, and the intracellular staining pattern was observed to be cytoplasmic. The cytoplasma of IFN-gamma-positive cells occasionally exhibited a fine granular appearance. Intracellular staining of IFN-gamma observed in the skin lesion is thought to be due to intracellular IFN-gamma production rather than IFN-gamma uptake [8].

In recent studies, the production pattern of cytokines has been investigated on the DTH response in dermatophytosis. We previously reported that peripheral blood mononuclear cells from patients with dermatophytosis produce a high level of IFN-gamma in response to stimulation with trichophytin [3, 4]. The presence of IFN-gamma mRNA in skin lesions of dermatophytosis has been detected by using reverse transcription-polymerase chain reaction [5, 6]. In agreement with these findings, we observed IFN-gamma-positive cells immunohistochemically in the upper dermis of the lesions. Our findings support the hypothesis that the skin lesions of dermatophytosis are associated with a Th1 response. The Th1 response, which is characterized by IFN-gamma release, is thought to be involved in the host defense against dermatophytes and to reflect cutaneous reaction in dermatophytosis.

In order to clarify the pathogenic significance of IFN-gamma-positive cells in dermatophytosis, further investigation of IFN-gamma-positive cells in other inflammatory skin disorders characterized by lymphocyte infiltration is needed.

In agreement with previous reports [11-14], we observed considerable numbers of CD1a-positive Langerhans cells in the upper dermis and epidermis of the dermatophyte lesions, supporting the notion of an antigen-presenting situation.

Langerhans cells have been shown to be capable of inducing a T cell response to trichophytin in dermatophytosis in vitro [15]. Our findings suggested that Langerhans cells are responsible for the uptake and processing of antigens in the lesional DTH response in superficial dermatophyte infection.

CONCLUSION

In conclusion, our comprehensive immunohistochemical detection of IFN-gamma-positive cells has provided histological evidence of lesional Th1 response in superficial dermatophyte infection.

Acknowledgements

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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