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Candida albicans induced interleukin 8 production by human keratinocytes
Journal of Dermatological Science (2003) 31, 233 /235
www.elsevier.com/locate/jdermsci
LETTER TO THE EDITOR Candida albicans induced interleukin production by human keratinocytes
8
To the Editor Candida albicans is the most common cause of superficial and systemic candidiasis. The characteristic pathological feature of cutaneous candidiasis is an infiltration of numerous neutrophils within the epidermis, especially beneath the stratum corneum where the fungal organisms are present. However, the precise mechanism and cause of neutrophilic infiltration within the epidermis is unknown at present, and no detailed interaction between yeast cells of C. albicans and human keratinocytes has not been studied. In this study, interleukin (IL-) 1b, 6 and 8, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF)-a levels in the medium where keratinocytes were co-cultured with C. albicans were determined by enzyme-linked immunosorbent assay (ELISA) in order to estimate the effect of C. albicans on the cytokines production by human keratinocytes. Three strains of C. albicans (TLD-0141, 0142 and 0143) used in this study, were clinical isolates originated from a cutaneous lesion of human candidiasis. C. albicans were harvested from the growth in Sabouraud’s liquid medium at 37 8C for 3 days. These yeast cells were washed twice in phosphate-buffered saline (PBS) and divided into two samples. The first sample was heat killed at 60 8C for 30 min. Both live and killed yeast cells were resuspended in serum-free keratinocyte growth medium of Clonetics (BioWhittaker, San Diego, CA), respectively. Normal human epidermal keratinocytes (NHEK) were obtained as cryopreserved first passage cells from Clonetics. Co-culture of NHEK with C. albicans was carried out according to the method by Watanabe et al. [1]. Cytoscreen Immunoassay kits (BioSource International, CA) was used for ELISA of cytokines and they were used according to the instruction manuals. After co-culture of NHEK with live or killed C. albicans for 1 /14 h, IL-1, IL-6, IL-8, MCP-1, and
TNF-a protein levels in the supernatants were determined by ELISA (Fig. 1a and b). IL-8 was detected in the supernatant and increased following co-culture, whereas the other cytokines (IL-1, IL-6, MCP-1, and TNF-a protein) were low or undetectable. IL-8 was detected at 3 h after NHEK were incubated with culture supernatants of C. albicans , and its level increased at 6 h after culturing (Fig. 1c), whereas the other cytokines could not be detected. After co-culture of keratinocytes with live C. albicans for 1 /6 h, IL-8 mRNA in the keratinocytes was determined by RT-PCR. IL-8 mRNA was detected in the keratinocyte co-cultured for 1 /6 h while undetectectable before co-cultured (Fig. 2). In the controls of culture supernatant of keratinocyte alone, keratinocyte lysate and C. albicans culture supernatant, IL-1b, IL-6, IL-8, MCP-1 and TNF-a were not detected at any time after culturing. LDH release as a measure of cell vitality was determined in supernatant of C. albicans with keratinocyte by the Cytotoxicity detection kit (LDH) (Roche, Mannheim, Germany). Growth and viability of NHEK, as assessed by the LDH assay and trypan blue exclusion tests, did not vary significantly and NHEK remained viable throughout the 14 h co-culture period. An increase of dead or damage cells in number resulted in an increase of LDH activity in the culture supernatant. Cytokines play important roles in inflammation and immunologic reactions. The data in this study revealed that C. albicans , depending on the numbers of yeast cells induced directly IL-8 production from human keratinocytes without activated macrophages. In addition, C. albicans could not induce the other cytokines examined in this study. These results suggested that IL-8 could be a main cytokine, released by keratinocytes in the infection sites of C. albicans . Since IL-8 is a potent chemoattractant for neutrophils, its production from keratinocytes infected by C. albicans may reflect the histopathological features of cutaneous candidiasis, which is characterized by prominent neutrophil infiltration in the epidermis, especially beneath the stratum corneum where the fungal organisms are present with or without a few
0923-1811/03/$30.00 – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0923-1811(03)00043-4
234
Letter to the Editor
Fig. 2 IL-8 mRNA production in human keratinocytes co-cultured with C. albicans . After co-culture of keratinocytes (5 /105 cells) with live C. albicans (5 /105) for 0, 1, 3 and 6 h, IL-8 mRNA levels in the keratinocytes were determined by RT-PCR. RT-PCR primers were prepared based on the sequences conserved in human IL-8 [2]. The primer sequences used for amplification of human IL-8 were 5?-ATGACTTCCAAGCTGGCCGT-3? (primer IL-8 1S; nt. 102 /122 in human mRNA for MDNCF in the Gene Bank accession nos.-Y00787) and 5?TCCTTGGCAAAACTGCACCT-3? (primer IL-8 1R; nt. 164 / 183). The PCR amplification was carried out for 30 cycles consisting of template denaturation (1 min, at 94 8C), primer annealing (1 min, at 55 8C) and polymerization (2 min, at 72 8C). Reaction products were run on 2% agarose gels. Although IL-8 mRNA in keratinocytes is undetectable at 0 h of co-culture, IL-8 mRNA was detected in the keratinocyte after 1 /6 h of co-culture. Ubiquitin is positive at transcriptional control.
Fig. 1 Effect of live, killed and culture supernatant of C. albicans on cytokine production by human keratinocytes. (a) After co-culture of keratinocytes (1 /104 cells per well) with the live C. albicans (1 /103 or 1/104 cells per well) for 1, 3, 6 and 14 h, IL-8 levels in the supernatants were measured by ELISA method. (b) After co-culture of keratinocytes with the killed C. albicans (1 /103 or 1/104 cells per well) for 1, 3, 6 and 14 h, IL8 levels in the supernatants were measured by ELISA method. (c) C. albicans (1 /108) were cultured in 10 ml serum-free keratinocyte growth medium (Clonetics) at 37 8C for 14 h. The culture medium was centrifuged at 300 /g for 5 min to remove the cells. The supernatant was harvested and filtrated by 0.22 mm of cellulose acetate filter. The keratinocytes (1 /104 cells) were incubated with 300 ml of the supernatant at 37 8C/5% CO2. After 1,3 and 6 h, the culture the supernatants were obtained and kept at /20 8C until used for cytokine assay. Results shown are representative of five experiments. In the control of culture supernatant keratinocyte alone, IL-8 proteins were undetectable at any time after culturing.
monocytes or lymphocytes [3]. In contrast, pustule formation in lesions of tinea versicolor and tinea corporis are not frequently in the epidermis. In previous study, these cytokines in medium where keratinocytes were co-cultured with Malassezia yeasts were determined by ELISA [1]. The culture supernatants of Malassezia yeasts could not induce any cytokine production [1]. On the other hand, the IL-8 level was increased in the supernatant where the keratinocytes were cultured with the culture supernatant of C. albicans for 3 /6 h. These results suggested that C. albicans might release stimulating factors to induce IL-8 production from keratinocytes. The stimulating factors released by C. albicans should be identified to control the inflammatory responses to the fungal infection. Since mannose receptor on human keratinocytes was reported to relate to the IL-8 production [4], mannose of C. albicans could be one of the stimulating factors. Although IL-8 levels were lower in the supernatant co-cultured with killed C. albicans than with live C. albicans , killed C. albicans could also induce IL-8 production by keratinocytes. Moreover, when NHEK were co-cultured with live C. albicans , the yeast cells produced hyphae, which might dramatically exert a pathological action, suggest-
Letter to the Editor
235
ing that physical contact might be influential to cause IL-8 production by keratinocyte. Further analysis is required to understand the process of IL-8 production by keratinocytes and the relation between C. albicans infection and the host defense.
References [1] Watanabe S, Kano R, Sato H, Nakamura Y, Hasegawa A. The effect of Malassezia yeasts on cytokine production by human keratinocytes. J Invest Dermatol 2001;116:769 /73. [2] Matsushima K, Morishita K, Yoshimura T, Lavu S, Kobayashi Y, Lew W, Appella E, Kung HF, Leonard EJ, Oppenheim JJ. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med 1988;167:1883 /93. [3] Longley BJ. Fungal disease. In: Elder D, editor. Lever’s Histopathology of the Skin, eighth ed. Philadelphia: Lippincott-Raven Publishers, 1997:517 /51. [4] Szolnoky S, Bata-Cso ¨rgo ¨ Z, Kenderessy AS, Kiss M, Pivarci A, Nova ´k Z, Newman KN, Michel G, Ruzicka T, Maro ´di L, Dobozy A, Keme ´ny L. A mannose-binding receptor is expressed on
.
human keratinocytes and mediates killing of Candida albicans . J Invest Dermatol 2001;117:205 /13.
Rui Kano, Atsuhiko Hasegawa Department of Pathobiology, Nihon University School of Veterinary Medicine, 1866, Kameino, Fujisawa, Kanagawa 252-8510, Japan E-mail address: [email protected] Shinichi Watanabe, Hiroko Sato, Yuka Nakamura Department of Dermatology, Teikyo University School of Medicine, 11-1, Kaga-2, Itabashiku, Tokyo 173-8605, Japan
www.elsevier.com/locate/jdermsci
LETTER TO THE EDITOR Candida albicans induced interleukin production by human keratinocytes
8
To the Editor Candida albicans is the most common cause of superficial and systemic candidiasis. The characteristic pathological feature of cutaneous candidiasis is an infiltration of numerous neutrophils within the epidermis, especially beneath the stratum corneum where the fungal organisms are present. However, the precise mechanism and cause of neutrophilic infiltration within the epidermis is unknown at present, and no detailed interaction between yeast cells of C. albicans and human keratinocytes has not been studied. In this study, interleukin (IL-) 1b, 6 and 8, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF)-a levels in the medium where keratinocytes were co-cultured with C. albicans were determined by enzyme-linked immunosorbent assay (ELISA) in order to estimate the effect of C. albicans on the cytokines production by human keratinocytes. Three strains of C. albicans (TLD-0141, 0142 and 0143) used in this study, were clinical isolates originated from a cutaneous lesion of human candidiasis. C. albicans were harvested from the growth in Sabouraud’s liquid medium at 37 8C for 3 days. These yeast cells were washed twice in phosphate-buffered saline (PBS) and divided into two samples. The first sample was heat killed at 60 8C for 30 min. Both live and killed yeast cells were resuspended in serum-free keratinocyte growth medium of Clonetics (BioWhittaker, San Diego, CA), respectively. Normal human epidermal keratinocytes (NHEK) were obtained as cryopreserved first passage cells from Clonetics. Co-culture of NHEK with C. albicans was carried out according to the method by Watanabe et al. [1]. Cytoscreen Immunoassay kits (BioSource International, CA) was used for ELISA of cytokines and they were used according to the instruction manuals. After co-culture of NHEK with live or killed C. albicans for 1 /14 h, IL-1, IL-6, IL-8, MCP-1, and
TNF-a protein levels in the supernatants were determined by ELISA (Fig. 1a and b). IL-8 was detected in the supernatant and increased following co-culture, whereas the other cytokines (IL-1, IL-6, MCP-1, and TNF-a protein) were low or undetectable. IL-8 was detected at 3 h after NHEK were incubated with culture supernatants of C. albicans , and its level increased at 6 h after culturing (Fig. 1c), whereas the other cytokines could not be detected. After co-culture of keratinocytes with live C. albicans for 1 /6 h, IL-8 mRNA in the keratinocytes was determined by RT-PCR. IL-8 mRNA was detected in the keratinocyte co-cultured for 1 /6 h while undetectectable before co-cultured (Fig. 2). In the controls of culture supernatant of keratinocyte alone, keratinocyte lysate and C. albicans culture supernatant, IL-1b, IL-6, IL-8, MCP-1 and TNF-a were not detected at any time after culturing. LDH release as a measure of cell vitality was determined in supernatant of C. albicans with keratinocyte by the Cytotoxicity detection kit (LDH) (Roche, Mannheim, Germany). Growth and viability of NHEK, as assessed by the LDH assay and trypan blue exclusion tests, did not vary significantly and NHEK remained viable throughout the 14 h co-culture period. An increase of dead or damage cells in number resulted in an increase of LDH activity in the culture supernatant. Cytokines play important roles in inflammation and immunologic reactions. The data in this study revealed that C. albicans , depending on the numbers of yeast cells induced directly IL-8 production from human keratinocytes without activated macrophages. In addition, C. albicans could not induce the other cytokines examined in this study. These results suggested that IL-8 could be a main cytokine, released by keratinocytes in the infection sites of C. albicans . Since IL-8 is a potent chemoattractant for neutrophils, its production from keratinocytes infected by C. albicans may reflect the histopathological features of cutaneous candidiasis, which is characterized by prominent neutrophil infiltration in the epidermis, especially beneath the stratum corneum where the fungal organisms are present with or without a few
0923-1811/03/$30.00 – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0923-1811(03)00043-4
234
Letter to the Editor
Fig. 2 IL-8 mRNA production in human keratinocytes co-cultured with C. albicans . After co-culture of keratinocytes (5 /105 cells) with live C. albicans (5 /105) for 0, 1, 3 and 6 h, IL-8 mRNA levels in the keratinocytes were determined by RT-PCR. RT-PCR primers were prepared based on the sequences conserved in human IL-8 [2]. The primer sequences used for amplification of human IL-8 were 5?-ATGACTTCCAAGCTGGCCGT-3? (primer IL-8 1S; nt. 102 /122 in human mRNA for MDNCF in the Gene Bank accession nos.-Y00787) and 5?TCCTTGGCAAAACTGCACCT-3? (primer IL-8 1R; nt. 164 / 183). The PCR amplification was carried out for 30 cycles consisting of template denaturation (1 min, at 94 8C), primer annealing (1 min, at 55 8C) and polymerization (2 min, at 72 8C). Reaction products were run on 2% agarose gels. Although IL-8 mRNA in keratinocytes is undetectable at 0 h of co-culture, IL-8 mRNA was detected in the keratinocyte after 1 /6 h of co-culture. Ubiquitin is positive at transcriptional control.
Fig. 1 Effect of live, killed and culture supernatant of C. albicans on cytokine production by human keratinocytes. (a) After co-culture of keratinocytes (1 /104 cells per well) with the live C. albicans (1 /103 or 1/104 cells per well) for 1, 3, 6 and 14 h, IL-8 levels in the supernatants were measured by ELISA method. (b) After co-culture of keratinocytes with the killed C. albicans (1 /103 or 1/104 cells per well) for 1, 3, 6 and 14 h, IL8 levels in the supernatants were measured by ELISA method. (c) C. albicans (1 /108) were cultured in 10 ml serum-free keratinocyte growth medium (Clonetics) at 37 8C for 14 h. The culture medium was centrifuged at 300 /g for 5 min to remove the cells. The supernatant was harvested and filtrated by 0.22 mm of cellulose acetate filter. The keratinocytes (1 /104 cells) were incubated with 300 ml of the supernatant at 37 8C/5% CO2. After 1,3 and 6 h, the culture the supernatants were obtained and kept at /20 8C until used for cytokine assay. Results shown are representative of five experiments. In the control of culture supernatant keratinocyte alone, IL-8 proteins were undetectable at any time after culturing.
monocytes or lymphocytes [3]. In contrast, pustule formation in lesions of tinea versicolor and tinea corporis are not frequently in the epidermis. In previous study, these cytokines in medium where keratinocytes were co-cultured with Malassezia yeasts were determined by ELISA [1]. The culture supernatants of Malassezia yeasts could not induce any cytokine production [1]. On the other hand, the IL-8 level was increased in the supernatant where the keratinocytes were cultured with the culture supernatant of C. albicans for 3 /6 h. These results suggested that C. albicans might release stimulating factors to induce IL-8 production from keratinocytes. The stimulating factors released by C. albicans should be identified to control the inflammatory responses to the fungal infection. Since mannose receptor on human keratinocytes was reported to relate to the IL-8 production [4], mannose of C. albicans could be one of the stimulating factors. Although IL-8 levels were lower in the supernatant co-cultured with killed C. albicans than with live C. albicans , killed C. albicans could also induce IL-8 production by keratinocytes. Moreover, when NHEK were co-cultured with live C. albicans , the yeast cells produced hyphae, which might dramatically exert a pathological action, suggest-
Letter to the Editor
235
ing that physical contact might be influential to cause IL-8 production by keratinocyte. Further analysis is required to understand the process of IL-8 production by keratinocytes and the relation between C. albicans infection and the host defense.
References [1] Watanabe S, Kano R, Sato H, Nakamura Y, Hasegawa A. The effect of Malassezia yeasts on cytokine production by human keratinocytes. J Invest Dermatol 2001;116:769 /73. [2] Matsushima K, Morishita K, Yoshimura T, Lavu S, Kobayashi Y, Lew W, Appella E, Kung HF, Leonard EJ, Oppenheim JJ. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med 1988;167:1883 /93. [3] Longley BJ. Fungal disease. In: Elder D, editor. Lever’s Histopathology of the Skin, eighth ed. Philadelphia: Lippincott-Raven Publishers, 1997:517 /51. [4] Szolnoky S, Bata-Cso ¨rgo ¨ Z, Kenderessy AS, Kiss M, Pivarci A, Nova ´k Z, Newman KN, Michel G, Ruzicka T, Maro ´di L, Dobozy A, Keme ´ny L. A mannose-binding receptor is expressed on
.
human keratinocytes and mediates killing of Candida albicans . J Invest Dermatol 2001;117:205 /13.
Rui Kano, Atsuhiko Hasegawa Department of Pathobiology, Nihon University School of Veterinary Medicine, 1866, Kameino, Fujisawa, Kanagawa 252-8510, Japan E-mail address: [email protected] Shinichi Watanabe, Hiroko Sato, Yuka Nakamura Department of Dermatology, Teikyo University School of Medicine, 11-1, Kaga-2, Itabashiku, Tokyo 173-8605, Japan