Amhives of

Arch Dermatol Res (1986)279:136-139

9 Springer-Verlag1986

Short communications

Candida albicans phagocytosis by separated human epidermal cells* M. Csat61, B. Bozdky z, j. Hunyadi 1, and A. Dobozy 1 1 Department of Dermatology and z Department of Pathology, University Medical School, Szeged, Hungary

Key words: Human epidermal cells albicans phagocytosis - Killing

Candida

That keratinocytes can perform phagocytosis is well established. They engulf and digest melanosomes daily [6]. Keratinocytes have been shown to ingest Thorotrast [11], synthetic melanin [10], charcoal particles [1], and erythrocytes [3]. Following skin incision, keratinocytes phagocytose extracellular debris and fibrin fragments [9]. Phagocytosis of parts of Langerhans cells after stripping of epidermal tape has also been demonstrated [8]. Furthermore, keratinocytes have been found to phagocytose latex beads injected into blister cavities [12]. Evidence of the in vitro phagocytosis of a living microorganism by separated human epidermal cells is, however, lacking. In the present work we report the in vitro phagocytosis and killing of Candida albicans cells by separated human epidermal cells. Epidermal cells in a single cell suspension c o n t a i n i n g 106 cells/ml obtained from ten healthy persons who underwent plastic surgery were separated by trypsin digestion. A reaction mixture of 106 freshly separated human epidermal cells/ml and of 2 x 106 C. albicans cells/ml - using 0656 CBS Delft and cultured on Sabouraud agar for 24 h at 37~ - was Dedicated to Prof. M. Simon, in honor of 70th birthday Offprint requests to: M. Csatt, M. D., Dept of Dermatology, Univ. Med. School Szeged, P.O. Box 480, H-6701 Szeged, Hungary * Presented as a poster at the XIIIth ADF Congress, 15--17 November 1985, in Vienna

incubated for 16 h at 37~ in an atmosphere of 5% COz in air and 100% relative humidity. Half of each sample was subjected to electron microscopy. In the other half, the C. albicans killing activity of the epidermal cells was studied, using modification of a method orginally introduced for blood leukocytes [4, 5, 71. In half of the samples, the phagocytosis of the yeast cells by epidermal cells was studied using electron microscopy. The keratinocytes and the C. albicans cells could readily be identified on the basis of their electron microscopic appearance. Some keratinocytes exhibited slight structural derangement, possibly due to the trypsin digestion. Although a large number of keratinocytes were examined, relatively few of them showed morphologic signs of phagocytosis. The phe' nomena observed, however, allow reconstruction of a phagocytic process. Candida cells were seen attached to the surface membrane of the keratinocytes. Protrusions of the keratinocytic cytoplasm surrounded and embraced the yeast cells (Fig. 1). In other cases, the Candida cells were already found within the cytoplasm of the cells. When intracellular, the Candida cells were always observed singly, sometimes entirely surrounded by tonofilaments (Figs. 2, 3). The structure of the yeast appeared to be intact. In the other half of the samples, the percentage of nonviable, killed Candida cells was counted under a light microscope. An increase in the percentage of killed yeast cells, as compared with the control tubes in which Candida cells were similarly incubated, in the absence of epidermal cells was found in all but one sample (Fig. 4). The difference indicates significant Candida killing activity of the epidermal cells (P < 0.005).

Fig. 1. Protrusion of the keratinocytic cytoplasm surrounding and embracing yeast cells, x 18,400 Fig. 2. A Candida cell within the cytoplasm of a keratinocyte, surrounded by tonofilaments, x 32,200

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M. Csat6 et al.: Candida phagocytosis in epidermal cells

Fig. 3. Two Candida cells within the cytoplasm of a keratinocyte. • 13,800 % of Candida ceils killed

15"

p .~ 0,005

10-

.

CONTROL

EPIDERMAL CELLS

Fig. 4. Candida albicans killing activity of the epidermal cells. Yeast cell suspension incubated similarly, but in the absence of epidermal cells served as a control (p < 0.005; Student's paired t-test)

In spite of the well-established ability of keratinocytes to pagocytose, the phagocytosis of living microorganisms by human epidermal cells in vitro has not been reported. After in vitro Candida albicans phagocytosis by freshly separated human keratinocytes, Candida albicans cells could clearly be recognized within the cytoplasm of the keratinocytes. The steps observed in the uptake of the yeast cells lead us to assume that the engulfment of the Candida cells is true phagocytosis. The phagocytic capacity of the epidermal cells is apparently weak. In a suspension containing epidermal cells at different stages of differentiation and keratinization, only a few of the cells showed electron microscopic signs of phagocytosis. In a similar system containing polymorphonuclear leukocytes ("professional phagocytes"), instead o f epidermal cells, almost all granulocytes take up yeast cells [4, 7]. This observation suggests that phagocytic capacity is not present in all epidermal cells, or all stages of differentiation. We were unable to find a Langerhans cell ingesting a Candida cell. This fact is consistent with the view that Langerhans cells are

M. Csat6 et al. : Candida phagocytosis in epidermal cells p o o r p h a g o c y t e s and are engaged in different i m m u n e functions. In spite o f the electron microscopic observation that the engulfed Candida cells were seemingly intact, in a functional test, a consequent and significant killing o f the yeast was found. A functional a p p r o a c h w o u l d a p p e a r to be m o r e a p p r o p r i a t e in this respect. The yeast is killed by an u n k n o w n mechanism. W o l f f and Schreiner [13] have observed that keratinocytes are capable o f degrading p h a g o c y t o s e d peroxidase protein. F u r t h e r m o r e , it has been d e m o n s t r a t e d quite recently t h a t epidermal cells possess an e n z y m e system which can p r o d u c e oxygen radicals, k n o w n to be involved in microbicidal processes [2]. A l t h o u g h the m e c h a n i s m o f the yeast killing requires elucidation, o u r results indicate that epidermal cells m a y play an active role in the elimination o f living microorganisms.

References 1. Bayoumi AHM, Gaskell S, Marks R (1978) Development of a model for transepidermal elimination. Br. J Dermatol 99:611-619 2. Bickers DR (1985) Drug and carcinogen metabolism in skin and its pharmacological modification. Paper delivered on the XIII ADF Congress, 15-17 November, Vienna 3. Boiron G, Surleve-Bazeille JE, Tamisier JM, Guillet G, Maleville J (1982) Phagocytosis of erythrocytes by keratinocytes in human and animal epidermis. Dermatologica 165:158-167

139 4. Csat6 M, Dobozy A (1981) A study on the Candida killing activity of polymorphonuclear leukocytes in patients with psoriasis vulgaris. Arch Dermatol Res 271:229-231 5. Csat6 M, Dobozy A, Raith L, Simon N (1985) The functions of polymorphonuclear leukocytes emigrating into the skin. Acta Derm Venereol (Stockh) 65 : 106-110 6. Konrad K, Wolff K (1973) Hyperpigmentation, melanosome size, and distribution patterns ofmelanosomes. Arch Dermatol 107 : 853 - 860 7. Lehrer RI, Cline MJ (1969) Interaction of Candida albieans with human leukocytes and serum. J Bacteriol 98:9961004 8. Mottaz JH, Zelickson AS (1970) The phagocytic nature of the keratinocyte in human epidermis after tape stripping. J Invest Dermatol 54: 272- 278 9. Odland G, Ross R (1968) Human wound repair. I. Epidermal regeneration. J Cell Biol 39:135--151 10. Potter B, Medenica M (1968) Ultramicroscopic phagocytosis of synthetic melanin by epidermal cells in vivo. J Invest Dermatol 51 : 300- 303 11. Wolff K, H6nigsmann H (1971) Permeability of the epidermis and the phagocytic activity of keratinocytes. Ulstrastructural studies with throrotrast as a maker. J Ultrastruct Res 36:176-190 12. Wolff K, Konrad K (1972) Phagocytosis of latex beads by epidermal keratinocytes in vivo. J Ultrastruct Res 39: 262280 13. Wolff K, Schreiner E (1969) Aufnahme, intracellul/irer Transport und Abbau exogenen Proteins in Keratinocyten. Eine elektronenmikroskopisch-cytochemische Studie mit Peroxidase als Markierungssubstanz. Arch Klin Exp Dermatol 235:203-220 Received March 27, 1986