1 Department of Dermatology and Allergology, University of Munich, Frauenlobstr. 911, D-80337 Munich, Germany
2 Department of Dermatology, Liebermeisterstr. 25, D-72076 Tuebingen, University of Tuebingen, Germany
3 Department of Parodontology, University of Munich, Goethestr. 70, D-80336 Munich, Germany
4 Robert Koch-Institute, NG4, Nordufer 20, D-13353 Berlin, Germany
Correspondence
Martin Schaller
Martin.Schaller{at}med.uni-tuebingen.de
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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Isolation of polymorphonuclear cells (PMNs).
PMNs obtained from healthy human volunteers were isolated from heparinized whole blood using Histopaque-1119 in combination with Histopaque-1077 (Sigma), according to the manufacturer's protocol. The cells recovered from the interface were washed three times and suspended at a concentration of 2x108 ml1 in RPMI 1640 medium (Sigma) in the presence and absence of 10 % fetal calf serum (FCS). Residual erythrocytes were removed by hypotonic lysis. Giemsa staining and light microscopy were used to ensure that a pure population of PMNs (>90 % purity) with typical morphology had been isolated. The cells were vital-stained using the trypan blue dye-exclusion method. Numbers of vital and non-vital leukocytes per sample were assessed using a Neubauer chamber. The viability was 95 % in all experiments.
Reconstituted human oral epithelium and model of oral candidosis.
The reconstituted human epithelium (RHE) for the in vitro model of oral candidosis was supplied by Skinethic Laboratory (Nice, France). It was obtained by culturing transformed human keratinocytes of the cell line TR146 derived from a carcinoma of the oral epithelium (Rupniak et al., 1985). Keratinocytes were incubated in serum-free conditions in a defined medium based on MCDB-153 medium (Clonetics), containing 5 µg insulin ml1, on a 0·5 cm2 microporous polycarbonate filter for 7 days at the airliquid interface. TR146 cells form a three-dimensional epithelial tissue resembling human oral mucosa in vivo (Fig. 1a
). The in vitro model and all culture media were prepared without antibiotics and antimycotics. Five infection experiments were performed for the C. albicans strain SC5314. RHE was infected with 2x106 Candida cells in 50 µl PBS for 12 and 24 h (Fig. 1b
). Non-infected controls contained 50 µl PBS alone.
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Assay of lactate dehydrogenase activity.
The release of lactate dehydrogenase (LDH) from epithelial cells into the surrounding medium was monitored as a measure of epithelial cell damage. LDH release in the maintenance media of the cultures from uninfected and infected epithelial cells was measured at 12 and 24 h. LDH activity was analysed spectrophotometrically by measuring the rate of NADH disappearance at 340 nm during the LDH-catalysed conversion of pyruvate to lactate, according to the WróblewskiLa Due method (Wróblewski & John, 1955). The LDH activity is given as U l1 at 37 °C.
Killing assay.
A killing assay was used to study the inhibitory effect of PMNs on C. albicans during RHE infection. It was performed by plating the infected samples 12 and 24 h after incubation, with and without PMN, on Sabouraud dextrose agar. Before plating, 2 ml PBS was added to the samples, and samples were thoroughly vortexed for 10 min. Furthermore, the solution was vigorously agitated by using a Pasteur pipette to separate big clumps of fungal cells into single cells, and then diluted 1 : 1000 and 1 : 10 000 in PBS. The presence of single cells was confirmed by light microscopy. Yeast cell viability was determined by assessment of the colony forming units produced after incubation for 24 h at 37 °C on Sabouraud dextrose agar.
Light microscopy.
Light-microscopy studies were performed to evaluate histological changes during infection. Part of each specimen was fixed, postfixed and embedded in glycide ether, and cut using an ultramicrotome (Ultracut). Semi-thin sections (1 µm) were studied with a light microscope after staining with 1 % toluidine blue and 1 % pyronine G (Merck). The histological changes of the mucosa were evaluated on the basis of 50 sections from five different sites for each infected epithelium.
RNA isolation and cDNA-synthesis by reverse transcriptase.
For the detection of mRNA, samples were rapidly removed and shock-frozen in liquid nitrogen. Total RNA from shock-frozen samples was isolated using RNAPure (Peqlab), according to the manufacturer's instructions. For assessing RNA concentration and purity, UV spectroscopy was used (Bio Photometer, Eppendorf). The absorbance of a diluted RNA sample was measured at 260 and 280 nm. cDNA synthesis was performed using Superscript II reverse transcriptase (Gibco), following the manufacturer's instructions.
Quantitative RT-PCR (QRT-PCR).
For cytokine expression, 20 ng of cDNA were amplified real-time in a LightCycler (Roche), using a FastStart DNA Master SYBR Green I kit (Roche) at 3 mM Mg2+ final concentration, and analysed with LightCycler Software 3.5. Annealing temperature and elongation time were optimized for each primer pair. The sequences of the primer pairs used were published recently (Schaller et al., 2002). Amplified DNA for each primer pair was serially diluted (six logs) and used to generate standard curves. Absolute quantification for these cDNAs was achieved with the LightCycler software.
Quantification of cytokine secretion by epithelial cells stimulated with C. albicans.
Epithelial tissues (with or without PMNs; see above) were infected with PBS-washed C. albicans S5314 or treated with PBS only. After 12 h and 24 h, samples of the maintenance medium surrounding the infected and uninfected epithelial tissues were collected and centrifuged. The amount of interleukin-1 (IL-1
), IL-6, IL-10, granulocyte-macrophages colony-stimulating factor (GM-CSF), interferon
(IFN-
) and tumour necrosis factor
(TNF-
) secreted into the supernatant was determined by fluorescence-activated cell sorting (FACS) analysis. Flow cytometric data were acquired on a Becton Dickinson FACSCan using the Human Inflammation Kit (Becton Dickinson). Data were analysed using Cellquest software (Becton Dickinson).
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RESULTS |
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Growth inhibition of Candida cells by PMNs was analysed by a quantitative plate-count method. Similar percentage inhibition values (12/24 h) were seen when PMNs were added to the apical layers (34±8/44±12 %), and to the basal side of intact (38±7/46±11 %) or perforated (36±9/39±11 %) layers of the infected epithelium.
Cytokine mRNA expression in response to mucosal C. albicans infection in the presence and absence of PMNs (QRT-PCR)
QRT-PCR of uninfected RHE 12 and 24 h after incubation with PBS demonstrated constant basal levels of mRNA expression for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IL-1, IL-1
, IL-8, TNF-
and GM-CSF (Table 1
). In contrast, C. albicans-infected RHE showed a strong increase of IL-8 and GM-CSF expression, and a moderate increase of gene expression for IL-1
, IL-1
and TNF-
after 12 and 24 h. (shown for 24 h in Table 1
).
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Cytokine secretion analysis in response to mucosal C. albicans infection in the absence and presence of PMNs (FACS analysis)
The modulation of the immune response was further characterized at the protein level by FACS analysis of the maintenance medium (Table 2). Non-infected mucosa produced only low levels of IFN-
, TNF-
and IL-1
, moderate levels of IL-6 and IL-10, but high IL-8 levels. In response to infection with C. albicans in the absence of PMNs, epithelial cells produced increasing concentrations of IFN-
, TNF-
, IL-1
, IL-6 and IL-8, whereas IL-10 secretion was not affected by epithelial infection. Addition of PMNs stimulated IFN-
and TNF-
production and reduced IL-10 secretion in all samples, while further increases in IL-1
, IL-6 and IL-8 production were only observed during transepithelial migration of the PMN cells.
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DISCUSSION |
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Recently published studies have shown induction of IL-8 synthesis in keratinocytes after stimulation with C. albicans (Dongari-Bagtzoglou & Kashleva, 2003; Schaller et al., 2002
). Since attachment and/or transepithelial migration of PMNs in this study was only seen after epithelial infection and concomitant strong expression of the chemoattractive cytokines GM-CSF, IL-6 and IL-8, we concluded that these cytokines contributed to the transepithelial migration of PMNs in our model. In vivo, PMNs accumulate rapidly at the site of infection in the oral cavity as a result of host signals (Borish et al., 1989
; Djeu et al., 1990
). IL-6 and IL-8 act as potent chemoattractants and promote degranulation and other antifungal activities of PMNs (Djeu et al., 1990
; Kullberg et al., 1999
). GM-CSF is known as a potent neutrophil growth factor, stimulating cytokine production and fungicidal activity of leukocytes (Tansho et al., 1994
). Therefore, our RHE bioassay system seems to accurately imitate oral candidosis in vivo.
Infection of RHE with C. albicans also induced expression of IFN- and TNF-
, which are linked with a protective Th1 response during systemic infection (Romani, 1999
). In contrast, IL-10, which is associated with a Th2-mediated susceptibility to candidosis (Romani, 1999
), was down-regulated in our bioassay system. PMNs, especially those migrating into epithelial tissue, enhanced this Th1 up- and Th2 down-regulation pattern and protected the mucosa against C. albicans infection by inhibiting fungal growth.
These data implicate a protective role for Th1-type cytokine release from epithelial cells during oral candidosis. Protection against tissue damage was observed by direct interaction of the PMN cells with C. albicans, either by apical application or by transepithelial migration of PMNs from the basal side. Interestingly, the protective effect was also seen in a modified experimental assay in which direct physiological contact between PMNs and C. albicans was prevented and only indirect interaction between PMNs, keratinocytes and C. albicans was possible. These results suggest that immunological cross-talk between epithelial cells and PMNs via cytokines, rather than direct phagocytosis of C. albicans cells by PMNs, may be of prime importance in initiating an epithelial-mediated protective anticandidal immune response in the oral cavity.
In summary, our model of oral candidosis supplemented by PMNs provides an attractive tool for studying the immunological cross-talk between keratinocytes and immune cells, and the chemoattraction of PMNs to the site of infection. Our results suggest an important role for PMNs in clearance of experimental oral candidosis. The protective effect was associated with a Th1-linked immune response, but was not necessarily connected to the direct interaction of PMNs with the pathogen.
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ACKNOWLEDGEMENTS |
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Received 15 March 2004;
revised 26 May 2004;
accepted 6 June 2004.
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