Interleukin-18 and Gamma Interferon Production by Oral ... · Oral candidiasis is a collective name...

INFECTION AND IMMUNITY, Dec. 2002, p. 7073–7080 Vol. 70, No. 120019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.12.7073–7080.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Interleukin-18 and Gamma Interferon Production by Oral EpithelialCells in Response to Exposure to Candida albicans or

Lipopolysaccharide StimulationMahmoud Rouabhia,1* Genevieve Ross,1 Nathalie Page,2 and Jamila Chakir2

Faculte de medecine dentaire, GREB,1 and Centre de recherche, Institut universitaire de cardiologie et depneumologie, Hopital Laval,2 Universite Laval, Quebec G1K 7P4, Canada

Received 5 June 2002/Returned for modification 29 July 2002/Accepted 27 August 2002

Oral candidiasis is a collective name for a group of disorders caused by the dimorphic fungus Candidaalbicans. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms andlocal oral mucosal epithelial cell defenses. Since oral epithelial cells secrete a variety of cytokines andchemokines in response to oral microorganisms and since C. albicans is closely associated with oral epithelialcells as a commensal organism, we wanted to determine whether interleukin-18 (IL-18) and gamma interferon(IFN-�) were produced by oral epithelial cells in response to C. albicans infection and lipopolysaccharide (LPS)stimulation. Our results showed that IL-18 mRNA and protein were constitutively expressed by oral epithelialcells and were down-regulated by Candida infections but increased following LPS stimulation. Both C. albicansand LPS significantly decreased pro-IL-18 (24 kDa) levels and increased active IL-18 (18 kDa) levels. Thiseffect was IL-1�-converting-enzyme dependent. The increase in active IL-18 protein levels promoted theproduction of IFN-� by infected cells. No effect was obtained with LPS. Although produced only at an earlystage, secreted IFN-� seemed to be a preferential response by oral epithelial cells to C. albicans growth. These resultsprovide additional evidence for the contribution of oral epithelial cells to local (direct contact) and systemic (IL-18and IFN-� production) defense against exogenous stimulation such as C. albicans infection or LPS stimulation.

Candida species are the most frequent cause of life-threat-ening invasive fungal infections in the immunocompromisedhost and are responsible for 10% of all nosocomial blood-stream infections (19, 27). The leading cause of candidiasis isCandida albicans. This fungus colonizes different body sites,including the oral cavity. C. albicans colonizes the oral mucosaof approximately 80% of normal individuals as a commensalorganism, causing no apparent damage and inducing no ap-parent inflammation in the surrounding tissue (1, 8). However,under a number of predisposing conditions, C. albicans multi-plies and penetrates the host tissue, causing inflammation andtissue destruction (43, 49). Host resistance mechanisms to C.albicans have been investigated in systemic and mucocutane-ous candidiases (10, 39), and although it is generally acceptedthat different mechanisms operate in systemic versus superfi-cial candidiasis, the mechanisms of host defense and the patho-genesis of candidiasis are not completely understood. Adher-ence of C. albicans to oral epithelial cells is the first step in theinitiation of infection, as this enables the organism to overcomethe normal flushing mechanisms of body secretions (14, 36).

The oral mucosal epithelium acts as the major barrier tophysical, microbial, and chemical agents that may cause localcell injury (38). Recent studies have shown that epithelial cellsmay function as immobile immunocytes (5, 34). During theevolution of the inflammatory response, leukocyte subsets arerecruited and extravasate through the endothelium, a processthat is promoted by local cytokine secretion (30). The activa-

tion of nonspecific and specific immune cells such as macro-phages and T cells during the inflammatory response elicits aconcomitant release of cytokines (50). A newly described cy-tokine, interleukin-18 (IL-18), also known as gamma inter-feron (IFN-�) inducing factor, has been recently shown to be apotent inducer of IFN-� production by activated T cells (35).Since its discovery, IL-18 has been found to contribute toprotective immunity against a variety of pathogens, includingCryptococcus, Leishmania, and Salmonella species and Myco-bacterium tuberculosis (33).

IL-18 is related to the IL-1 family both structurally andfunctionally (3). Like IL-1, IL-18 is synthesized as a biologicallyinactive 24-kDa precursor protein. Cleavage to the active formis mediated by the IL-1 converting enzyme (ICE), also calledcaspase-1 (18, 20). Cleavage of IL-18 by ICE is essential forIL-18 to become biologically active. This protein is producedby activated macrophages, dermal keratinocytes, osteoblasts,adrenal cortex cells, and intestinal epithelial cells (12, 17, 44),implying that it plays other physiological roles in addition toimmune regulation.

Oral epithelial cells are involved in the proinflammatoryprocess through the production of cytokines either constitu-tively or after a variety of stimuli (26), implying that they maypotentially participate in controlling oral infections through aninflammatory (5, 34) process involving different interleukins,such as IL-1� and IL-18. Given the biological role of oralepithelial cells as active immunocytes (5, 32) and the role ofIL-18 as a proinflammatory cytokine, we hypothesized that oralepithelial cells, through IL-18, maintain the dynamic equilib-rium between the oral microbial community (free microorgan-isms and dental plaque bacteria) and the host. We looked atwhether oral epithelial cells constitutively expressed IL-18 and

* Corresponding author. Mailing address: Faculte de medecine den-taire, Pavillon de medecine dentaire, Local 1728, Universite Laval,Quebec G1K 7P4, Canada. Phone: (418) 656-2131, ext. 16321. Fax:(418) 656-2861. E-mail: [email protected].

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its converting enzyme (ICE). We determined whether IL-18production was potentiated when oral epithelial cells werestimulated with LPS or live C. albicans. We also assessed theIFN-� induction potential of the IL-18 (18 kDa) produced bythe oral epithelial cells. In this study, we obtained evidence for thefirst time that human oral epithelial cells act against C. albicansthrough an IFN-� pathway in an IL-18-dependent manner.

MATERIALS AND METHODS

Isolation and culture of oral epithelial cells. Small pieces of palatal mucosawere biopsied from gingival graft patients after obtaining their informed consent.Biopsy specimens were treated with thermolysin (500 �g/ml) to separate theepithelium from the lamina propria. Epithelial cell suspensions were obtainedfollowing treatment with a 0.05% trypsin–0.01 M EDTA solution. Freshly iso-lated epithelial cells (9 � 103 cells/cm2) were cultured in Dulbecco’s ModifiedEagle-Ham’s F-12 (3:1) (DMEM) (Flow Laboratories, Mississauga, Ontario,Canada) supplemented medium (40). Before their use, cells were characterizedusing specific antibodies (40). After being characterized, oral epithelial cells wereused at passage 3 to realize this study.

Candida. One C. albicans isolate was used in this study. It is an original clinicalisolate (Candida-associated stomatitis) that has been characterized very thor-oughly using numerous experimental approaches (4) and was kindly provided byN. Deslauriers (GREB, Laval University, Quebec, Canada). The yeast was grownon Sabouraud dextrose agar (Becton Dickinson, Cockeysville, Md.) at 30°C. ForC. albicans suspensions, one colony was used to inoculate 10 ml of phytone-peptone medium (Becton Dickinson) supplemented with 0.1% glucose. Theculture was grown to the stationary phase for 18 h at 25°C in a shaking waterbath. The blastoconidia were collected, washed with phosphate-buffered saline(PBS), and enumerated using a hemacytometer (Reichert, Buffalo, N.Y.) andtrypan blue dye exclusion (24). Viable cells were adjusted to 108 C. albicanscells/ml and used to infect gingival epithelial cell cultures.

Stimulation of epithelial cells with LPS or C. albicans. Epithelial cells weredetached from 75-cm2 culture flasks using trypsin. They were washed twice inculture medium, counted, seeded into six-well tissue culture plates (Falcon,Becton Dickinson, Lincoln Park, N.J.) at 2.5 � 105 cells/well, and then incubatedin an 8% CO2 atmosphere at 37°C. The epithelial cells were cultured to about90% confluence, which was obtained after 5 days of culture. They were thenstimulated with C. albicans (105 cells/cm2) or lipopolysaccharide (LPS) (5 �g/ml)extracted from Porphyromonas gingivalis. LPS was extracted and purified asdescribed previously (11, 37). The LPS was kindly provided by D. Grenier(GREB, Laval University, Quebec, Canada). Stimulated and unstimulated cellswere cultured for 3, 6, 12, and 24 h. At the end of each contact period, the culturesupernatants were harvested and stored at �80°C. The cells were detached asdescribed above and used to extract either total mRNA or proteins.

RNA extraction and reverse transcription (RT)-PCR analysis. Total cellularRNA was prepared from unstimulated and LPS- and C. albicans-stimulatedepithelial cells using the RNeasy Total RNA kit (Qiagen Inc., Valencia, Calif.)and quantified by fluorescence using Ribogreen (Molecular Probes Inc., Eugene,Oreg.). Total RNA was reverse transcribed into cDNA using the Moloneymurine leukemia virus reverse transcriptase (Canadian Life Technologies, Gaith-ersburg, Md.) and random hexamers (Amersham Pharmacia Biotech Inc., Que-bec, Canada). One microliter of each cDNA product was added to a 50-�l PCRmixture containing Taq polymerase (Qiagen Inc.) and forward and reverse prim-ers (Keystone Laboratories Inc., Camarillo, Calif.). All reactions were performedin a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) over 35 cycles,each consisting of 1 min at 95°C, 30 s at 52°C, and 1 min at 72°C for. Glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) cDNA was measured in eachsample as a control. The following primers were used for the PCR: IL-18(forward), 5�-GCTTGAATCTAAATTATCAGTC-3�; IL-18 (reverse), 5�-GAAGATTCAAATTGCATCTTAT-3�; ICE (forward), 5�-ATCCGTTCCATGGGTGAAGGTACA-3�; ICE (reverse), 5�-CAAATGCCTCCAGCTCTGTAATCA-3�; GAPDH (forward), 5�-ATGCAACGGATTTGGTCGTAT-3�; and GAPDH(reverse), 5�-TCTCGCTCCTGGAAGATGGTG-3�. The predicted sizes of thePCR products were 320 bp for pro-IL-18, 620 bp for ICE, and 220 bp forGAPDH. After the PCR procedure, 10 �l of the products were separated on a 2%agarose gel containing ethidium bromide and visualized by UV light. The gels werephotographed, and the relative intensity of the bands was measured on digitizedimages using a Macintosh computer and the public domain NIH Image program.

Western blotting and immunodetection of intracellular IL-18. Western blot-ting analyses were performed as described previously (31). Unstimulated and

LPS- and C. albicans-stimulated oral epithelial cells were lysed in Tris buffercontaining 2 mM EDTA, 2% Triton X-100, and an antiprotease mix. The lysateswere boiled in sample buffer (200 mM Tris [pH 6.8], 20% glycerol, 2% SDS,0.1% bromophenol blue, and 10% �-mercaptoethanol), and equal amounts oftotal protein were loaded onto a sodium dodecyl sulfate-10% polyacrylamide gel.Recombinant human IL-18 and monocyte lysate were used as positive controlsfor IL-18 and ICE protein detection. After electrophoretic separation, the pro-teins were transferred to a nylon polyvinylidene difluoride membrane (Millipore,Bedford, Mass.), blocked overnight with TBS-T (100 mM Tris [pH 7.5], 0.9%NaCl, and 0.1% Tween 20), and complemented with 4% skim milk (BLOTTO).The membrane was incubated for 1 h at room temperature with goat anti-humanIL-18 monoclonal antibody (MBL, Naka-ku Nagoya, Japan) diluted 1/1,000 inBLOTTO or with affinity-purified anti-human ICE rabbit polyclonal antibody(Santa Cruz Biotechnology, Santa Cruz, Calif.) diluted 1/1,000 in BLOTTO.Anti-human IL-18 monoclonal antibody reacts with pro-IL-18 and IL-18. Anti-human ICE polyclonal antibody reacts with a 45-kDa precursor, procaspase-1,also designated ICE. The membrane was washed (once for 10 min and once for5 min) in TBS-T and then incubated for 30 min at room temperature withhorseradish peroxidase-labeled sheep anti-mouse immunoglobulin G (CedarlaneLaboratories Ltd., Ontario, Canada). After washing with TBS-T (three times for5 min and once for 2 min), the blots were developed using an enhanced chemi-luminescence kit (Amersham International, Little Chalfont, United Kingdom).

Measurement of IL-18 and IFN-� secreted by oral epithelial cells. The super-natants collected from unstimulated and LPS- and C. albicans-stimulated oralepithelial monolayer cultures were used to determine the levels of secreted IL-18and IFN-�. Cytokine measurements were performed in triplicate samples usingan enzyme-linked immunosorbent assay (ELISA) for IL-18 (Medical & Biolog-ical Laboratories Co., Ltd., Nagoya, Japan) and IFN-� (Cell Sciences, Norwood,Mass.). Supernatants were filtered through 0.22-�m-pore-size filters and used toquantify IL-18 and IFN-� according to the manufacturer’s instructions. A 50-�lvolume of undiluted supernatant was used to perform the quantitative analysis.Plates were read at 450 nm and analyzed using an EAR-400 Easy Reader(SLT-Lab Instrumentation, Grodig, Austria). The sensitivity of the IL-18 ELISAkit was better than 13 pg/ml and the sensitivity of the IFN-� ELISA kit was betterthan 1 pg/ml according to the manufacturer. The experiments were repeatedthree times, and the means � standard deviations (SD) were plotted.

Effect of C. albicans on epithelial cell viability and growth. Immediately aftereach contact period with C. albicans, the epithelial cell cultures were washedthree times with culture medium and then detached using a 0.05% trypsin–0.01M EDTA solution for 20 min at 37°C. After the enzyme treatment, the cells werewashed twice with DMEM-supplemented culture medium. The pellets wereresuspended in 1 ml of DMEM, and cell viability was determined using a dyeexclusion test (41). A 100-�l volume of each epithelial cell suspension was mixedwith the same volume of trypan blue solution and incubated on ice for 5 min.This step was performed three times for each cell suspension. The total numberof cells in each sample and their viability were then determined by trypan blueexclusion. The percentage of viable cells was then calculated, and the resultswere reported as means � SD of six different experiments.

Evaluation of C. albicans adherence to and entry into epithelial cells. Todetermine whether C. albicans adheres to and enters the cytoplasm of epithelialcells, glass coverslips were placed in each well of six-well tissue culture platesprior to seeding with epithelial cells. The cells were grown in the tissue cultureplates for 72 h at 37°C in an 8% CO2 atmosphere as described above. Aftercontact with C. albicans for 3, 6, 12, and 24 h, the monolayers were washed sixtimes with PBS and fixed with 40% methanol–60% acetone for 10 min. The cellswere washed twice with PBS and stained with Masson trichrome. Stained sam-ples were examined under an optical microscope (Leitz), and photographs weretaken using Kodak color ISO 100 film. To quantify the number of C. albicanscells adhering to the monolayer and entering the cells, infected cultures werewashed six times with Hanks’ balanced salts solution. The cells then were lysedwith 1% (vol/vol) Triton X-100 in PBS, and C. albicans viability and numberswere estimated by trypan blue dye exclusion as described previously (24). Thisassay quantifies the total number of fungus cells bound to the outside of andinternalized by the epithelial cells. The adherence rates are the means of sixdifferent experiments.

Statistical analysis. All experiments in this study were performed at least threetimes. The data shown are representative results. Experimental values are givenas means � SD. The statistical significance of differences between two means wasevaluated by Student’s unpaired t test. Results were considered significant if Pwas 0.05.

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RESULTS

IL-18 mRNA expression by human oral epithelial cells mod-ulated by C. albicans and LPS. We first examined the profile ofconstitutive and induced IL-18 mRNA expression by culturedhuman oral epithelial cells. Oral epithelial cells displayed ro-bust constitutive IL-18 mRNA expression (Fig. 1). There was asignificant increase in the IL-18 message following a 12-h stim-ulation with LPS (Fig. 1). Interestingly, C. albicans down-reg-ulated the IL-18 mRNA expression(Fig. 1). Indeed, in 24-h-infected tissue, we found a significant (P � 0.05) reduction ofIL-18 mRNA expression.

IL-18 production by human oral epithelial cells decreasedby C. albicans and LPS. Since IL-18 mRNA is constitutivelyexpressed by epithelial cells, and since the gene expression mayor may not be modulated by stimulating agents, we thus lookedat whether IL-18 mRNA was translated into IL-18 protein.Western blot analyses using cell lysates showed that unstimu-lated oral epithelial cells constitutively expressed IL-18 protein(Fig. 2). Unstimulated oral epithelial cells contained pro-IL-18(24 kDa) but not IL-18 (18 kDa). In stimulated cultures, bothC. albicans and LPS induced an important decrease in pro-IL-18 protein. This down-regulation of pro-IL-18 by C. albi-cans and LPS was time dependent. Oral epithelial cells seemed

to have stable high levels of IL-18 mRNA and Pro-IL-18 pro-tein that dropped following contact with C. albicans or LPS.These results suggested that the drop in pro-IL-18 in stimu-lated cells may be due to the cleavage of inactive IL-18 toactive IL-18 and that this mobilizes the cell to produce highlevels of IL-18 mRNA in order to renew its stock of protein.These observations raised the question as to whether oralepithelial cells express the converting enzyme involved in pro-cessing inactive IL-18 to active IL-18 protein.

ICE mRNA and protein modulation by C. albicans and LPS.To be converted to active IL-18 protein, inactive pro-IL-18, a24-kDa protein, has to be cleaved by a specific ICE, also knownas caspase-1. Inactive-IL-1 and IL-18 have been identified assubstrates for ICE (18, 20). Since oral epithelial cells expressthe IL-18 protein, we next looked at whether cultured humanoral epithelial cells expressed ICE mRNA. Total RNA analy-ses using RT-PCR showed (Fig. 3) that oral epithelial cellsconstitutively expressed the converting enzyme. The basal lev-els of ICE expression were significantly (P � 0.01, P � 0.05)modified by C. albicans or LPS stimulation. The question wasthus whether the cells expressed the ICE protein involved inpro-IL-18 cleavage. Western blot analyses of cell lysatesshowed (Fig. 4) that oral epithelial cells constitutively ex-

FIG. 1. IL-18 mRNA is transiently modulated by LPS and C. albi-cans stimulation. (A) Total cellular RNA (1 �g) from unstimulatedand C. albicans (105 cell/cm2)- and LPS (5 �g/ml)-stimulated oralepithelial cells was analyzed by RT-PCR with primers specific for IL-18(320 bp) and GAPDH (220 bp). The stimulation period is indicated atthe top of each lane. Lane Ctrl contains unstimulated oral epithelialcell cultures. Expression of GAPDH mRNA is shown as a loadingcontrol for the experiments. The gel shown is a representative of threedifferent experiments. (B) The increase in IL-18 was assessed by thepublic domain NIH Image software package as the mean � SD ofthree different experiments.

FIG. 2. IL-18 protein is constitutively expressed by oral epithelialcells but significantly decreased by LPS and C. albicans stimulation.(A) Total protein (100 �g) from lysates of unstimulated (Ctrl) and C.albicans (105 cell/cm2)- and LPS (5 �g/ml)-stimulated oral epithelialcells was analyzed by Western blotting for the presence of inactive-IL-18 (24 kDa) protein. The stimulation period is indicated at the topof each lane. The position of IL-18 protein is indicated. Recombinanthuman IL-18 (10 ng) was used as a positive control. The gel shown isa representative of three different experiments. (B) The decrease ininactive IL-18 protein was determined by band scanning using thepublic domain NIH Image software program. Data are the means �SDs (error bars) of three different experiments. The statistical differ-ences (��, P � 0.01) were obtained by comparing the value obtained at3 h to the value obtained at 6 h or the value obtained at 6 h to thatobtained at 12 h, etc., for the same stimuli (LPS or C. albicans).

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pressed the pro-ICE protein (45 kDa). When the cells werestimulated with LPS, no significant changes were noticed rel-ative to ICE protein expression. Interestingly, when cells wereinfected with C. albicans, ICE protein levels decreased in atime-dependent manner, nearing almost zero after a 24-h con-tact between the epithelial cells and C. albicans. The ICEdecrease seemed to follow the pro-IL-18 decrease reported inFig. 2, suggesting that pro-IL-18 was cleaved by ICE to theactive form (18 kDa). It was therefore important to determinewhether oral epithelial cells secrete the active form of IL-18into the culture medium.

Detection of active IL-18 in culture supernatants of humanoral epithelial cells stimulated with C. albicans or LPS. Todetermine whether oral epithelial cells are able to produceactive IL-18 protein, culture supernatants of unstimulated andLPS- and C. albicans-stimulated cells were analyzed by sand-wich ELISA using anti-IL-18-specific reagents. A very lowbasal production of active IL-18 protein was detected in 24-h

oral epithelial cell culture supernatants (Fig. 5). These basallevels increased following a C. albicans infection. In infectedcultures, IL-18 protein levels reached a maximum after a 12-hcontact. The concentration of IL-18 protein varied from 124 �7 pg/ml after 3 h to 519 � 15 pg/ml after 12 h in infected cellcultures. In LPS-stimulated oral epithelial cell cultures, theamount of IL-18 protein increased in a time-dependent man-ner, reaching 300 � 11 pg/ml after 3 h and climbing to amaximum of 696 � 5 pg/ml after 24 h (Fig. 5). Thus, both C.albicans and LPS up-regulated active IL-18 protein productionby oral epithelial cells.

IFN-� production by human oral epithelial cells stimulatedwith C. albicans or LPS. It is well known that IL-18 is a potentinducer of IFN-� production by activated cells such as T lym-phocytes (33). To determine whether the IL-18 protein se-creted by oral epithelial cells is functional (by stimulating theseepithelial cells to produce IFN-�), a specific IFN-� ELISA wasperformed using oral epithelial cell culture supernatants. Con-trol cells did not express IFN-� at a level that was detectable byour assay. When cells were stimulated with LPS they produceda limited amount of IFN-� that varied from 10 to 20 pg/ml.

FIG. 3. ICE mRNA is significantly modulated by LPS and C. albi-cans stimulation. (A) Total cellular RNA (1 �g) from unstimulatedand C. albicans (105 cell/cm2)- and LPS (5 �g/ml)-stimulated oralepithelial cells was analyzed by RT-PCR with primers specific for ICE(620 bp) and GAPDH (220 bp). The stimulation period is indicated atthe top of each lane. Lane Ctrl contains unstimulated oral epithelialcell cultures. The gels shown is a representative of three differentexperiments. (B) The degree of ICE modulation (increase or decrease)was determined by band scanning using the public domain NIH Imagesoftware package. Expression of GAPDH mRNA is shown as a loadingcontrol for the experiments. Data are the means � SDs (error bars) ofthree different experiments. The statistical differences (��, P � 0.01; �,P � 0.05) were obtained by comparing the value obtained at 3 h to thevalue obtained at 6 h or the value obtained at 6 h to that obtained at12 h, etc., for the same stimuli (LPS or C. albicans).

FIG. 4. ICE protein is constitutively expressed by oral epithelialcells but significantly decreased by LPS and increased by C. albicansstimulation. (A) Total protein (100 �g) from lysates of unstimulated(Ctrl) and C. albicans- and LPS-stimulated oral epithelial cells wasanalyzed by Western blotting using an anti-ICE (45 kDa) affinity-purified rabbit polyclonal antibody. The stimulation or infection periodwith C. albicans is indicated at the top of each lane. Total protein (50�g) from a rat monocyte lysate was analyzed for ICE protein as apositive control. The gel shown is a representative of three differentexperiments. (B) The degree of ICE modulation (increase or decrease)was determined by band scanning using the public domain NIH Imagesoftware program. Data are the means � SDs (error bars) of threedifferent experiments. The statistical differences (��, P � 0.01; �, P �0.05) were obtained by comparing the value obtained at 3 h to thevalue obtained at 6 h or the value obtained at 6 h to that obtained at12 h, etc., for the same stimuli (LPS or C. albicans).



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When oral epithelial cell cultures were infected with C. albi-cans, they produced significant amounts of IFN-� after a 3-hcontact (Table 1). Surprisingly, this apparent release of IFN-�was not temporally correlated with the accumulation of extra-cellular IL-18 despite the well-characterized role of IL-18 asthe major IFN-� inducing factor. Indeed, during the subse-quent contact periods of oral epithelial cells and C. albicans,IFN-� production was very weak after 6, 12, and 24 h (Table 1).Two conclusions can be reached from these results: (i) LPSmodulated weakly the oral epithelial cell response via an IL-18independent mechanism, and (ii) C. albicans stimulated oralepithelial cells to produce IFN-� via an IL-18-dependentmechanism at early events but blocked IFN-� production lateron to escape its toxic effect.

Epithelial cell viability after infection with C. albicans. Todetermine whether epithelial cells were still alive 3 h after

being infected with C. albicans and whether the high amount ofIFN-� obtained after 3 h was not the result of cell death andlysis-inducing protein liberation in the culture medium, weassessed the cell viability of C. albicans-infected cultures. Asreported in Table 2, no significant differences were detectedbetween infected and uninfected cultures, either in terms ofcell viability or total numbers of viable cells. This indicated thatthe increase level of IL-18 observed after a 3-h contact be-tween oral epithelial cells and C. albicans was not promoted bythe C. albicans infection. The high level of IFN-� producedafter a 3-h infection period was thus a biological response ofthe oral epithelial cells to the C. albicans infection.

Effect of human oral epithelial cells on C. albicans adher-ence, transformation, and growth. The above observationraised a question concerning the effect of epithelial cells on theadherence, transformation, and growth of C. albicans. We thusseparated infected oral epithelial cells from nonadhering C.albicans, and observed and photographed them using an opti-cal microscope. This revealed that C. albicans adhered to theepithelial cells and changed its blastospore form to a hyphalform (Fig. 6). The transformation of C. albicans from blasto-spores to hyphae was time dependent. The percentage of thehyphal form rose from 22% after a 3-h contact to more than80% after a 24-h contact (Table 3). Viability and growth anal-yses showed that all C. albicans that adhered to and penetratedinto epithelial cells were alive However, their numbers weresignificantly reduced, varying from 1.2 � 105/cm2 after a 3-hinfection to around 103/cm2 after a 24-h infection. These re-sults suggested that oral epithelial cells inhibited the growth ofC. albicans. C. albicans adheres to the cells and changes itscommensal form (blastospore) to a virulent form (hyphal),which may help C. albicans escape epithelial cell inhibitoryeffect. The transformation to the hyphal form requires closecontact between C. albicans and oral epithelial cells since freeC. albicans cells only change to a pseudohyphal form.

DISCUSSION

The various properties that make Candida species opportu-nistic pathogens and the multiple oral diseases they cause arestill a matter of debate. The systemic immune mechanismsinvolved in the control of candidiasis have been thoroughlyresearched. Nonetheless, the contribution of oral mucosalcells, especially epithelial cells, to immune surveillance re-mains to be investigated. During the inflammation process,epithelial cells are capable of secreting a variety of proinflam-matory cytokines. These cytokines play critical roles in the

FIG. 5. Enhanced production of active IL-18 protein by oral epi-thelial cells stimulated with LPS and C. albicans. Oral epithelial cellswere isolated from gingival biopsy specimens of healthy persons. Cellswere cultured in six-well plates alone or in the presence of C. albicans(blastospore form; 105 C. albicans cells/cm2) or LPS (5 �g/ml) fordifferent periods of time as indicated at the bottom of the figure.Culture medium was collected from each experiment, filtered througha 0.22-�m-pore-size filter, and used to determine the amount of theactive form of IL-18. IL-18 concentrations were measured by ELISA.Data are the means � SD (error bars) (n 3). Note the basal level ofIL-18 secretion by oral epithelial cells and the time-dependent increaseof this cytokine caused by C. albicans infection and LPS stimulation oforal epithelial cells. The levels of significance for C. albicans andLPS-stimulated oral epithelial cell cultures compared to unstimulatedcultures were P 0.05 (�) and P 0.01 (��), respectively. Ctrl-0 is oralepithelial cell culture medium, and Ctrl-24 is supernatant from un-stimulated oral epithelial cells cultured for 24 h.

TABLE 1. IFN-� produced by epithelial cells following contact withC. albicans or LPS

Contact time (h)

IFN-� (pg/ml) produced (mean � SD)a followingcontact with:

C. albicans (105

Candida cells/cm2) LPS (5 �g/ml)

Controlb 0.0 � 0.0 0.0 � 0.03 264.5 � 10.07 10.40 � 2.56 3.5 � 0.3 19.60 � 1.9

12 31.5 � 2.09 15.40 � 1.324 0.0 � 0.0 13.90 � 0.7

a (n 3).b Cultured supernatant of unstimulated oral epithelial cells.

TABLE 2. Total oral epithelial cell number and viability followingcontact with C. albicansa

Contact time (h) Mean % cellviability � SD

Mean total no.of cells � SD

(106)

Control 95 � 2.5 1.17 � 0.0513 94 � 3.5 1.21 � 0.0726 95 � 2.8 1.45 � 0.092

12 95.6 � 2.01 1.57 � 0.02424 95.5 � 2 1.67 � 0.08

a Results are based on six different experiments.



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development of protective immunity against intracellularpathogens (7, 30). IL-18 is a new entry in the vast epithelialcytokine bank. IL-18, which has pleiotropic immunomodula-tory functions, is involved in a variety of human inflammatoryconditions, including Crohn’s disease (32), rheumatoid arthri-tis (29), and tuberculoid leprosy (16). IL-18 restores defectiveTh1 immunity to C. albicans in infected caspase-1-deficientmice (18, 48). Oral epithelial cells may contribute to local andsystemic immune defenses against oral microorganisms, in-cluding C. albicans through IL-18. The present study indicatedthat oral epithelial cells produced IL-18 mRNA. As reported

FIG. 6. C. albicans adhesion to oral epithelial cells and the transition to the hyphal phase from the blastospore phase. Oral epithelial cells werecultured as monolayers in six-well plates. When they reached 80% confluence, they were infected with C. albicans (105 cells/cm2) for 3 and 24 h.The culture supernatants were harvested, observed under an optical microscope, and photographed. The monolayer cultures were stained usingMasson trichrome, observed under an optical microscope, and photographed. Phase-contrast photomicrographs show C. albicans harvested fromthe supernatants of oral epithelial monolayer cultures after 3 h (a) and 24 h (b), when a large number of blastospores can be seen changing to thepseudohyphal (arrows) form. Also shown are C. albicans as a hyphal form adhering to an oral epithelial cell monolayer after 3 h (c) and 24 h (d).(e) Uninfected oral epithelial monolayer culture (e). Note the switch of C. albicans from a budding to a hyphal form when it adheres to theepithelial cells (arrows). Magnification, �300.

TABLE 3. Viability, form, and number of C. albicans cells adheringto cultured oral epithelial cells.

Contacttime(h)

Mean no. of C.albicans � SDa

Mean % viability ofC. albicans � SD

Mean % estimatedhyphae � SD

3 1.2 � 0.7 98 � 0.01 22 � 36 0.7 � 0.03b 99 � 0.02 30 � 4

12 0.2 � 0.09b 97 � 0.32 50 � 124 0.06 � 0.01b 99 � 0.37 80 � 7

a C. albicans was seeded onto epithelial cell culture at 105 cells/cm2.b P � 0.01 is the level of significance for growth inhibition of C. albicans in

infected oral epithelial cell cultures compared to the initial C. albicans seedingconcentration.



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previously using Salmonella enterica (47), we showed that oralepithelial cells expressed significant basal levels of IL-18mRNA signals that were increased by LPS stimulation anddecreased by C. albicans stimulation, respectively. This is thefirst time that the interaction between C. albicans and oralepithelial cells via IL-18 has been addressed. The fact that C.albicans has a slight decreasing effect on IL-18 mRNA expres-sion may be explained by the presence of saturating levels ofIL-18 transcripts in both infected and uninfected epithelialcells that overcome the transcription inhibitory effect of C.albicans. As to whether IL-18 transcripts expressed by oralepithelial cells are translated into IL-18 protein, Western blotanalyses using epithelial cell lysates showed that basal expres-sion of inactive IL-18 protein (24 kDa) was significantly de-creased by stimulation with both LPS and C. albicans.

The presence of IL-18 in epithelial cells, which constitute theprimary host-pathogen interface, suggests that IL-18 may playan important role in fighting C. albicans infections. Severalstudies have demonstrated that IL-18 plays a key role in anti-microbial defenses by protecting against cryptococcal, myco-bacterial, and Salmonella infections. Furthermore, IL-18 gene-disrupted mice are susceptible to mycobacterial infections (33,45, 46). However, some pathogens may limit or interfere withhost IL-18 responses (15). This may explain the decrease inIL-18 protein production by C. albicans-infected oral epithelialcells. A second explanation could be that the cleavage of theinactive form of IL-18 to the active form (18 kDa) was notdetected on the Western blots of the cell lysates. However, thisraises the question as to whether oral epithelial cells possessthe ICE. Our data indicate that oral epithelial cells expressbasal levels of both ICE mRNA and protein. ICE proteinlevels decreased only when the epithelial cells were infectedwith C. albicans. Similar results were published by Barsig et al.(2), who reported that macrophages infected with Listeriamonocytogenes released smaller quantities of IL-1� than thoseinfected with Shigella flexneri, indicating that the activation ofICE was diminished or absent in macrophages harboring Lis-teria monocytogenes (2). Interestingly, the drop in the level ofICE protein that we observed (Fig. 4) paralleled the decreaseof IL-18 protein expression by C. albicans-infected cells (Fig.2), suggesting that epithelial cells produce enough ICE proteinto convert inactive IL-18 to the active form. The involvementof ICE in infections reported here confirms similar resultsreported in other situations. Indeed, Brennan et al. (6) haveshown that Salmonella-infected macrophages are killed by anICE-dependent mechanism (6). The decrease in inactive IL-18protein that we observed (Fig. 2) in infected cell lysates mightbe due to the conversion of pro-IL-18 to the active form.Specific immunoassays revealed that the production of activeIL-18 (18 kDa) was promoted by both LPS stimulation and C.albicans infection. These results are in accordance with thosepreviously reported regarding other infections. In a mouseinfection model, genetically susceptible BALB/c mice ex-pressed less IL-12 and IL-18 and had a diminished IFN-�/Th1response. In contrast, resistant DBA/2 mice exhibited an in-creased expression of IL-18 (23), indicating a good correlationbetween clearance of Mycobacterium avium and levels of IL-18.IL-18-deficient C57BL/6 mice have more severe infections, andpathological changes are significantly suppressed by treatmentwith exogenous IL-18 (46). Our results may then suggest that

oral epithelial cells elaborate a defensive strategy against C.albicans infections through an IL-18 pathway. The vital biolog-ical role of IL-18 requires the production of IFN-� (33). How-ever, the much increased production of the active form ofIL-18 (Fig. 5) by C. albicans-infected oral epithelial cells couldnot be linked to the IFN-� production by these cells (Table 2)in order to control the pathogenic potential of C. albicans. Thiscontrol seems to occur at an early stage of the infection (3 h orless), and then C. albicans dominates the cells via a completeinhibition of IFN-� secretion by infected oral epithelial cellseven if these cells continue to produce active IL-18. This mayexplain the high levels of active IL-18 produced by C. albicans-infected epithelial cells after more than 3 h as well as theabsence of IFN-� production by these cells in response to theIL-18 protein. Such a hypothesis needs to be investigated. Theearly significant production of IFN-� could be a transient ep-ithelial cell defense mechanism elaborated by these cells tocontrol fungal pathogenesis. This study establishes an indepen-dent relationship between the capacity of oral epithelial cells tosecrete active IL-18 protein and IFN-� protein and the controlof Candida infection.

In vitro studies have shown the stimulatory effects of IFN-�on the phagocytosis and the killing of C. albicans by neutro-phils and macrophages (13, 28). The administration of IFN-�to mice infected with C. albicans has a beneficial effect on theoutcome of the infection (25). The important role of endoge-nous IFN-� in resistance against both gastrointestinal and sys-temic candidiases has also been demonstrated by the increasedsusceptibility of knockout mice deficient in IFN-� or IFN-�receptors infected with the yeast (9, 22). We found that C.albicans growth was reduced in infected oral epithelial cellcultures (Table 3), but the C. albicans cells that overcame theepithelial cell effect changed from a commensal (blastospore)to a virulent (hyphal) form. This situation may mimic thephysiological and pathological situation in the oral cavity.

The identification of Candida as the etiologic agent of can-didiasis, especially in immunocompromised hosts, has resultedin a vast range of studies to assess its virulent properties, witha view to elucidating the pathogenesis of the disease. Muchprogress has been made in our understanding of some features,such as the mechanisms for adherence to host tissue, cell sur-face hydrophobicity, form switching, secretion of aspartyl pro-teinases, and phospholipase production (21, 42). Nonetheless,more research is needed to elucidate and fully understand thekey events involved in host-yeast interactions and the mecha-nisms that eventually lead to candidal infections. Studying therole of the epithelial cell in host defenses against Candidainfections is crucial to finding new insights into the complexbiological and pathological behavior of C. albicans and non-C.albicans Candida species. In such cases, oral epithelial cellsmay help us unravel the opportunistic process by which thisubiquitous organism becomes a pathogen.

ACKNOWLEDGMENTS

This work was supported by grants from the Natural Sciences andEngineering Research Council (M.R.), the Medical Research Council(M.R. and J.C.), and the Fonds de la Recherche en Sante du Quebec(M.R.). M.R. and J.C. are research scholars (junior and senior, re-spectively) supported by the FRSQ program.



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