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Bioactive interleukin-1a is cytolytically released from

Candida albicans-infected oral epithelial cells

A. DONGARI-BAGTZOGLOU*, H. KASHLEVA & C. CUNHA VILLAR

*University of Connecticut, School of Dental Medicine, Farmington, CT, USA

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and

may regulate the inflammatory host response to this pathogen. This investigation

studied the mechanisms underlying interleukin-1a (IL-1a) release by oral epithelial

cells and the role of IL-1a in regulating the mucosal inflammatory response to

C. albicans. Infected oral epithelial cells released processed IL-1a protein in culture

supernatants. The IL-1a generated was stored intracellularly and was released upon

cell lysis. This was further supported by the fact that different C. albicans strains

induced variable IL-1a release, depending on their cytolytic activity. IL-1a from

C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine

secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our

studies suggest that production of IL-1a, IL-8 and GM-CSF may take place in the

oral mucosa in response to lytic infection of epithelial cells with C. albicans. This

process can act as an early innate immune surveillance system and may contribute

to the clinicopathologic signs of infection in the oral mucosa.

Keywords C. albicans, cytokines, epithelial cells, fibroblasts

Introduction

Epithelial cells lining the oral and gastrointestinal

mucosa constitute an integral component of the innate

mucosal immune system and may be involved in theinitiation and regulation of inflammation as well as in

the clearance of mucosal infections [1,2]. Oral candi-

diasis is a superficial mucosal infection caused primar-

ily by Candida albicans [3], which may predispose

severely immunocompromised patients to invasive dis-

ease [4]. In recent years, due to the fast spread of HIV

infection in developing countries and the common use

of immunosuppressant therapy in industrialized na-

tions, the incidence of this infection is rising [5].

Mucosal sites appear to be a major portal of entry

for C. albicans in human infections, thus it has been

suggested that the type of mucosal inflammatory

response to this opportunistic pathogen may determine

resistance or susceptibility to invasive infection by

regulating local immune cell function [6,7]. In oral

mucosal infections, C. albicans organisms are found in

the uppermost layers of epithelium, rarely invading

past the spinous cell layer [4,8]. As a result, the oral

mucosa is chronically inflamed with intense intrae-

pithelial and subepithelial infiltration by leukocytes

[4,8]. Although the role of epithelial cells as an

infection barrier against Candida is well recognized

[9], there is a paucity of information about the role of

these cells in orchestrating the oral mucosal inflamma-

tory response to this pathogen, which in turn may

regulate the antifungal functions of leukocytes re-

cruited to these lesions.

Epithelial cells respond to infection with the release

of a number of proinflammatory cytokines [10], which

can initiate and perpetuate mucosal inflammation.Interleukin-1a (IL-1a) is a major constitutive and

inducible proinflammatory product of epithelial cells

[10] that can act as a key cytokine to amplify the

inflammatory response by neighboring mucosal cells

[11], or activate local leukocyte antifungal activities

[12,13]. Several animal studies have established a

protective role for IL-1a in lethal, disseminated

Correspondence: Anna Dongari-Bagtzoglou, University of

Connecticut, School of Dental Medicine, Department of

Periodontology, 263 Farmington Avenue, Farmington, CT 06030-

1710, USA. Tel: '/860 679 4543; Fax: '/860 679 1673; E-mail:

[email protected]

Received 27 August 2002; Accepted 5 January 2003

2004 ISHAM DOI: 10.1080/1369378042000193194

Medical Mycology December 2004, 42, 531/541

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C. albicans infections [14,15] and they have also

supported a crucial role for IL-1 as an endog-

enous adjuvant in the initiation of protective immunity

[16].

Studies screening cell supernatants or lysates of

C. albicans- infected epithelia for various proinflamma-

tory cytokines, have identified IL-1a as one of the

major cytokines upregulated at both the mRNA and

protein levels [2,17,18]. Epithelial cell IL-1a has

also been found to be upregulated in human oral

mucosal candidiasis lesions [8]. Given the importance

of this cytokine for host defense against Candida

infections, we extended these studies by testing the

IL-1a responses of a large number of primary oral

keratinocyte cultures and cell lines, and provided new

information on the mechanisms that lead to IL-1a

release by C. albicans-infected oral mucosal epithelial

cells. We have shown for the first time that mature IL-

1a protein is released as a result of loss of epithelial cell

membrane integrity during infection with liveC. albicans. In addition, we have demonstrated that

epithelial cell-derived IL-1a can induce further proin-

flammatory cytokine responses in uninfected oral

epithelial and stromal cells. These studies identify an

important early signaling system through which acute

inflammation can be initiated and perpetuated in the

oral mucosa in response to the cytolytic pathogen

C. albicans.

Materials and methods

Organisms

Candida albicans strain SC5314, isolated from a patient

with disseminated candidiasis [19] was provided by

Dr Aaron Mitchell (Columbia University). C. albicans

strains 28366 and 32077 are human oral isolates and

they were obtained from the American Type Culture

Collection (ATCC; Rockville, MD). The organisms

were routinely propagated in YPD agar (Difco La-

boratories) at 258C.

Oral mucosal cell cultures

Oral keratinocyte cell lines, primary gingival keratino-

cytes and primary gingival fibroblasts were used in this

study. Cell lines SCC4 and SCC15 (both available at

ATCC) originated from well differentiated squamous

cell carcinomas of the floor of the mouth and the

ventral tongue, respectively [20]. Cell lines OKF6/

TERT-1 and OKF6/TERT-2 represent normal oral

mucosal epithelium (floor of the mouth) immortalized

by forced expression of telomerase via retroviral

transduction [21], and were provided by Dr D. Wong

(UCLA). Cell lines were maintained in Keratinocyte

Serum Free Media (KSFM; Invitrogen, Carlsbad, CA),

supplemented with 0.4 mmol/l CaCl2, 0.1 ng/ml epi-

dermal growth factor, 50 mg/ml bovine pituitary extract

(Invitrogen) and antibiotics (penicillin/streptomycin,

100 U/ml and 100 mg/ml, respectively).

Primary oral mucosal cell cultures were establishedfrom discarded gingival tissues obtained anonymously

from systemically healthy donors, undergoing

periodontal surgical procedures, following local IRB

protocol (IRB#: X10015). Within 4 h after excision the

tissues were washed exhaustively in antibiotics and

fungizone-supplemented media and were further pro-

cessed as described previously [6,22]. Briefly, to estab-

lish oral epithelial cell cultures, tissues were incubated

overnight in 0.4% dispase at 48C. Subsequently, the

epithelial layer was enzymatically and mechanically

separated from the underlying connective tissue and it

was further incubated in 0.05% trypsin/0.53 mmol/l

EDTA for 5/7 min with vigorous pipetting to achieve

cell dispersion. After trypsin neutralization and wash-

ing, the cell suspension was seeded in 25-cm2 flasks at a

density of 0.25)/106 cells in complete EpiLifeTM media

(Cascade Biologics, Portland OR), supplemented with

0.06 mmol/l CaCl2 and 0.05 mg/ml gentamycin

(Mediatech, Herndon, VA). Purity of these cultures

with respect to epithelial cell origin ranged within 92 /

98% as assessed by intracellular stain with a FITC-

conjugated mouse monoclonal anti-human cytokeratin

antibody, following the manufacturers instructions

(clone MNF116; Dako, Carpinteria, CA) and subse-

quent FACS analysis (not shown). This antibody

recognizes an epitope that is present in a wide range

of cytokeratins expressed by human gingival epithelium

[20]. Primary epithelial cells were used between pas-

sages three and five.

Explant oral fibroblast cultures were established

from gingival tissues as previously described [23,24]

and they were grown in Dulbeccos Modified Eagles

medium (DMEM; GIBCO, Grand Island, NY) supple-

mented with 2 mmol/l L-glutamine (GIBCO), penicil-

lin/streptomycin (GIBCO; 100 U/ml and 100 mg/ml,

respectively) and 10% heat-inactivated fetal bovine

serum (Mediatech). The cells were harvested byEDTA (0.53 mmol/l) Trypsin (0.05%) (Sigma Chemical

Company, St Louis, MO) and were transferred to 25-

cm2 tissue-culture flasks. Purity of these cultures, with

respect to stromal cell origin, was confirmed by indirect

immunofluorescence staining for vimentin. Fibroblast

cultures were used in this study between passages five

and 12.

2004 ISHAM, Medical Mycology, 42, 531/541

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Co-culture of C. albicans with oral epithelial cells

Epithelial cells were seeded in six-well (5)/105

cells/well) or 12-well (2)/105 cells/well) plates and

incubated overnight in complete KSFM. The following

day the media were discarded and the cells were

challenged with suspensions of stationary phase viable

blastospores at varying fungal cell to epithelial cellratios (0:1/100:1), for 24 h. Stationary phase yeast

organisms were prepared by growth for 18 h at 228C in

YPD broth (Difco Laboratories), supplemented with

2% (wt/vol) dextrose. The blastospores were then

harvested by centrifugation and washed twice in PBS.

Subsequently the yeast cells were counted in a hema-

cytometer and further adjusted to their final concen-

trations in complete KSFM before adding to epithelial

cells. Negative controls for these experiments included

uninfected cultures and C. albicans alone. In certain

experiments a protease inhibitor cocktail containing

aprotinin, bestatin, E-64, leupeptin and pepstatin A

(Sigma) was added during co-culture, at 1:200 dilution

(based on preliminary titration experiments), to pre-

vent the proteolytic cleavage of extracellular cytokines.

At the end of the experimental period, supernatants or

cell lysates were collected and stored at(/708C until

assayed.

Studies determining the bioactivity of IL-1a

IL-1a can induce IL-8 and GM-CSF responses by oral

epithelial cells [25] and fibroblasts [24], therefore we

tested whether IL-1a released from the interaction of

C. albicans with oral epithelial cells can act on

uninfected oral epithelial or stromal cells to upregulatethese cytokines. In these experiments, SCC15 cells were

challenged with viable C. albicans at 1:1 yeast to

epithelial cell ratio (strain SC5314) and 24-h super-

natants were collected and filter-sterilized. Uninfected

SCC15 cells and primary oral fibroblasts were then

challenged with these supernatants (diluted 1:2 in

KSFM and DMEM, respectively) and IL-8 and GM-

CSF secretion was measured after 24 h. Because IL-8

and GM-CSF are present in the supernatants used to

challenge uninfected cells [6,22], cytokine release by

these cells was calculated after subtracting the existing

IL-8 and GM-CSF amounts. To demonstrate that the

cytokine-inducing activity of these supernatants was

related to the presence of bioactive IL-1a, a neutraliz-

ing anti-IL-1a monoclonal antibody (10 mg/ml; BD

PharMingen, San Diego, CA) or IL-1 receptor antago-

nist (IL-1ra, 1 mg/ml; R&D Systems, Minneapolis, MN)

were added in these experiments. Preliminary titration

experiments confirmed that maximum effects of these

agents in this in vitro system are attained at these

concentrations. Isotype control antibody (IgG1; BD

PharMingen) was used as negative control at the same

concentration as the neutralizing anti-IL-1a antibody.

Exogenously added rhIL-1a (1 ng/ml; Sigma) was used

as positive control in these experiments. Negative

controls included media conditioned with live

C. albicans or with epithelial cells alone for 24 h.

Supernatants used to challenge uninfected cells con-

tained less than 0.01 endotoxin units per ml.

Detection of cytokines by ELISA

The concentrations of cytokines were quantified by

sandwich ELISA using commercially available mono-

clonal antibody pairs (Endogen MiniKit; Pierce, Rock-

ford, IL) according to the manufacturers protocols. In

each experiment, samples from two to four replicate

wells were pooled and assayed by duplicate ELISA.

The sensitivity of the assays were 8 pg/ml for IL-1a and

IL-8, and 4 pg/ml for GM-CSF. The absorbance values

and corresponding cytokine concentrations were deter-

mined with an Opsys MR microplate reader (Dynex

Technologies, Chantilly, VA) using the Revelation

QuickLink software (Thermo Labsystems, Chantilly,

VA). In some experiments, cytokine levels in each

sample were normalized over basal, unstimulated

secretion levels and were expressed as the Stimulation

Index (SI0/stimulated cytokine divided by constitutive

cytokine in pg/ml of sample).

Detection of full length and processed IL-1a

Full-length and processed forms of IL-1a were detected

in cell supernatants or lysates by Western blotting. Cellsupernatants were freed of cell debris by centrifugation

prior to testing. Cell lysates were prepared on ice by

adding a buffer containing 1% IGEPAL CA-630, 1

mmol/l EDTA, 0.02% NaN3, 0.5 mol/l PMSF, 1 mg/ml

aprotinin, and 2 mg/ml pepstatin in PBS. In some

experiments, IL-1a from culture supernatants or lysates

was first immunoprecipitated using a monoclonal

mouse anti-human IL-1a antibody (clone M421AE;

Pierce), which recognizes both the mature processed

and the unprocessed forms of the protein [11]. Subse-

quently, samples were incubated with ImmunoPure

Immobilized Protein G beads (Pierce). The beads were

then collected by centrifugation (5000 r.p.m. for 5 min),

washed and IL-1a was eluted in sample buffer at 1008C.

For immunodetection, equal amounts of samples

were loaded on a 12% acrylamide gel and fractionated

by electrophoresis. Proteins were then electrophoreti-

cally transferred onto a nitrocellulose membrane

(NitroBind; Osmonics, Minnetonka, MN). Mem-

branes were probed using a rabbit anti-human IL-1a


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polyclonal antibody (1 mg/ml; Pierce), followed by a

peroxidase-labeled secondary antibody at 1:15 000

dilution (Immun-Star Goat Anti Rabbit; BioRad,

Hercules, CA). To reduce nonspecific antibody binding

membranes were blocked using 5% non-fat dry milk in

PBS. Bands were visualized using a chemoluminescence

western blotting detection system (ECL Plus; Amer-

sham Biosciences, Piscataway, NJ). Band intensities

were compared using a densitometer.

Assessment of cytotoxicity

Trypan blue exclusion

Trypan blue dye exclusion was used to quantify the

number of viable cells at the end of the infection period.

Following collection of supernatants, 10% trypan blue

solution (in PBS) was added and the epithelial cells

were assessed under a light microscope. The number of

trypan blue positive cells per 50 cells was counted in

triplicate and expressed as a percentage.

LDH release

Cell lysis was assessed by the CytoTox-96 assay

(Promega), which measures release of lactate dehydro-

genase (LDH) from dying cells, according to the

manufacturers protocol. Total LDH release was esti-

mated by treating control cultures with Triton X-100

(9% solution in KSFM, Sigma) for 18 h. Experimental

values were then expressed as percent of the total LDH

release.

Statistical analyses

All data are presented as means9/SEM of measure-ments in three or more independent cultures (each

sample tested in duplicate). The statistical significance

of the differences in cytokine secretion in control and

infected cultures, or in the presence and absence of IL-1

inhibitors, was determined by two-tailed t-test assum-

ing unequal variances between groups. Differences were

statistically significant at PB/0.05.

Results

(1) Infection of oral epithelial cells with C. albicans induces

the release of mature IL-1a in culture supernatants

In the first series of experiments we determined whether

infection of oral epithelial cell monolayers with viable,

germinating C. albicans blastospores would induce

release of IL-1a. This was important since the existing

information about Candida -induced IL-1a synthesis in

oral epithelial cells is based on different experimental

systems, such as three-dimensional multilayered oral

epithelium reconstituted from cell lines [18], or short-

lived keratinocytes contained in human saliva [2]. To

determine the optimal conditions for infection of the

monolayers, epithelial cell lines were co-cultured with

increasing doses of yeast (strain SC5314) for 2/24 hand cell supernatants were analyzed for IL-1a by

ELISA. These preliminary experiments (data not

shown) indicated that yeast to epithelial cell ratios

ranging between 0.1 and 1.0 caused significant IL-1a

release after 8 h of infection, which is consistent with

prior reports in endothelial cells [26] and monocytes

[27]. The oral carcinoma cell lines responded to

infection with strains SC5314 and ATCC28366 (1:1

yeast to oral epithelial cell ratio) with an increase in IL-

1a in culture supernatants ranging approximately

between 7- and 37-fold above basal levels (PB/0.05

for SCC4 and PB/0.005 for SCC15 cells) (Table 1). The

retrovirally transformed cell lines OKF6/TERT-1 andOKF6/TERT-2 responded with a 16-fold increase in

IL-1a in culture supernatants above basal levels, which

was also statistically significant (Table 1).

To verify that the IL-1a response to C. albicans was

not limited to transformed epithelial cell lines, we next

Table 1 Interleukin-1-alpha (IL-1a) responses of oral epithelial cell lines to Candida albicans strains SC5314 and ATCC28366

Cell line Basal C. albicans

SC5314

C. albicans

ATCC28366

IL-1a in culture supernatants, pg/ml (fold over basal)

SCC4 4.69/0.8 168.79/41.6 (36.6)* 51.69/7.2 (11.2)*

SCC15 99.79/44.8 2370.09/168.0 (23.7)** 670.09/45.1 (6.7)**

OKF6/TERT1 120.09/11.0 1970.0 (16.4) 1930.0 (16.1)

OKF6/TERT2 89.39/44.9 1480.09/37.9 (16.5)*** 1440.0 (16.1)

Oral keratinocyte cell lines were cultured for 24 h in the presence or absence (basal) of live C. albicans added at 1:1 yeast to oral epithelial cell

ratio, and IL-1a was measured in culture supernatants. Basal and stimulated levels are expressed as mean IL-1a values from three separate

experiments9/SEM. Strain ATCC28366 was only tested once with the OKF6/TERT1 and OKF6/TERT2 cell lines. Asterisks indicate statistically

significant differences in infected cultures as compared to uninfected (basal) controls (* PB/0.05, **PB/0.005, ***PB/0.0005).



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determined whether primary gingival epithelial cell

cultures from seven different human donors responded

similarly to infection with C. albicans strains SC5314

and ATCC28366. To better standardize these experi-

ments all primary cultures were screened at passage 3.

Uninfected primary cultures secreted variable amounts

of IL-1a (ranging approximately between 100 and 600pg/ml). As seen in Table 2, although the seven primary

cultures had a relatively variable magnitude of response

to C. albicans challenge, all responded to infection with

the release of IL-1a in culture supernatants. The mean

response of these cultures to strain SC5314 was 11.1

(9/2.8)-fold above basal levels and the median response

8.2-fold above basal levels. The response to oral strain

ATCC28366 was 9.2(9/1.8)-fold and 7.5-fold above

basal levels, for mean and median respectively.

C. albicans-conditioned epithelial cell media did not

contain IL-1a-like molecules detectable by ELISA

(data not shown).

As a significant IL-1a response to C. albicans

infection was observed with all primary oral epithelial

cells and cell lines, our subsequent studies focused on

the mechanisms that underlie the process of IL-1a

release in culture supernatants. Because keratinocytes

are known to store large amounts of pre-formed,

unprocessed IL-1a intracellularly [10,28], we first

determined whether the cytokine released in culture

supernatants due to infection was the 33-kDa unpro-

cessed protein. As expected, uninfected oral epithelial

cell lysates contained IL-1a with a molecular mass of

33 kDa, assessed by western blot analysis, consistent

with the presence of the unprocessed cytokine. Incontrast, supernatants from SCC15 cells co-cultured

with C. albicans for 24 h contained the 17-kDa

processed form (Fig. 1A). In order to further define

whether some or all of this processing was occurring

extracellularly, a protease inhibitor cocktail was used

during co-culture, which inhibits the extracellular

degradation of secreted IL-1a [29] and has broad

specificity for the inhibition of serine, cysteine, aspartic,

thiol and aminopeptidases. Immunoprecipitation of

infected cell supernatants, generated in the presence

of protease inhibitors, followed by western blot analy-

sis, revealed a reduction of the 17-kDa signal by almost

40% and the appearance of a band with a molecular

mass of 33 kDa, as well as the intensified appearance of

a band between 33 and 17 kDa. This is consistent with

the presence of the full-length protein and an inter-

mediate IL-1a cleavage product, respectively (Fig. 1B).

Intermediate IL-1a cleavage products between 33 and

17 kDa frequently appear on IL-1a blots and are

assumed to result from alternate calpain cleavage

events [30]. Collectively these findings suggest that the

majority of the IL-1a released by oral epithelial cellsfollowing infection with C. albicans is the mature 17-

kDa protein and that less than 40% of the processing of

this protein is due to extracellular proteolysis.

(2) Cytolysis accompanies IL-1a release triggered by

C. albicans infection

We and others have previously shown that only live

germinating blastospores ofC. albicans in contact with

oral epithelial cells are capable of generating cytokine

responses [6,22], including triggering IL-1a mRNA

synthesis [18]. Although contact between oral epithelial

cells and live C. albicans is required for cytokineresponses, trypan blue staining of the oral epithelial

cells while in contact with germinating C. albicans

Table 2 Interleukin-1-alpha (IL-1a) responses of primary oral epithelial cells from seven individuals to Candida albicans strains SC5314 and

ATCC28366

Culture # Basal C. albicans

SC5314

C. albicans

ATCC28366

IL-1a in culture supernatants, ng/ml (fold over basal)

45 0.159 3.930 (24.7) 2.180 (13.7)

51 0.395 2.410 (6.1) 2.970 (7.5)

53 0.109 1.970 (18.1) 1.930 (17.7)

57 0.177 1.550 (8.8) 1.440 (8.1)59 0.307 1.240 (4) 1.060 (3.5)

60 0.605 4.960 (8.2) 4.330 (7.2)

61 0.342 2.750 (8.0) 2.240 (6.5)

Primary gingival keratinocytes were cultured for 24 h in the presence or absence (basal) of live Candida albicans added at 1:1 yeast to oral

epithelial cell ratio, and IL-1a was measured in culture supernatants. Each primary culture was tested once, at passage 3. Mean Basal IL-1 a for

the seven cultures: 0.2999/0.064 ng/ml (SEM). Mean IL-1a with strain SC5314 (n0/7): 2.6879/0.505 ng/ml (SEM). Mean IL-1a with strain

ATCC28366 (n0/7): 2.3079/0.408 ng/ml (SEM). The mean increases in IL-1a in cultures infected with either strain, as compared to uninfected

controls, were statistically significant (PB/0.005).



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SC5314 revealed that epithelial cells lying in close

proximity to hyphal structures stained positive, indicat-

ing advanced cell injury or necrosis (Fig. 2A,B).

In a series of kinetic experiments, we next character-

ized the relationship between cell injury, as assessed by

LDH release, and the release of IL-1a in culture

supernatants. A high correlation was shown between

the levels of LDH and IL-1a in culture supernatants at

each time point (R20/0.987; Fig. 3). Candida damage

to oral epithelial cells was detectable after 6 h of co-

culture and increased with longer exposure to the

organism. Most importantly, the time course of cell

injury paralleled the increase in IL-1a release in culture

Fig. 1 (A).Western blot analysis of IL-1a present in epithelial cell lysates and supernatants. Uninfected cell lysates were prepared by lysing

confluent SCC15 cell monolayers, grown in complete keratinocyte serum free media (KSFM), as described in materials and methods. Infected

SCC15 cells were exposed to viable Candida albicans SC5314 at 1:1 fungal cell to epithelial cell ratio, for 24 h. Samples were gel fractionated,

transferred onto nitrocellulose membranes and probed with a rabbit anti-human IL-1a polyclonal antibody. For reference, rhIL-1a was also run

in parallel. A representative blot from three independent experiments is shown. (B) Effect of extracellular protease inhibitors in the processing of

IL-1a released by infected cells. SCC15 cells were infected with C. albicans (strain SC5314, 1:1 infectivity ratio) in the presence or absence of

protease inhibitors for 24 h. IL-1a in supernatants was immunoprecipitated as described in materials and methods. As a positive control for the

detection of the unprocessed 33-kDa form, uninfected cell lysates, prepared as described above, were immunoprecipitated and were run in

parallel. Immunoreactive bands were detected with a polyclonal rabbit anti-human IL-1a polyclonal antibody and were quantified using

scanning densitometry. A representative blot using immunoprecipitated supernatants from two independent experiments is shown.

Fig. 2 Candida albicans triggers oral epithelial cell death. SCC15

cells were seeded on glass slides and incubated (A) alone or (B) in

contact with live, germinating C. albicans (strain SC5314, 1:1 yeast to

epithelial cell ratio) for 6 h. At the end of this co-culture period,

cultures were stained with 10% trypan blue in phosphate-buff-

ered saline (PBS) and were examined by phase-contract microscopy.

Bar0/10 mm.

Fig. 3 Linear regression analysis between epithelial cell injury (LDH

release) and the increase of IL-1a in culture supernatants. SCC15 cells

were co-cultured with Candida albicans SC5314 (1:1 infectivity ratio)

for 0, 3, 7, 12 or 24 h, and cell supernatants were analyzed for IL-1a

(y axis) and LDH presence (x axis), using colorimetric assays as

described. Each data point represents LDH and IL-1a release at each

time point, and is an average of duplicate determinations from two

independent experiments. The bars represent 1 SEM.



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supernatants, with maximal release of IL-1a taking

place at 24 h of co-culture, when approximately 100%

of the monolayer was lysed (Fig. 3). To further support

the hypothesis that IL-1a was released by cell lysis

during C. albicans infection, cell monolayers were co-

cultured with increasing numbers of yeast for 8 h and

IL-1a concentrations were determined in cell super-natants and cell lysates by ELISA. In parallel, cell

viability was assessed by trypan blue dye exclusion or

by the LDH release assay using culture supernatants.

Uninfected cell lysates contained approximately 1.5 ng/

ml of pre-formed IL-1a. As a result of infection with

lower yeast inocula (1:10 and 1:1 yeast to epithelial cell

ratios) the intracellular IL-1a concentration increased

by approximately two-fold (PB/0.05), while the IL-1a

concentration in culture supernatants remained at or

close to basal, uninfected levels (Fig. 4). At these low

infectivity ratios cell viability was maintained at high

levels (90/

100%), as assessed by trypan blue exclusion(Fig. 4) or the LDH release assay (not shown). The

highest yeast inocula (10:1 and 100:1 yeast to epithelial

cell ratios) caused a drop in cell viability (35/55%),

accompanied by increase of IL-1a in culture super-

natants and a significant decrease in the intracellular

IL-1a concentration.

Taken together, these results suggest that IL-1a in

oral keratinocytes remains intra-cellular and that

C. albicans -induced cell lysis is responsible for the

release of pre-formed as well as newly synthesized IL-

1a in culture supernatants.

(3) Different strains ofC. albicans vary significantly in their

cytolytic activity as well as in their ability to trigger IL-1a

release by oral epithelial cells

To further substantiate a relationship between

C. albicans cytolytic activity and the potential for

stimulating IL-1a release, strains exhibiting different

degrees of cytotoxicity to oral epithelial cells (as

evidenced by the differential release of LDH from

oral epithelial cells during co-culture), but similar

growth rates in KSFM (data not shown), were exam-

ined. The ability of C. albicans strain SC5314 to trigger

IL-1a responses by oral epithelial cells was compared

to that of strains ATCC28366 and ATCC32077, when

added at increasing yeast to epithelial cell ratios, and

levels of LDH in culture supernatants were quantified

in parallel. When SCC15 cells were tested, strain

SC5314 was the most potent inducer of IL-1a release,

as well as the most potent inducer of cell lysis, causing

approximately 100% cell lysis after 24 h at the highest

inocula tested. Strain ATCC28366 induced moderate

release of IL-1a, paralleled by moderate LDH release

in culture supernatants. Strain ATCC32077 did not

trigger a significant IL-1a increase, even when higher

yeast inocula were tested, consistent with the low levels

of cell injury induced by this strain after 24 h of co-

culture (Fig. 5). Strains SC5314 and ATCC28366exerted similar degrees of cytotoxicity when co-cultured

with primary oral keratinocytes (not shown), consistent

with their ability to stimulate similar release of IL-1a in

culture supernatants of these cells (Table 2).

(4) IL-1a released byC. albicans-infected oral epithelial cells

can stimulate other pro-inflammatory cytokines in

uninfected oral epithelial or stromal cells

Because IL-1a is a potent stimulus of IL-8 and GM-

CSF in oral epithelial cells [25] and fibroblasts [24], the

possibility was tested that bioactive IL-1a released by

infected epithelial cells can increase secretion of these

proinflammatory cytokines by neighboring uninfected

cells. To test this hypothesis, uninfected oral epithelial

cells and fibroblasts were challenged with supernatants

derived from the interaction of oral epithelial cells

(SCC15 cells) with C. albicans (strain SC5314), and the

GM-CSF and IL-8 content was analyzed in culture

supernatants. These experiments showed that the

released IL-1a was bioactive since supernatants of

Fig. 4 Relationship between Candida albicans inoculum, IL-1a

release and epithelial cell viability. SCC15 cells were infected withC. albicans SC5314 at different infectivity ratios, as indicated, for 8 h.

Cell lysates (/) and supernatants (") were analyzed for the presence

of IL-1a by ELISA. In parallel, the number of viable cells remaining

in the monolayers at the end of the co-culture period (^) was

determined in adjacent wells by trypan blue dye exclusion, and is

expressed as a percentage of viable cells in a total of 50 cells counted.

A representative of two independent experiments is shown. Each data

point is the average of duplicate determinations. The bars represent 1

SEM.



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C. albicans infected cell cultures stimulated the secre-

tion of GM-CSF and IL-8 by uninfected cells, and this

activity was inhibited by the addition of IL-1ra or anti-

IL-1a but not by isotype control antibody (Table 3).

Fibroblast responses to these supernatants were com-

pletely abrogated with IL-1ra, indicating that these

responses were entirely IL-1-dependent. In contrast,

partial inhibition (60/70%) was accomplished when

anti-IL-1a or IL-1ra were used to block cytokine

secretion by oral epithelial cells, suggesting that in

addition to IL-1a and IL-1b other mediators were also

responsible for triggering cytokine secretion in these

cells. Media conditioned by epithelial cells alone or by

live germinating organisms did not activate IL-8

secretion in both uninfected cell types, or GM-CSF

secretion by fibroblasts (Table 3). A statistically sig-

nificant (PB/0.05) effect of supernatants conditioned

with live C. albicans (supernatants C) was noted on

uninfected epithelial cell GM-CSF secretion, which

suggests that C. albicans -derived products, may be

partly responsible for the GM-CSF stimulation in

epithelial cells.

These data strongly suggest that C. albicans induces

the release of bioactive IL-1a by oral epithelial cells and

that IL-1a can act as an early amplification signal in

the mucosal inflammatory response to this pathogen.

Discussion

Oral candidiasis is characterized by a recurrent,

persistent, acute inflammatory reaction to Candida

infection, which is limited to the uppermost epithelial

layers of the oral mucosa. The inflammatory response

to this pathogen elicits chronic pain and discomfort

upon mastication, but it may also be responsible for the

prevention of invasive infection. The mechanisms that

trigger this acute inflammatory response in the oral

Table 3 Cytokine responses of uninfected oral epithelial cells and fibroblasts to conditioned media

Oral epithelial cells Oral fibroblasts

Stimulus IL-8 (pg/ml) GM-CSF (pg/ml) IL-8 (ng/ml) GM-CSF (pg/ml)

None 181.09/28.2 4.09/3.1 1.49/0.1 22.09/1.2

IL-1a 865.09/49.5 34.69/2.7 72.59/2.1 500.09/23.4

IL-1a'/anti-IL-1a 155.59/31.8* 8.09/2.2* 10.59/3.5* 36.39/4.6**

IL-1a'/ctrlAb 945.09/7.1 39.39/3.2 75.09/9.9 ND

IL-1a'/IL-1ra 159.59/4.9* 9.09/1.3** 0.99/0.6** ND

B supernatants 1595.09/49** 45.79/3.4* 36.69/1.1** 577.09/10.9

B'/anti-IL-1a 490.09/113* 16.59/2.1* 10.69/1.2* 87.59/4.6**

B'/ctrlAb 1445.09/162.6 34.59/2.4 37.99/0.4 470.09/42.3

B'/IL-1ra 650.09/23.0** 12.69/1.1** 1.29/0.4** 26.49/4.5**

A supernatants 101.59/64.3 6.59/1.3 0.359/0.1 149/3.3

C supernatants 221.59/48.7 11.29/2.5* 0.479/0.4 259/2.7

Oral epithelial cells (SCC15) and fibroblasts were challenged with media conditioned by: oral epithelial cells (SCC15) alone (A supernatants);

oral epithelial cells co-cultured with C. albicans SC5314 at 1:1 yeast to oral epithelial cell ratio (B supernatants); and C. albicans SC5314 alone

(C supernatants), for 24 h and IL-8 and GM-CSF were measured in culture supernatants. Basal and stimulated levels are expressed as means

9/SEM of three independent experiments. Statistical comparisons were performed as follows: the effects of B supernatants were compared to

those of C; the effects of anti-IL-1a and IL-1ra when used in conjunction with rhIL-1a or B supernatants were compared to the effects of IL-1a

and B supernatants alone. The IL-8 and GM-CSF concentrations in the supernatants used to challenge the cells were as follows: supernatants A:

1939/24.2 and 49/4.1 pg/ml; supernatants B: 980.79/67.6 and 11.59/2.3 pg/ml; supernatants C: none detectable. (*PB/0.05, **PB/0.005, ND,

not determined).

Fig. 5 The ability of different Candida albicans strains to induce IL-

1a release is closely related to their cytolytic activity. SCC15 cells were

infected with C. albicans (strains SC5314, ATCC28366 or

ATCC32077) at increasing infectivity ratios (1:10, 1:1, and 10:1 yeastto epithelial cell ratio). IL-1a (lines) and LDH (bars) were assessed in

culture supernatants as described. Results are expressed as the mean

LDH and IL-1a value for each condition, obtained by analysis of

three separate experiments. The bars represent 1 SEM.



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mucosa are unknown. However, dissection of this

process is critical to the understanding of the patho-

genesis of this fungal infection and may be important

for the development of strategies to prevent invasive

infection in immunocompromised hosts. The findings

presented here suggest a novel mechanism for candidal

pathogenesis in the oral mucosa, whereby the acute

host response to infection is initiated and perpetuated

by oral epithelial cells, the first and principal targets of

infection. As shown herein, Candida -infected oral

epithelial cells synthesize significant amounts of the

proinflammatory cytokine IL-1a and release this cyto-

kine in a bioactive form in their microenvironment

upon cell lysis. Furthermore, we showed that IL-1a

released by injured cells can increase the proinflamma-

tory cytokine production by neighboring mucosal and

stromal cells. Such a mechanism could serve to amplify

and extend the local inflammatory response, even in the

absence of direct fungal invasion of the deeper mucosal

and submucosal tissues. The local release of cytokinessuch as IL-1a, IL-8 and GM-CSF by oral epithelial

cells and fibroblasts could explain the histopathologic

finding of neutrophilic microabscesses in these lesions

[4,8], as these cytokines are potent chemoattractants

and/or activators of PMN [31/34].

Our findings suggest that the cytolytic release of IL-

1a may have a central role in the inflammatory

response to this pathogen. Lysis of the host cells may

be mediated by Candida cysteine proteases [35], kera-

tinases [36], aspartyl proteases [37] or phospholipases

expressed at the tips of hyphal organisms [38], which

invade into and germinate within the epithelial cell

cytoplasm [39]. We and others have shown that onlylive, germinating organisms are capable of stimulating

proinflammatory cytokine responses in non-immune

cells [6,22,40]. Our findings here further support the

hypothesis that epithelial cells from mucosal sites that

normally harbor a great number of commensal organ-

isms, such as colon, vagina or oral cavity, require

contact with the microorganism and active invasion of

the host cell cytoplasm, possibly via destruction of the

plasma membrane, for a proinflammatory response. In

agreement with our findings, it has been shown that

lytic infection of colon or vaginal epithelial cells with

live organisms, such as Entamoeba histolytica , Chla-

mydia trachomatis, or invasive strains of bacteria

induces the expression of an array of proinflammatory

cytokines, and that the release of IL-1a is an important

regulator of other coordinately expressed cytokines,

including IL-8 and GM-CSF [11,41,42]. Epithelial cells

are not unique in this regard, as candidal induction of

endothelial cell cytokine and adhesion molecule re-

sponses also requires physical contact with the organ-

ism, and is closely associated with endothelial cell

injury [26,40].

It has been reported that different strains of

C. albicans are able to induce different cytokine signals

in PBMC and monocytes and that the magnitude of the

cytokine signal is also dependent on the individual cell

donor [27]. Oral epithelial cells function similarly in

this regard since C. albicans strains differed in their

capacity to trigger IL-1a responses, and cells derived

from different donors exhibited variable magnitudes in

these responses. Our data also support the hypothesis

that the lack of the ability of certain commensal strains

to trigger a proinflammatory response in the oral

mucosa may be related in part to their inability to

cause a breakdown of the host cell membrane integrity,

when in contact with oral epithelium.

Although keratinocytes were among the first cell

types described to synthesize and store large amounts

of IL-1a [28], the mechanism of IL-1a export from

these cells is still unclear. Like IL-1b, IL-1a lacks theaminoterminal signal peptide required for efficient

secretion [10], but can be secreted by monocytic cells

in the processed form in response to infectious and

other stimuli [43]. Cell fractionation and immunoelec-

tron microscopic analyses of IL-1a have localized the

nascent pro-peptide on polysomal or plasma mem-

branes [44,45]. Also, in contrast to IL-1b, mature IL-1a

is almost never observed intracellularly in these cells

[30]. Recent studies have suggested that physical injury

to cells, regardless of the insult, and not a unique

secretory pathway, may be responsible for the majority

of the processing and release of the mature IL-1a

protein [46,30]. Because the cytolytic actions of severalagents (nigericin, ATP, LPS) are accompanied by a

dramatic release of mature IL-1a in some cell systems

[43,46], it has been hypothesized that processing of the

newly synthesized IL-1a requires a second stimulus,

which may be associated with the breakdown

of membrane integrity. The enzyme responsible for

IL-1a processing is a neutral protease known as calpain

[29]. Activation of calpain occurs at the inner surface

of the plasma membrane in the presence of calcium and

released phospholipids [47]. Based on this information

and our finding that the majority of the IL-1a

in culture supernatants is fully processed, we propose

that most of the IL-1a processing in our co-culture

system takes place at the plasma membrane where the

cytolytic actions of C. albicans phospholipases and

proteases trigger a release of membrane phospholipids.

This in turn leads to calpain activation coupled

with digestion of membrane-associated pro-IL-1a and

the release of the mature protein in culture super-

natants.



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In view of this suggested processing and release

mechanism in our system, our finding that the presence

of protease inhibitors during co-culture reduces the

signal of the fully processed protein, may be explained

in two ways. First, some of these protease inhibitors

(E64 and leupeptin) are known to inhibit calpain [29]

and may gain access to this enzyme when membrane

damage is advanced. Second, protease inhibitors such

as pepstatin A are successfully inhibiting some of the

extracellular processing of this protein due to the

presence of C. albicans aspartyl proteases, which can

cleave IL-1 and generate 17/19-kDa protein fragments

[48].

The IL-1a released in our in vitro co-culture system

was bioactive, as tested on human gingival fibroblasts

and epithelial cells. This is an important finding since in

certain IL-1 bioassays activity of IL-1a and IL-1b

is known to be hampered by soluble metabolic products

released by C. albicans [48]. Using a similar co-culture

system, we previously reported that IL-1a resultingfrom the interactions of oral epithelial cells with

C. albicans autoregulates part of the IL-8 secretion

in response to this pathogen [22]. Our present

work extends those findings by showing that rel-

eased IL-1a can also regulate proinflammatory cyto-

kine responses by neighboring uninfected oral mucosal

cells.

Our studies suggest that production of IL-1a, IL-8

and GM-CSF may take place in the oral mucosa in

response to C. albicans infection. Expression of these

cytokines in the oral epithelium can act as an early

innate immune surveillance and warning system for

leukocytes in the underlying stroma. Induction of thisspecific group of cytokines may have important con-

sequences for the pathogenesis of invasive candidiasis

since resistance or susceptibility to invasive infection

may correlate closely with the presence and activity of

these cytokines. All of these cytokines are key host-

response molecules in the protection against fungal

infection as they have the ability to promote PMN,

monocyte and keratinocyte antifungal activities [32/

34]. Increased production of proinflammatory cyto-

kines by oral epithelial cells and fibroblasts combined

with the cytolytic activity of the inflammation-inducing

C. albicans strains are also likely responsible for the

clinical findings of redness and surface ulceration of the

oral mucosa during this superficial oral infection.

Future animal studies are needed to determine whether

mitigation of these proinflammatory cytokine re-

sponses and the ensuing acute inflammation would

ameliorate the clinical symptoms of this superficial

infection, and/or promote invasion into the submucosal

tissues.

Acknowledgements

This study was supported by USPHS Research Grants

RO1 DE13986 and RO3 DE12668 to A.D.B. from the

National Institute of Dental and Craniofacial Re-

search, National Institutes of Health, Bethesda, MD

20892. The authors thank Dr Stephen Wikel, Director

of the Center for Microbial Pathogenesis at UCHC, forhis thoughtful review and suggestions.

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