This study investigated cytokine and chemokine production in response to C. albicans
by enriched populations of epithelial cells isolated from the oral and vaginal mucosa and by human epithelial cell lines. Since Candida
is in contact with epithelial cells more than any other cell type at mucosal sites and since epithelial cells secrete cytokines (20
), the potential is high for epithelial cells to contribute to the local cytokine milieu present during asymptomatic or symptomatic mucosal Candida
colonization. Although C. albicans
exists as an asymptomatic commensal at both the oral and the vaginal mucosa, we investigated cytokine and chemokine production under conditions that simulated a C. albicans
mucosal infection (i.e., predominantly hyphae from the natural transformation of C. albicans
during the 96-h culture) to ascertain the potential role of epithelial cells in in vivo mucosal immune responses against the pathogenic form of the organism. In addition, we confirmed that C. albicans
did not contribute to or interfere with the detection of the cytokines.
The cytokines examined in this study were chosen based on the fact that they represent important members of proinflammatory, regulatory, T-helper, and chemotactic cytokine classes. The coculture design in this study employed a variety of controls in order to best determine the ability of epithelial cells to produce cytokines constitutively and in response to Candida. Constitutive cytokine production by the cells may represent a homeostatic mechanism of innate immunity at mucosal tissues or consequences of the in vitro culture conditions.
Results showed that primary oral and vaginal epithelial cells produced considerable levels of TNF-α and IL-1α both constitutively and in response to C. albicans
as early as 24 h postculture. The exception was primary vaginal epithelial cells that produced little to no Candida
-specific IL-1α. IL-6, IFN-γ, IL-12, IL-10, MCP-1, and IL-8 were also variably produced in undetectable to low concentrations constitutively with little to no additional production in response to C. albicans
through the 96-h culture. For the epithelial cell lines, high constitutive levels of IL-1α, IL-6, and IL-8 were observed as early as 24 h postculture, but there was little to no Candida
-specific production of IL-6, IL-12, IL-10, MCP-1, and IL-8 and only moderate production of TNF-α. On the other hand, IL-1α was produced in relatively high concentrations by the vaginal cell line, but not by the oral/cervical cell line, in response to C. albicans
, a result similar to that seen with primary oral epithelial cells. Interestingly, IL-1α was the one cytokine that was not consistently produced by either primary cells or the cell lines. A potential explanation is that IL-1α can be released from intracellular stores when the cell expires (32
). Although we did not observe vast changes in cell viability between pre- and postculture, it is possible that IL-1α was released from dying cells that were variable in each culture. Finally, TGF-β was not produced at detectable levels by either primary or epithelial cell lines alone or in response to C. albicans
The use of epithelial cell lines enabled us to predict the source of the cytokines produced in the enriched primary populations. Accordingly, since the cytokines in most cases were produced by both types of primary epithelial cells and the epithelial cell lines, and since the primary populations were >95% pure, our data suggest that the cytokines were indeed epithelial cell derived. We recognize, however, that the immortalized characteristics of the epithelial cell lines employed in these studies may not accurately predict the outcome of nonimmortalized epithelial cells (i.e., primary epithelial cells). Our results are also consistent with the high levels of IL-1α, IL-6, and IL-8 produced by the cell lines in response to TNF-α as an integrity control for the cells and the culture conditions (1
). It is unclear why the primary epithelial cells did not similarly produce cytokines in response to TNF-α, although the state of differentiation may not be permissive for cytokine stimulation without the presence of the appropriate receptors. In contrast, the high concentrations of IL-6 and IL-8 produced by the cell lines, but not by the primary cells, may be due to the transformed characteristics of the epithelial cell lines. In any event, our results are consistent with results previously reported for these cell lines (7
It has been known for some time that epithelial cells of the gastrointestinal and urinary tracts produce cytokines in response to microorganisms such as Neisseria gonorrhoeae
, Shigella dysenteriae
, and Chlamydia trachomatis
). C. albicans
can now be added to this list as the first fungal organism with similar properties. In fact, the concentrations of IL-1α and TNF-α produced in response to C. albicans
were similar to those induced in these other epithelial cell populations. The only other attempt to stimulate cytokine production by epithelial cells in response to C. albicans
was through the confluent culture of gingival epithelial cells or with the oral/cervical epithelial cell line employed in this study. Under these conditions, both IL-6 and IL-8 were produced in high concentrations by both types of epithelial cells (7
). Although it is unclear why the epithelial cell line in our study produced lower levels of IL-6 and IL-8 in response to C. albicans
than did the previous study, culture conditions (i.e., tissue culture media, cell preparations, and effector/target ratios, etc.), the state of the epithelial cell line (i.e., prior to contamination with HeLa cells), and a different C. albicans
strain may have contributed to the differences despite the similar levels produced in the presence of TNF-α.
Based on the documented role for T-helper responses in protection (Th1) or susceptibility (Th2) to Candida
), Th1 and Th2 cytokines have been detected in mucosal secretions from both the oral and the vaginal mucosa (11
). However, data from our study showed low concentrations of both Th1 (IFN-γ and IL-12) and Th2 (IL-10) cytokines produced by oral and vaginal epithelial cells, with little to no modulation in response to C. albicans
. Therefore, our data suggest that cells other than epithelial cells most likely contribute to these T-helper cytokines present during mucosal C. albicans
Also interesting was the lack of TGF-β production by oral and vaginal epithelial cells. Clinical (P. L. Fidel, M. Barousse, T. Espinosa, R. R. Chesson, and K. Dunlap, submitted) and experimental (38
) studies from our laboratory show the presence of TGF-β in vaginal secretions. As such, TGF-β has been postulated to play a downregulatory or tolerance role in vaginal immunity (38
). However, data from this study suggest that, as with the Th1 and Th2 cytokines, cells other than epithelial cells are responsible for the presence of TGF-β in vaginal secretions. In contrast, the level of TGF-β is low in the saliva of healthy individuals as well as in those with OPC (P. L. Fidel, unpublished results) and thus would not be expected to be produced by oral epithelial cells.
Although the presence of proinflammatory cytokines in mucosal secretions has not been investigated during OPC or VVC, our study clearly shows the production of IL-1α and TNF-α by oral and vaginal epithelial cells in response to C. albicans
. If similar proinflammatory cytokines are produced in vivo, an inflammatory condition would be expected. Indeed, edema and erythema are common symptoms of both OPC and VVC (19
). Interestingly, TNF-α and IL-1α have been reported to play an active role in the immune response to C. albicans
) and have been used therapeutically during experimental infection (24
Chemokines are considered critical for migration of cells to sites of pathogenic insults. Accordingly, some chemokines (MCP-1 in particular) have been constitutively present at the vaginal mucosa in mice and are elevated during infection (33
). If a similar scenario occurs in human tissue, the moderate amounts of IL-8 and MCP-1 produced by oral and vaginal epithelial cells, together with little to no additional production in response to C. albicans
, suggests that epithelial cells are not a primary source of these chemokines.
Additional considerations are the similarities in cytokine production between oral and vaginal epithelial cells with few exceptions. These results suggest that these epithelial cells, although anatomically distinct, are similar in their capacity to produce cytokines and respond to Candida. We recognize, however, that the studies and interpretations discussed here are from in vitro culture conditions and may not reflect fully the activity of the cells in the intact mucosal microenvironment.
In summary, this is the first comprehensive evaluation of cytokine production, albeit in vitro, by primary oral (buccal) and vaginal epithelial cells both constitutively and in response to C. albicans. Accordingly, our findings suggest that oral and vaginal epithelial cells are an important source of proinflammatory cytokines (TNF-α and/or IL-1α) in response to C. albicans that may contribute to the local immune response at either mucosal site. This, together with future studies examining additional cytokines and chemokines present in mucosal secretions and in epithelial cell-Candida cocultures from other patient populations (e.g., HIV-positive individuals with or without OPC or women with RVVC), will undoubtedly provide a more complete picture of the local host response to mucosal C. albicans infections.