Candida albicans-infected oral epithelial cells augment the anti-fungal activity of human neutrophils in vitro.

Oropharyngeal candidiasis (OPC) is the most common opportunistic infection in immunosuppressed patients. In OPC, Candida albicans persists intraepithelially triggering inflammatory events, without generally causing invasive infection. Since neutrophils play an important role in preventing invasive infection and since they establish contact with the microorganisms only within the epithelial cell layer, we examined the ability of Candida-infected oral epithelial cells to augment neutrophil-mediated hyphal damage in vitro. We found that challenge of neutrophils with hyphal organisms in the presence of C. albicans-infected oral epithelial cell supernatants resulted in a significantly greater suppression of hyphal cell metabolic activity compared to basal neutrophil anti-fungal function. Anti-hyphal activity in response to these supernatants was partly inhibited by neutralizing anti-IL-1alpha antibody and IL-1 receptor antagonist. Control supernatants from uninfected oral epithelial cells, as well as C. albicans conditioned-medium had a much less pronounced effect on neutrophil anti-fungal activity, which was not inhibited by these cytokine antagonists. We conclude that oral epithelial cells can act as activators of neutrophil anti-hyphal function, an effect that can be partly attributed to the generation of immunomodulatory cytokines during the interaction of oral mucosal cells with the pathogen.     

Candida albicans triggers interleukin-8 secretion by oral epithelial cells

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha.     

Intercellular adhesion molecule 1-dependent activation of interleukin 8 expression in Candida albicans-infected human gingival epithelial cells

Increased induction of interleukin 8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by oral epithelial cells may play a role in the host defense mechanism in oropharyngeal candidiasis; however, little is known about the expression feature of these molecules on human gingival epithelial cells (HGECs) during Candida albicans infection. In this report we present evidence that neutralization with antibody against ICAM-1 inhibited both the adherence of C. albicans to HGECs and the Candida-induced production of IL-8, suggesting a role for ICAM-1 in recognition and signaling in HGECs to express IL-8 upon infection with C. albicans.     

Autocrine regulation of interleukin-8 by interleukin-1alpha in respiratory syncytial virus-infected pulmonary epithelial cells in vitro.

Respiratory epithelial cells infected with respiratory syncytial virus (RSV) produce interleukin-8 (IL-8); however, the mechanisms of RSV-induced regulation of IL-8 are poorly understood. In the present study, the regulation of IL-8 by RSV was evaluated using pulmonary type II-like epithelials (A549). Live purified RSV (pRSV) induced a significant increase in IL-8 after 8 hr of exposure, while conditioned supernatants from pRSV-infected A549 cells (cRSV) induced IL-8 production in fresh A549 cultures within 4 hr of infection. Furthermore, cRSV that had been rendered non-infectious by ultraviolet-irradiation (UV-cRSV) or ribavirin treatment also induced an increased production of IL-8 in fresh A549 cells, suggesting that RSV induced the synthesis of a soluble mediator(s) which in turn enhanced the synthesis of IL-8. We have previously shown that RSV-infected A549 cells produce IL-1alpha, IL-1-beta and tumour necrosis factor-alpha (TNF-alpha), which by themselves are known to induce the synthesis of IL-8. Preincubation of UV-cRSV or simultaneous incubation of pRSV with recombinant IL-1 receptor antagonist almost completely blocked (95-98%) the production of IL-8 by A549 cells. Furthermore, incubation with neutralizing antibodies against IL-1alpha, IL-1beta and TNF-alpha showed that IL-1alpha was the predominant soluble mediator that enhanced the mRNA expression and synthesis of IL-8. IL-1beta and TNF-alpha induced the synthesis of IL-8 at 24 hr, but partially inhibited the synthesis at 48 hr. In summary, these experiments provide direct evidence for an autocrine mechanism of enhanced IL-8 production in RSV-infected epithelial cells that is primarily mediated by IL-1alpha. In clinical settings, inhibitors of IL-1alpha may be useful in suppressing inflammation due to IL-1alpha as well as IL-8.     

Adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining

OBJECTIVE: To examine the relative adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining. METHODS: Oral epithelial cells were collected from 10 healthy adults (five male, five female) and counted. Equal volumes of oral epithelial cells and candida were mixed and incubated. The epithelial cells from this mix were collected by filtration through 10 microns polycarbonate membrane filters. Cells retained on the membrane filters were stained with crystal violet followed by Papanicolaou stain. The number of yeast attached to each of 100 red, orange, and green staining oral epithelial cells was determined by direct microscopic examination. RESULTS: C albicans had a higher level of adherence (p < 0.001) to red staining oral epithelial cells (mean (SD) number of candida attached to 100 oral epithelial cells 562 (159)) than to cells staining either orange (105 (47)) or green (161 (66)). CONCLUSIONS: Oral epithelial cell variability for candidal adherence is confirmed. The technique provides an opportunity to examine the relation between oral epithelial cell type and oral candidosis in specific groups, such as tobacco smokers, where increased epithelial cell keratinisation and candidal colonisation has been reported.     

Interleukin-6 production by thyroid epithelial cells. Enhancement by interleukin-1.

Interleukin-1 is a potent inhibitor of thyroglobulin and cAMP production in human thyroid cells and the inhibitory effect is enhanced by tumor necrosis factor-alpha and interferon-gamma. In the present study secondary cultures of human thyroid cells produced interleukin-6 and the production was significantly increased after exposure of the cells to recombinant interleukin-1 alpha and -1 beta. This increase was dose-dependent and concomitant of the IL-1 induced decrease in cAMP and thyroglobulin production. Both tumor necrosis factor-alpha and -beta also augmented interleukin-6 production, but less potently than interleukin-1. Interferon-gamma did not affect the production of interleukin-6. The rat thyroid cell line FRTL-5 produced interleukin-6 spontaneously, and the production was enhanced after addition of recombinant interleukin-1 beta. A pathogenetic role of interleukin-6 in autoimmune thyroid disease is suggested.     

Human interleukin-1 alpha is chemotactic for normal human keratinocytes

Interleukin-1 (IL-1) has been shown to have mitogenic and chemotactic properties for a variety of cell types includes keratinocytes. These studies suggested that keratinocytes possess receptors for IL-1. In this study, the chemotactic properties of IL-1 for keratinocytes were confirmed and IL-1 receptors were demonstrated on keratinocytes using a radio receptor assay. Crosslinking studies with IL-1 alpha identified two major bands of Mr 97 kDa and 133 kDa. Thus, keratinocytes possess high affinity IL-1 receptors and respond to IL-1 by directed migration.     

Enumeration of interleukin-1 alpha and beta producing cells by flow cytometry.

A technique for intracytoplasmic immunofluorescence staining to detect and quantify human interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in CD4, CD8, and CD14 positive lymphoid cells is described. Mononuclear cells stimulated in vitro with PHA to produce IL-1, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic and surface staining of both forms of IL-1 were demonstrated by indirect fluorescence using IL-1 beta and IL-1 alpha specific mouse monoclonal antibodies and quantified with flow cytometry.     

Adherence of Candida albicans and other Candida species to mucosal epithelial cells.

To study the possible involvement of candidal adherence in mucosal colonization, we examined the in vitro adherence capabilities of seven Candida species. Adherence was evaluated by direct microscopic examination and by a quantitative radiometric adherence test. The results indicate that C. albicans adheres to vaginal and buccal epithelial cells to a significantly greater degree (P less than 0.01) than the other species tested. C. tropicalis and C. stellatoidea demonstrated moderate adherence capabilities, while C. parapsilosis adhered only to a slight degree. Other species failed to interact with isolated mucosal cells. These findings suggest that there is a relationship between the adherence capabilities of the Candida species and their abilities to colonize mucosal surfaces, since those species which adhere are those which most frequently colonize mucosal surfaces. C. albicans was found to be adherent under a variety of environmental conditions. Stationary-phase blastospores of C. albicans were found to be more adherent than logarithmic-phase yeasts, and larger blastospore cell-to-epithelial cell ratios resulted in greater adherence values. The actual number of adherent yeasts varied considerably when epithelial cells were obtained from different donors.     

Natural killer cells do not play a dominant role in CD4+ subset differentiation in Candida albicans-infected mice.

The effects of in vivo administration of monoclonal antibodies against NK-1.1-bearing cells on the early production of gamma interferon (IFN-gamma) in vitro and development of Th1-associated immunity were studied in mice infected with a live vaccine strain of Candida albicans. At 1 and 4 days postinfection, natural killer (NK) cell-enriched fractions from the spleens of antibody-treated mice displayed a dramatic reduction in 5E6+ lymphocytes and negligible anti-YAC-1 cytotoxic activity in vitro. Nevertheless, the frequency of IFN-gamma-producing cells in those fractions was reduced by less than half, on average, by anti-NK-1.1 treatment in vivo. In addition, the antibody-treated and infected mice demonstrated unchanged T helper cell responses, as measured by yeast-specific footpad reactions, resistance to reinfection, occurrence of antibodies of different isotypes, and production in vitro of interleukin-2 (IL-2), IFN-gamma, IL-4, and IL-10 by CD4+ cells. Therefore, although NK cells may contribute to early IFN-gamma production in Candida-vaccinated mice, these cells apparently do not play a dominant role in the qualitative development of yeast-specific T helper responses.     

HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.     

Follicle epithelial M cells are a source of interleukin-1 in Peyer's patches.

The production of interleukin-1 (IL-1) by rabbit Peyer's patch M cells populating the follicle-associated epithelium (FAE) was studied. Sorted 5D9+ phagocytic epithelial M cells synthesized IL-1 after stimulation with lipopolysaccharide (LPS) in vitro, as evidenced by the ability of culture supernatants to induce the proliferation of the T-cell line D10.G4.1. Fixed LPS-stimulated M cells were less effective at mediating T-cell proliferation than supernatants from LPS-activated M cells. The magnitude of T-cell proliferation was M-cell concentration dependent, and proportional to the dose of LPS. The M-cell-mediated D10.G4.1 cell proliferation was inhibited > 75% with anti-IL-1 alpha, but < 50% with similar concentrations of anti-IL-1 beta. The results show that M cells secrete IL-1, and suggest the participation of M cells in the delivery of a localized co-stimulatory signal for T-cell and B-cell proliferation in the microenvironment of gut-associated lymphoid tissue (GALT).     

Recombinant interleukin-1 alpha inhibits the growth of rat mesangial cells in culture.

We have investigated the effect of interleukin-1 (IL-1) on the growth of mesangial cells isolated from rat kidney. Recombinant IL-1 alpha inhibited 3H-thymidine uptake by mesangial cells in a dose-dependent manner in the presence of either 0.5% or 5% fetal bovine serum (FBS). In the presence of high concentration of FBS (10%), the effect of IL-1 was not prominent. The inhibitory effect of IL-1 on the growth of mesangial cells was also confirmed by a change in cell numbers and measurements of protein synthesis with 3H-leucine. IL-1-induced inhibition of mesangial cell growth was not affected by indomethacin. IL-1 showed no effects on intracellular Ca2+ levels in mesangial cells. These observations indicate that IL-1 inhibits the growth of mesangial cells, and thus may play a protective role for mesangial cells from abnormal proliferation in some pathophysiological states.     

Adherence of Candida albicans to human buccal epithelial cells.

The adherence of Candida albicans to human buccal epithelial cells after 2 h at 37 degrees C was significantly greater in human saliva than in phosphate-buffered saline. in saliva, viable fungi adhered much better than did nonviable fungi, and this adherence was greater at 37 than at 25 degrees C. Viable yeasts, preincubated in saliva for 90 min at 37 degrees C before being washed and mixed with epithelial cells in phosphate-buffered saline, adhered better than nonviable yeasts or yeasts preincubated in phosphate-buffered saline. Enhanced adherence in saliva appeared to be associated with germination of the yeast cells. Conditions permitting germination (growth in tissue culture medium 199 at 37 degrees C but not at 25 degrees C) also supported enhanced adherence. After germination had occurred, the fungi could be killed with Formalin without interfering with their rapid and efficient adherence to epithelial cells. These data indicate that the enhanced adherence of C. albicans observed after incubation in saliva is related to changes in the fungi, rather than to a requirement for prolonged interaction between fungi and epithelial cells.     

Expression of interleukin-1 alpha, interleukin-1 beta and interleukin-6 in chronic B lymphocytic leukaemia (B-CLL) cells from patients at different stages of disease progression.

We have previously found that isolated B-CLL cells from progressive disease produce less interleukin-1 beta (IL-1 beta) as compared with cells from patients with indolent disease. Here we extend that finding to include measurements of IL-1 beta mRNA and secretion of IL-1 alpha and interleukin-6 (IL-6). As before, a lower production of IL-1 beta was found in cells from progressive disease. IL-6 was produced by cells from patients at all stages, with a tendency to follow the IL-1 beta production. Low secretion of IL-1 alpha was noted. When viable cells were permeabilized and analysed at the single cell level with monoclonal antibodies, most B-CLL cells were found to contain IL-1 alpha. A minor fraction of non-permeabilized cells expressed IL-1 alpha at the cell membrane. However, only small fractions of cells were positive for intracellular IL-1 beta (less than 1%) and almost no IL-6-positive cells were found. We conclude that either IL-1 beta and IL-6 are produced by a minor population of undefined cells, or that a more sensitive in situ method is needed to detect production of these cytokines in B-CLL cells. The possible biological significance of secreted, and membrane-expressed helper factors in B-CLL is discussed.     

Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells.

We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells. Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively). Prematurity and serious illness did not alter adherence in newborns. The increased susceptibility of premature newborns to E. coli K1 cannot be explained by enhanced epithelial cell adherence.     

Autophagy is required for toll-like receptor-mediated interleukin-8 production in intestinal epithelial cells

Autophagy is an evolutionarily conserved process that maintains cellular homeostasis via synthesis, degradation, and subsequent recycling of cellular products under various physiological conditions. However, the link between autophagy and the innate immune system remains unknown. In the present study, we evaluated Toll-like receptor (TLR)-mediated autophagy induction in intestinal epithelial cells (IECs) and its relationship to interleukin (IL)-8 production. IEC-6, HCT-15, RAW264.7, and THP-1 cells were cultured with or without various TLR ligands, followed by evaluation of the expressions of pro-inflammatory cytokines [IL-8, cytokine-induced neutrophil chemoattractants (CINC)-2β, macrophage inflammatory protein (MIP)-2] by real-time PCR and ELISA. To reveal the status of autophagy in IECs and macrophages, light chain 3 (LC3)-II expression was examined using Western blotting and immunofluorescence with confocal microscopy. Also, to evaluate the influence of TLR ligands on autophagy-mediated innate-immune responses, autophagy-related gene (Atg)7 specific siRNA was transfected into intestinal epithelial cells and IL-8 expression was determined following exposure to various TLR ligands. Cells treated with the TLR ligands produced considerable amounts of pro-inflammatory cytokines (IL-8, CINC-2β, MIP-2). Furthermore, the basal levels of LC3-II were markedly higher in IECs as compared to those in macrophages. Our findings indicated that autophagy induction following TLR ligand stimulation was not significantly evident in IECs as compared to macrophages. In addition, Atg7 gene expression silencingled to down-regulation of TLR-mediated IL-8 expression in IECs, which indicates a potential role of autophagy in generating innate-immune responses. In conclusion, autophagy may be an important intracellular machinery for inducing the innate immune system in IECs.