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Immunopathogenesis of Oropharyngeal Candidiasis in Human Immunodeficiency Virus Infection

Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal,¹ Sainte-Justine Hospital,² Laboratory of Molecular Biology, Clinical Research Institute of Montreal Montreal, Quebec, Canada³

SUMMARY

INTRODUCTION

OROPHARYNGEAL AND ESOPHAGEAL CANDIDIASES IN THE SETTING OF HIV INFECTION

Clinical Features and Pathology

Epidemiology

Correlation with Progression of HIV Infection

Impact of Antiretroviral Therapy

HISTOLOGY OF THE ORAL MUCOSA

ALTERATIONS IN MECHANISMS OF ORAL INNATE RESISTANCE TO C. ALBICANS IN HIV INFECTION

ORAL MUCOSAL IMMUNE SYSTEM AND HOST DEFENSES AGAINST C. ALBICANS

Cells with Immune Potential in the Oral Mucosa

Lymphoid cells.

Langerhans' cells.

Keratinocytes.

Macrophages and PMNs.

Mechanisms of Protective Cellular Immunity to C. albicans in the Oral Mucosa

PATHOGENESIS OF OROPHARYNGEAL AND ESOPHAGEAL CANDIDIASES IN HIV/AIDS

Evidence Implicating C. albicans Virulence Factors

Perturbed Mucosal Immune Defense Mechanisms against C. albicans in HIV-Infected Patients

Humoral immune response.

Cellular immune response.

AIDS-Like Disease in Transgenic Mice Expressing HIV-1

Structure and expression of HIV-1 in CD4C/HIV Tg mice.

Clinical and pathological features of AIDS-like disease in CD4C/HIV Tg mice.

(i) Kidneys.

(ii) Lungs.

(iii) Heart.

(iv) Bones.

Immune defects in CD4C/HIV Tg mice.

(i) Thymus.

(ii) Peripheral lymphoid organs.

(iii) CD4+ T cells.

(iv) CD8+ T cells.

(v) B cells.

(vi) Macrophages.

(vii) Dendritic cells.

Oroesophageal Candidiasis in CD4C/HIV Transgenic Mice: a New Tool To Study Pathogenesis

FUTURE DIRECTIONS AND CONCLUSION

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Oropharyngeal candidiasis (OPC) is the most frequent opportunistic fungal infection among human immunodeficiency virus (HIV)-infected patients, and it has been estimated that more than 90% of HIV-infected patients develop this often debilitating infection at some time during progression of their disease (374, 375). Although the incidence of OPC in HIV infection has been significantly reduced since the introduction of highly active antiretroviral therapy (HAART) (280), it remains a common opportunistic infection in HIV-infected patients. Clinically, OPC in HIV infection has been classified as exhibiting pseudomembranous and erythematous variants, or angular cheilitis (2). The pseudomembranous form of HIV-associated OPC is characterized by the presence of multifocal smooth white papular lesions that can usually be rubbed away, leaving a red surface, and surface pseudohyphae can be readily detected. The erythematous form of OPC typically presents as diffuse and multiple foci of macular erythema involving the palate, oropharynx, buccal mucosa, and dorsal tongue, but hyphae are frequently absent. OPC is frequently complicated by esophageal candidiasis, which may limit food consumption and lead to weight loss, threatening the general health and well-being of HIV-infected patients (428). Furthermore, clinical and in vitro resistance to antifungal azoles frequently occurs in OPC when CD4⁺ cell counts fall to <200 cells/mm³ of blood, either by selection or acquisition of resistant strains of Candida albicans or by infection with inherently resistant species of Candida other than C. albicans (273, 332, 343, 344, 354, 380, 445). Of added concern, the full potential impact of antiretroviral therapy on the ability to reconstitute immunity (10, 21, 22, 55, 111, 292, 323, 324, 395) and therefore to reduce the incidence of OPC and esophageal candidiasis (67, 68, 470) has been hampered (362, 429) by suboptimal adherence (235, 441, 443), toxicity (125), and resistance (316, 337) to antiretrovirals, as well as the limited availability of these treatments in developing countries where most HIV-infected patients reside (104).

The leading cause of candidiasis, C. albicans, is an imperfect diploid dimorphic fungus that resides as a commensal of the mucosae and the gastrointestinal tract. Intraoral C. albicans is found in 40% of healthy humans (16). However, colonization often leads to opportunistic mucosal or life-threatening deep-organ infection in immunocompromised hosts. Invasion of the human gastrointestinal mucosa by C. albicans and its passage across the bowel wall into the bloodstream is an important portal of entry for this opportunistic pathogen into the neutropenic host, leading to systemic or disseminated candidiasis (114, 451). Hematogenous candidiasis is a frequent complication in the treatment of patients with acute leukemia (2, 278). In contrast, Candida fungemia is infrequent in HIV-infected patients and is confined mainly to the late stage of HIV infection (247, 333, 438).

The predisposition for OPC and esophageal candidiasis among HIV-infected patients, initially attributed to T-cell impairment, is enigmatic (72, 74, 240, 446). Colonization of oral mucosal surfaces and symptomatic disease are closely correlated with the development and progression of the cellular immunodeficiency of HIV infection (230, 311, 414). However, because Candida colonization of the keratinocyte surface occurs without invasion of the submucosa, the occurrence of this superficial fungal disease in a T-cell-poor environment has not been adequately explained. The onset of lesions depends on imbalances between Candida virulence attributes and progressively impaired host mucosal defenses in the sequential development of HIV infection, but the exact pathways leading to this imbalance are still unclear. The enhanced risk of OPC and esophageal candidiasis in HIV infection stands in striking contrast to the unenhanced incidence of vaginal candidiasis in HIV-infected women (255, 389), indicating that mucosal immune defense mechanisms and/or their perturbations which favor candidiasis in HIV infection are anatomically compartmentalized (158, 160, 250).

A large body of work conducted with experimentally infected intact or congenitally immunodeficient mice has provided a foundation for understanding the critical roles of Th1 CD4⁺ T cells, CD8⁺ T cells, T cells, macrophages, and polymorphonuclear leukocytes (PMNs) in host defense against mucosal and systemic candidiasis (19, 29, 149, 150, 213, 446). The results of these investigations indicated that protection against mucosal candidiasis involves the recruitment and interactive collaboration of several cell populations which, together, can prevent invasion of mucosal surfaces by C. albicans in the normal host. It is thus evident that multiple, rather than single, defects in host defense mechanisms potentially underlie mucosal candidiasis in HIV-infection.

In this review, the salient clinical features of OPC and esophageal candidiasis are correlated with mucosal immune defense mechanisms against C. albicans and their perturbations in HIV infection. We also describe how a novel experimental model of oroesophageal candidiasis in transgenic (Tg) mice expressing HIV and developing an AIDS-like disease (116, 363) can be used as a new and powerful tool to investigate critical issues regarding innate and acquired immunity at the level of the oral and esophageal mucosa.

OROPHARYNGEAL AND ESOPHAGEAL CANDIDIASES IN THE SETTING OF HIV INFECTION

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At least 75% of HIV-infected patients with OPC have concurrent AIDS-associated (70a) esophageal candidiasis (263) confirmed by histopathology of biopsy material obtained at endoscopic examination (355). While 30 to 43% of these patients may not have symptoms of esophageal involvement, a majority have symptoms including dysphagia and odynophagia (80, 428). For this reason, the combination of OPC and these symptoms of esophagitis is highly predictive of esophageal involvement, and these patients can receive empirical antifungal therapy without confirmation of the diagnosis by endoscopy (15, 349, 428). However, patients who fail to respond to antifungal treatment require esophageal biopsy to assess the possibility of azole-resistant Candida, other opportunistic pathogens including herpes simplex virus and cytomegalovirus, and lymphoma or Kaposi's sarcoma.

Because procurement of oral tissue samples is restricted for ethical reasons (358), only a limited number of studies have been conducted to determine the histopathologic and ultrastructural features of OPC in HIV infection (147, 357, 358, 367). In erythematous candidiasis, Candida hyphae are few while blastoconidia may be found on an atrophic epithelial surface. In contrast, hyphae are numerous and extend into the spinous cell layer in pseudomembranous candidiasis, accompanied by parakeratosis, acanthosis, and spongiosis of the infected superficial epithelium (357). Of interest, hyphae have been observed to penetrate through intercellular spaces, suggesting that Candida can engage in thigmotropism (contact guidance), a phenomenon commonly seen in plant fungi and also recognized in C. albicans in vitro (398). In some cases, hyphae are seen to traverse spinous cells and display appressoria-like appendages at their extremities, another common feature in plant fungi which enhances the strength of attachment of the exploring fungal tip (357). Intercellular penetration of hyphae is also facilitated by the detachment of epithelial cell desmosomes, presumably by C. albicans secretory aspartyl proteinases (SAPs) and/or phospholipase (357). This particular feature is also observed in non-HIV-infected patients with OPC (294). In addition to the marked contrast in penetration of the epithelium by C. albicans in pseudomembranous and erythematous candidiasis, these two forms of OPC are distinguished by the nature and intensity of the mucosal inflammatory cell response (147, 357, 358, 367). The erythematous form in both HIV-infected and uninfected patients is characterized by abundant neutrophilic microabcesses in the parakeratin layer of the epithelium, while microabcesses are rarely found in pseudomembranous candidiasis, even underneath foci of extensive hyphal colonization of the parakeratin layer (147, 358, 367). Indeed, some HIV-infected patients with pseudomembranous candidiasis have almost no epithelial inflammatory response (147, 357). In both clinical forms, however, an abundant mononuclear cell response is observed in the submucosa with no significant difference between HIV-infected and -uninfected patients with the exception of an enhanced infiltration in HIV-infected compared to HIV-uninfected patients with pseudomembranous candidiasis. Immunohistochemical analysis has demonstrated that the inflammatory response in both forms of OPC consists predominantly of CD8⁺ T cells and CD1a⁺ Langerhans cells (367). The mechanisms which govern the more intense inflammatory response in erythematous compared to pseudomembranous candidiasis remain unknown but are probably independent of HIV infection and its progression since these differences are also observed in patients who are not infected with HIV (147).

Although HIV-infected women may develop both OPC and vaginal candidiasis, the risk of OPC alone is enhanced by HIV-infection (255, 389). Molecular typing of C. albicans colonizing HIV-infected women revealed that concurrent oral and vaginal isolates were in all cases dissimilar, suggesting that the dominant strains of C. albicans colonizing these different mucosal sites are distinct (102). These differences may indicate an ability of specific genotypes of C. albicans to colonize different ecological niches or may result from interhuman transmission of different genotypes to separate mucosal sites.

The mechanisms underlying the dramatic impact of HAART on the incidence of OPC and esophageal candidiasis have received close attention (10, 17, 21, 39, 67, 68, 127, 292) and provide valuable insights into understanding the perturbations of mucosal defense mechanisms against C. albicans in HIV-infection. Several observations indicate that increases in CD4⁺ cell counts in response to HAART confer immunologic reconstitution and a decreased incidence of opportunistic infections. Episodes of OPC and esophageal candidiasis that continue to occur despite HAART have done so at low CD4⁺ cell counts, and patients whose CD4⁺ cell counts have increased in response to HAART are at lower risk (17, 127, 222, 280), establishing a correlation between CD4⁺ cell recovery and a decreased incidence of mucosal candidiasis. A three-phase T-cell reconstitution has been demonstrated after HAART, with an early rise in the number of memory CD4⁺ cells, an improved CD4⁺ cell reactivity to recall antigens, and a late rise in the number of naive CD4⁺ cells (21, 22, 292). In addition, proliferative responses to the mitogen phytohemagglutinin develop in the majority of patients in whom responses were absent at baseline (10, 292), and there is increasing interleukin-2 (IL-2), IL-12, and IL-10 production (10). It could therefore be hypothesized that HAART reduces the incidence of mucosal candidiasis by reconstituting delayed-type hypersensitivity to C. albicans antigens and a protective mucosal Th1 response to C. albicans (42, 70, 211) and rectifying the shift to a nonprotective Th2 response resulting from HIV infection (83). However, in contrast to the frequent recovery of a proliferative response to phytohemagglutinin, treatment with HAART results only in late and inconsistent recovery of anticandidal cellular immunity, as assessed either by skin test reactivity for delayed-type hypersensitivity or by a proliferative response to C. albicans antigens (17, 67, 68, 292). These findings, associated with the resolution of refractory OPC in some HAART-treated patients well before the recovery of CD4⁺ cell counts and response to Candida antigens (67, 68), indicate that the decreased incidence of OPC in patients receiving HAART cannot be fully accounted for by reconstitution of Candida cell-mediated immunity (67, 68). Indeed, decrease of the viral load after HAART therapy (10) may also ameliorate mucosal candidiasis by correcting a dysfunction of neutrophils induced by HIV envelope glycoprotein gp41 (143, 168, 454) or by increasing the neutrophil count in HIV-infected patients with neutropenia (127, 471). Evidence has also been presented that HAART has an early, immune reconstitution-independent inhibitory effect on C. albicans Saps in the oral cavities of HIV-infected patients (67), and that HIV protease inhibitors attenuate adherence of C. albicans to epithelial cells in vitro (39). It has been shown that C. albicans strains from HIV-infected patients with OPC have increased expression of Saps (107, 321), possibly enhanced by HIV envelope gp160 and gp41 binding to C. albicans (180). Therefore, inhibition of C. albicans Saps by HIV protease inhibitors may also contribute to the amelioration of OPC and esophageal candidiasis in HIV-infected patients treated with HAART.

HISTOLOGY OF THE ORAL MUCOSA

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The oral mucosa has histological features in common with the vaginal and esophageal mucosas, including a superficial stratified squamous epithelium and an underlying lamina propria of dense collagenous connective tissue, separated by a basal membrane. However, the mucosa of the oral cavity varies in cellular layer composition, depending on the position and function (145, 369, 408). The lining mucosa, including that found on the cheeks, floor of the mouth, underside of the tongue, and soft palate, represents 60% of the surface area of the oral mucosa. The stratified squamous epithelium in these areas contains a germinating layer (stratum basale) overlying the basal membrane, a spinous layer (stratum spinosum), and a superficial granular layer (stratum granulosum) and is generally nonkeratinized and therefore similar to the esophageal epithelium. The cells undergo structural differentiation as they migrate from the stratum basale to the epithelial surface (408). The masticatory mucosa, found on the gingiva and hard palate, represents 25% of the surface area of the oral mucosa and has an additional keratinized or parakeratinized surface layer resembling that of the skin but lacking a stratum lucidum (145, 369). The specialized stratified squamous epithelium of the dorsum of the tongue (15% of the surface area of the oral mucosa) contains abundant lingual papillae differentiated into four different types: filiform, fungiform, circumvallate, and foliate. The outer surface of the papillae is covered by keratinized epithelium and thus resembles the hard palate, while the interpapillary region is covered by nonkeratinized epithelium similar to that of the lining mucosa (388). The oral epithelium thus varies in the degree of keratinization, cornification, and orthokeratinous and parakeratinous layer thickness found in areas (gingiva, hard palate, and dorsal surface of the tongue) where frictional forces created by biting, chewing, or movement of food occur. Although the thickness of the human oral stratified squamous epithelium shows regional variation ranging from 190 ± 40 µm (floor of the mouth) to 580 ± 90 µm (cheeks) (388), the width of the epithelium is three to five times less in the mouse oral mucosa at each site (197).

Keratinocyte proliferation is stimulated by epidermal growth factor, transforming growth factor , platelet-derived growth factor, and IL-1 (408). The switch between proliferation and differentiation is modulated by extracellular calcium, phorbol esters, retinoic acid, and vitamin D₃ (408). To ensure a 14- to 20-day median turnover time of oral epithelial cells (408), the keratinocytes attached to the basal membrane lose integrin expression, leading to progressive morphologic changes during migration to the mucosal surface (408). Interestingly, the turnover times of mouse palate and cheek epithelia are slightly shorter than that of tongue epithelium, and the times for all of these tissues are threefold that for epidermis (197). Keratinocytes are linked by desmosomes, which increase in number from the basal to the superficial layer of the epithelium, and by nexus-like (gap) junctions (388, 392). Polygonal and more flattened, upwardly migrating cells discharge the contents of membrane-coating granules by an exocrine process into the intercellular space, forming broad lipid lamellae containing ceramides and acylceramides which serve as a permeability barrier in the keratinized stratified squamous epithelium (399, 408, 455). In nonkeratinized epithelium, intercellular lipid is nonlamellar, contains mainly cholesterol and glycosphingolipids but no acylceramides and only small amounts of ceramide, and provides a less efficient permeability barrier (399, 408, 455). Continuous desquamation of surface keratinocytes of the oral epithelium plays a pivotal role in maintaining a healthy oral mucosa and in limiting candidal colonization and infection (378).

In several regions of the oral cavity, there are nodules of lymphoid tissue consisting of crypts formed by invagination of the epithelium into the lamina propria. These areas are extensively infiltrated by lymphocytes, which play an important role in host defense against oral infections.

ALTERATIONS IN MECHANISMS OF ORAL INNATE RESISTANCE TO C. ALBICANS IN HIV INFECTION

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The skin and mucosal tissues represent the primary portal of entry for opportunistic pathogens, leading to life-threatening systemic dissemination in the immunocompromised host. In the normal host, however, several redundant immunological and nonimmunological defense mechanisms directly limit the proliferation of pathogenic microorganisms, thus reducing the burden of organisms available for binding to potential attachment sites. In the oral cavity, the flow and composition of saliva establish a dynamic equilibrium between C. albicans (361) and other members of the commensal microbiota, preventing the establishment of oral candidiasis in the normal host (37). Salivary flow protects the oral cavity by dislodging yeasts and bacteria, which are then removed by swallowing, and studies have provided evidence that this process may be facilitated by binding of C. albicans to salivary mucins (139, 140) or to a nonmucin proteoglycan (198, 199). In patients with Sjögren's syndrome, however, decreased salivary flow and buffering capacity are associated with an increased frequency of carriage of C. albicans (361) and of oral candidiasis (7). The prevalence of oral candidiasis in these patients has been estimated at about 35% (7). A similar effect is observed in patients with advanced HIV infection, in whom the salivary flow rate is reduced by 40% and is also correlated with enhanced recovery of Candida from saliva (258). The incidence of oral candidiasis is also enhanced in patients with acidic saliva (373), and a low pH increases the adherence of C. albicans to epithelial surfaces in vitro (377). Glucose supplementation of saliva augments the growth rate of C. albicans in vitro, and the resulting acidic pH provides the required environment for activity of Candida Saps, which enhance virulence by degrading a wide variety of host substrates including the mucins, which play an important role in lubrication of epithelial surfaces and host defense (89, 115, 253, 307, 363, 376, 384). The pH of the mucosa also regulates the expression of the C. albicans virulence genes PHR1 and PHR2 (108) and is thus a significant environmental signal in determining the virulence capacity of Candida and/or modulation of the host defenses (372). Finally, biofilm formation by C. albicans has been implicated in the ability of the fungus to cause persistent infection on bioprosthetic materials, including denture acrylic, as well as mucosal surfaces (75, 76). However, there were no significant quantitative differences in biofilm formation between C. albicans oral isolates from HIV-infected and noninfected patients, indicating that the biofilm-forming ability of C. albicans is unlikely to contribute to high levels of oral yeast carriage in cases of HIV infection (212).

Several salivary anticandidal proteins, including lysozyme, lactoferrin, the histatins, calprotectin, and antileukoprotease, inhibit the growth of C. albicans and its attachment to the oral epithelium. Because saliva from HIV-infected patients shows decreased anticandidal activity (258), several investigations have focused on identifying putative defects in salivary antimicrobial proteins which may favor oral candidiasis in HIV infection. Lysozyme and lactoferrin are two major nonimmunological antimicrobial proteins in saliva which possess concentration-, time-, and strain-dependent fungicidal activity against C. albicans in vitro (379, 466). Lysozyme is found at a concentration range of 1.5 to 57 µg/ml of saliva (350, 419), and its antifungal properties are thought to be mediated by the enzymatic hydrolysis of N-glycosidic linkages in the microbial cell wall and injury to the cytoplasmic membrane following direct cationic-protein binding (279). Interestingly, concentrations of salivary lysozyme are increased in HIV-infected patients with or without oral candidiasis (20, 199, 274, 467), and a trend toward progressive in vitro resistance to lysozyme has been observed in genetically similar, sequential oral C. albicans isolates from patients infected with HIV (379). Because the concentration of lysozyme is increased in HIV-infected patients while the anticandidal activity of saliva is decreased, the contribution of salivary lysozyme to limiting the proliferation of C. albicans in the oral cavity of these patients appears doubtful.

Lactoferrin is a member of the transferrin family of nonheme iron-binding glycoproteins and is found at the mucosal surface, where it functions as a prominent component of the first line of host defense against infection (452). The concentration of lactoferrin in unstimulated saliva is about 7 to 20 µg/ml (126, 371), and its fungicidal activity against C. albicans (404) has been attributed not only to sequestration of ferrous ions (284) but also to structural damage to the fungal cell wall (313) and activation of intracellular autolytic enzymes (243). Salivary concentrations of lactoferrin in patients with HIV infection have been variously reported to be increased (20, 258), unchanged (276), or decreased (266, 300). These variable results have been at least partly ascribed to the source of the saliva, because increased concentrations of lactoferrin in HIV infection have been found in submandibular but not parotid saliva (20, 258, 274). The predisposition to oral candidiasis in HIV-infected patients is thus not convincingly associated with defective production of lactoferrin. In contrast to lysozyme, serial genotypically identical oral isolates of C. albicans from HIV-infected patients did not develop progressive in vitro resistance to lactoferrin (379). The therapeutic potential of lactoferrin for the treatment of OPC has recently led to the development of mucoadhesive lactoferrin tablets with fungicidal activity against C. albicans and C. glabrata (239). This novel approach to the treatment of mucosal candidiasis will require further validation in clinical trials.

The family of salivary histatins consists of at least 12 low-molecular-weight, structurally related, histidine-rich, cationic proteins, which also contribute to nonimmunological host defense of the oral mucosa (138, 437). The histatins have broad fungicidal activity against pathogenic fungi, including C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus (194), and are present at 50 to 425 µg/ml (244) in the saliva of healthy adults. Histatin 5, which exerts potent candidacidal activity (138), is internalized by C. albicans, inhibits the respiration of mitochondria, and induces the formation of reactive oxygen species leading to mitochondrial and cytoplasmic membrane damage, efflux of ATP and other nucleotides, and cell death (183, 194). The mechanism of action of the histatins is thus distinct from that of other cationic peptides such as the defensins, which can directly insert into and disrupt cell membranes because of the strongly amphipathic nature of their -helical structures (138). The concentration of histatins in the saliva of HIV-infected patients has been determined to be increased (20) unchanged (258), or decreased (244, 274), and these seemingly discordant results may have been caused by the different stages of HIV infection among the patients under study as well as by the analytical methods employed. However, decreased concentrations of histatins appeared to correlate with an increased tendency to oral candidiasis in a subgroup of HIV-infected patients (274), suggesting that decreased histatin concentrations and/or an inability of these proteins in saliva to interact with C. albicans may contribute to the defective salivary anticandidal activity seen in HIV-infected patients (244). Interestingly, transfer of the gene encoding histatin 3 in the salivary glands of rats by using recombinant adenovirus vectors resulted in its expression at up to 1,045 µg/ml of saliva, suggesting that a gene transfer approach to overexpression of naturally occurring antifungal proteins may be potentially useful in the management of mucosal candidiasis (317).

The heterodimeric calcium- and zinc-binding protein calprotectin is produced by PMNs, monocytes, macrophages and mucosal keratinocytes (54, 73, 403). In vitro, calprotectin quantitatively inhibits the growth of C. albicans by depriving the fungus of zinc, which is essential for microbial growth (138). Salivary calprotectin concentrations and oral keratinocyte expression of calprotectin are augmented in response to oral candidiasis, in both HIV-infected and -uninfected patients (146, 231, 424). However, the results of two independent studies demonstrated that salivary concentrations of calprotectin are deficient in HIV-infected patients with oral candidiasis or high salivary Candida counts compared to those in HIV-infected patients without oral candidiasis or with low salivary Candida counts (298, 424). These results suggested that a diminution of this antimicrobial factor may predispose to oral candidiasis in HIV infection. On examination of the oral mucosa of HIV-infected patients with OPC, however, Candida hyphae were found to penetrate through the epithelial parakeratin layer yet did not extend beyond the zone of spinous-layer keratinocyte calprotectin expression (147). Further studies are required to determine whether reduced salivary calprotectin is not simply associated with but directly contributes to the predisposition to oral candidiasis in HIV-infected patients.

Antileukoprotease (436), also known as secretory leukocyte protease inhibitor (141), is produced by keratinocytes (457) and constitutes the last member of the family of antimicrobial proteins involved in nonimmunological defense against C. albicans at mucosal sites. Like other cationic antimicrobial polypeptides, the antimicrobial activity of antileukoprotease is limited to conditions of low ionic strength. In addition to its inhibition of leukocyte-derived proteinases, antileukoprotease has fungicidal activity by an unknown mode of action against C. albicans which is localized primarily in the NH₂-terminal domain (436) and it may thus play an important role in the innate mucosal defense. Interestingly, antileukoprotease exhibits anti-HIV-1 activity in vitro and may contribute to the antiviral activity of saliva associated with the infrequent oral transmission of HIV-1 (287).

ORAL MUCOSAL IMMUNE SYSTEM AND HOST DEFENSES AGAINST C. ALBICANS

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Lymphoid cells. The oral mucosal immune system possesses features in common with both the skin immune system and the mucosal immune system (442). The normal oral mucosa shares with normal skin an absence of B lymphocytes, which are present in the mucosal immune system (50, 442). In contrast to the skin, however, T lymphocytes in the normal human oral mucosa are not organized in rows around postcapillary venules of the superficial and deep vascular networks (50) but are distributed singly or in small clusters on both sides of the basal membrane (442). In addition, T lymphocytes are only rarely found in the more superficial layer of the epithelium. The oral mucosal epithelium contains about 37 times as many T lymphocytes as the epidermis of normal skin (442). The vast majority of T lymphocytes in the oral mucosa express the memory CD45R0⁺ phenotype (86, 340). T lymphocytes within the oral epithelium are not activated (CD25^–), in contrast to CD25⁺ T cells in the underlying stroma (86). The conversion of T cells from the naive CD45RA⁺ to the memory CD45R0⁺ phenotype requires repeated antigenic stimulation, suggesting that apoptotic CD25^–, CD45RA⁺ intraepithelial T cells die after unsuccessful antigen presentation by Langerhans' cells (86).

The CD4/CD8 ratio of 1:2 in the human oral epithelium and 1:4 in the skin indicates the preferential presence of the CD8⁺ subset in both sites, but CD4⁺ cells are proportionately more frequent in the oral mucosa than in the skin (442). However, a CD4/CD8 ratio of 1 within the epithelium of the normal human gingiva indicates regional variation in the oral cavity (86). In contrast to normal humans, CD4⁺ cells are twice as numerous as CD8⁺ cells in the normal murine oral mucosa (47). CD4⁺ T cells are required for a Th1-type protective response against oral candidiasis in mice (149) and therefore play a central role in host defense against OPC.

Of direct relevance to host defense against OPC in HIV infection, the buccal epithelium is an inductive site for the generation of cytotoxic T-lymphocyte responses mediated by major histocompatibility complex (MHC) class I-restricted CD8⁺ T cells, independent of CD4⁺ cell help (119). It has been suggested that CD8⁺ T lymphocytes are attracted to the epithelium by IL-8 produced by keratinocytes (267, 442). Moreover, IL-2 (but not gamma interferon [IFN-])-activated CD8⁺ cells exert direct growth inhibition against the hyphal form of C. albicans (40). However, CD8⁺ cells may not be in proximity to C. albicans hyphae, which are usually confined to the upper layers of the epithelium (72, 147, 357). Alternatively, CD8⁺ cells may produce cytokines which enhance the antimicrobial activity of macrophages and neutrophils against C. albicans. In addition, MHC class I molecules expressed constitutively on keratinocytes may represent a target for CD8⁺ cytotoxic T lymphocytes after internalization by keratinocytes of microbial pathogens (448).

T cells represent at most 2% of the T-cell population in the human oral epithelium (331). Oral mucosal T cells display ultrastructural features typical of large granular lymphocytes, also found in cytotoxic CD8⁺ and NK cells (267), and probably represent a first immunologic line of defense. T cells are located within the epithelium in both normal and inflamed gingiva, often in close proximity to CD1a⁺ and/or CD1c⁺ Langerhans' cells and keratinocytes (268). In inflamed mucosa the T cells show the phenotype of activated cells (CD45RO⁺, CD8⁺, or CD4⁺), whereas in normal mucosa the cells are CD4^– CD8^– and express CD45RA (268). In the connective tissue, under the basal membrane, V2⁺ T cells are predominant, whereas the epithelium contains mostly V1⁺ T cells (206, 331). T cells participate in the immune response to microbial pathogens including C. albicans by producing cytokines such as IFN- (213, 267) or by direct cell-to-cell contact leading to cytotoxicity (267, 303). Increases in the numbers of T cells have been found in the oral mucosa soon after mice are colonized and infected with C. albicans (71), coinciding with resolution of infection.

Finally, natural killer (NK) cells are large granular lymphocytes which represent 6 to 39% of human gingival (268) and 3% of lower lip (283) mononuclear cells. NK cells are cytotoxic in vitro to certain tumor cell lines and to virally infected cells (72) and have direct antimicrobial activity against Cryptococcus neoformans (256) but little or no effect against C. albicans hyphae in vitro (40).

Langerhans' cells. Langerhans' cells develop from bone marrow stem cells as one of three distinct subsets of dendritic cells (DCs) which home in to selected tissues (30, 99, 448). The bone marrow stem cells appear to be common precursors of both macrophages and DCs (30). Serving as an essential link between innate and acquired immunity, dendritic cells function as antigen-presenting cells (APCs) that patrol all tissues of the body, capturing pathogens for processing and presentation to T cells in the secondary lymphoid organs. Two subsets of human DCs, Langerhans' cells and interstitial (or dermal) DCs, belong to the myeloid lineage, while the third subset is composed of lymphoid DCs (99). Culture of human CD34⁺ hematopoietic progenitors in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-) gives rise to CD1a⁺ DCs related to Langerhans' cells and CD14⁺ DCs closely related to interstitial DCs, which can differentiate into macrophages in the presence of M-CSF (69). DCs are thus phenotypically and functionally heterogeneous depending on their specific differentiation pathways (11, 468, 469). Serving as sentinels for pathogen entry at the epithelium of the skin and mucosa, Langerhans' cells, identified by expression of CD1a, Lag, and langerin, are localized on the basal and suprabasal layers and represent 2 to 4% of the cells in the epithelium (6, 34, 49, 59, 96, 99, 103, 216, 245, 262, 351, 356, 365, 390, 391, 442). These cells express MHC class II molecules and are also CD11b⁺ and CD11c⁺. Langerhans' cells have a pronounced dendritic shape and contain rod-shaped organelles called Birbeck granules. Immature epithelial Langerhans' cells are equipped to capture antigens by phagocytosis, macropinocytosis, and receptor-mediated absorptive endocytosis, including the macrophage mannose receptor, DEC-205, as well as Fc and Fc receptors (30). The loose interaction of DC-specific, ICAM-3 grabbing, nonintegrin (DC-SIGN) with ICAM-3 establishes the initial contact of the Langerhans' cell with a resting T cell in the apparent absence of foreign antigen (416). To successfully present antigens for T-cell activation, Langerhans' cells must undergo a maturation process (334) triggered by whole bacteria, bacterial lipopolysaccharide, cytokines such as TNF- and IL-1ß, or the T-cell CD40 ligand (CD40L) (30, 99, 216, 458). Mature Langerhans' cells lose the ability to take up antigens but express surface molecules required for communication with T cells at the immunologic synapse (416). During maturation, Langerhans' cells express high levels of surface MHC class I and II and the costimulatory molecules CD54, CD58, and CD86 that interact with receptors on T cells to enhance adhesion (30, 99, 416). In addition, high CD40 expression on mature Langerhans' cells favors binding to CD40L on T cells, which in turn up-regulates the expression of CD80 and CD86, secretion of IL-12, and release of chemokines such as IL-8 and macrophage inflammatory proteins 1 and 1ß (MIP-1 and MIP-1ß) (30). Antigen presentation via MHC class II molecules in the presence of IL-12 and collaborating IL-18 induces CD4⁺ cells to differentiate into IFN--producing Th1 cells, leading to activation of the antimicrobial properties of macrophages, and is therefore critical to the induction of a protective acquired cell-mediated immune response (30, 99, 308). Furthermore, Langerhans' cells receiving T-cell help mediated by CD40-CD40L interactions (385) can also present antigen on MHC class I molecules to cytotoxic CD8⁺ cells, which can be loaded through both endogenous and exogenous pathways (30, 99). More recent work, however, indicates that Langerhans' cells do not need to receive a signal from T cells to become fully mature DCs capable of stimulating CD4⁺ T cells and cytotoxic CD8⁺ T cells (110). Interestingly, the murine oral mucosa is an inductive site for priming class I-restricted CD8⁺ cytotoxic T cells in vivo (119). The expression of MIP-3 by TNF--stimulated keratinocytes in the spinous layer (77, 435) and the production of defensins (464), which both recognize the CCR6 chemokine receptor in immature DCs, may explain the positioning of Langerhans' cells in the epidermis and their ready access to microbial pathogens (99). The mobilization of Langerhans' cells and their migration via afferent lymphatics to draining lymph nodes for antigen presentation (208) is governed by the upregulation of the chemokine receptor CCR7 and the production of MIP-3ß (405). In addition, IL-18 produced by Langerhans' cells and keratinocytes also contributes to the regulation of Langerhans' cell migration by a TNF- and IL-1ß-dependent mechanism (98).

In normal humans, the density of Langerhans' cells in nonkeratinized oral mucosa is apparently the same as in the skin, but keratinized oral mucosa has fewer Langerhans' cells (96, 103, 442). Although murine palate implants are repopulated by Langerhans' cells within 2 weeks (365), the numerical densities of Langerhans' cells in old mice is reduced compared with that in young mice (364). At the ultrastructural level, murine and human Langerhans' cells in the oral mucosa exhibit no significant differences (59). In the normal human oral mucosa, however, Langerhans' cells present a highly variable morphology according to their epithelial location (391). In contrast to the upper epithelium, where CD1a⁺ Langerhans' cells have long dendrites forming a network, Langerhans' cells in the basal layer are more rounded and have very few short dendrites. Functionally, the well-developed dendritic morphology of Langerhans' cells in the upper epithelium could reflect optimal immune surveillance (391). Conventionalization of germfree mice with a bacterial flora results in enhanced migration of Langerhans' cells to the oral epithelium (49), and the densities of oral epithelial Langerhans' cells are increased in patients with chronic periodontitis compared to healthy controls (216), demonstrating that Langerhans' cells are recruited to the oral epithelium in response to a bacterial challenge. Purified human (35) or rat (192) oral mucosal Langerhans' cells can serve as APCs in vitro and are more efficient than skin Langerhans' cells in providing costimulatory signals to T cells (193). C. albicans-specific T-cell activation by human epidermal Langerhans' cells (85, 223) requires not only the ligation of the T-cell receptor to the antigen-MHC complex but also costimulation by the combination of adhesion molecules CD54 and CD58 with CD11a and CD2 on T cells, respectively (433). As described in further detail below, productive infection of oral mucosal Langerhans' cells by HIV-1 may contribute to their selective depletion (81) and perturb their ability to generate a primary immune response (44), which may impair protective mucosal immunity against colonization and infection by opportunistic microbial pathogens. In addition, Langerhans' cells serve as the portal of entry for HIV-1 at mucosal sites and are critical to the initiation and subsequent spread of infection to draining lymphoid tissue (340).

Keratinocytes. Keratinocytes are of primary importance in the pathogenesis of OPC since they constitute a physical barrier after adhesion of C. albicans to the epithelial surface. In addition to their role as a mechanical barrier, epithelial keratinocytes function as fixed or immobile immunocytes and are capable of producing a number of soluble factors and expressing receptors that are involved in up-regulating and down-regulating immune responses (179, 415, 440). The major growth factors produced include basic fibroblast growth factor, platelet-derived growth factors, transforming growth factors and ß, and TNF-. Keratinocytes also produce several cytokines including IL-1, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, and IL-20, and a number of CSFs such as GM-CSF, G-CSF, and M-CSF (14, 45, 179, 440, 448). Under normal conditions, most of these mediators are not constitutively produced (162), but their gene expression and release is up-regulated during inflammation by a variety of external stimuli derived from leukocytes, Langerhans' cells, and keratinocytes themselves, including IFN-, TNF- and IL-17 (14, 241, 257, 282, 432, 448). Interestingly, mRNA expression of IL-1, IL-1ß, IL-8, GM-CSF, and TNF- is up-regulated in experimental cutaneous C. albicans infection with reconstituted human epidermis, demonstrating that the fungus induces a brisk cytokine response by host keratinocytes (383). C. albicans also triggers the production of IL-1 and TNF- (410), as well as GM-CSF (131), by primary oral epithelial cells and oral epithelial cell lines in vitro. In addition, proteolytic activation of the IL-1ß precursor by C. albicans Sap (38) suggests that candidal proteinases may contribute to the activation and maintenance of the inflammatory response at the epithelial surface. IL-1, IL-8, and IL-12 possess attractant effects on PMNs, macrophages, and lymphocytes (448). In addition, some of the cytokines produced by keratinocytes (IL-1 and TNF-) promote the maturation of DCs and therefore could differentially modify the ability of Langerhans' cells to respond to antigens (448). Keratinocyte-derived IL-7 and IL-15 are involved in epidermal T-cell trafficking (179, 448). In addition to the production of soluble factors, keratinocytes express the adhesion molecules CD54 and CD58, and CD54 expression is increased by IFN- (136, 448). MHC class I molecules are expressed constitutively and may be a target for CD8⁺ T cells (448). MHC class II molecules are not expressed constitutively but can be induced by IFN- produced by infiltrating T lymphocytes (448). Keratinocytes may function as accessory cells in antigen presentation and interact with lymphocytes to produce a Th2 cytokine response (448).

Of potential relevance to the fragile equilibrium between epithelial colonization and infection, IFN- promotes expression of the glycoprotein desquamin, a cell adhesion molecule in the stratum corneum of the human epidermis which possesses lectin-like properties for amino sugars (58), as well as trypsin-like serine proteinase (57) and RNase (393) activity. Desquamin may thus play a crucial role in desquamation and shedding of Candida from the superficial portion of the epithelium.

In addition to these indirect mechanisms, keratinocytes possess several potential antimicrobial mechanisms which may directly contribute to host defense against Candida. (i) Keratinocytes have been shown to express inducible nitric oxide synthase activity (43), and NO has been associated with candidacidal activity and resistance to mucosal candidiasis (213). (ii) Human oral keratinocytes produce numerous antimicrobial peptides, including ß-defensins 1 to 3 (134, 135, 191, 261, 281, 387), cathelicidins (132, 166, 167, 182, 465), adrenomedullin (220, 221), calprotectin (54, 73, 146, 147, 231, 298, 370, 403, 424), and bactericidal/permeability-increasing protein (BPI) (61), which, as natural antibiotics, contribute to the innate immunity of the epithelium (170, 453). ß-Defensins exhibit potent antimicrobial activity against Candida (387), and their expression by keratinocytes at the mRNA and protein level is enhanced by TNF-, IL-1ß, whole bacteria, and bacterial lipopolysaccharide (191, 261, 281, 387). Although epithelial injury or inflammatory disorders augment the expression and release of the human cathelicidin LL-37 from keratinocytes (132, 166), its antimicrobial activity in vitro has so far been demonstrated only against bacteria and the MBCs against Candida species are >100 µg/ml (182). In addition to their direct antimicrobial properties, human ß-defensins and the cathelicidin LL-37 are chemotactic for immature DCs and neutrophils and for monocytes and T cells, respectively (465). Secretion of the vasoactive peptide adrenomedullin from oral keratinocytes is stimulated by IL-1, IL-1ß, TNF-, LPS, and live bacteria but not by C. albicans (220, 221). Although adrenomedullin possesses antimicrobial properties, it is not yet known whether it contributes to host defense against oral candidiasis. As outlined previously in this review, oral keratinocytes also express calprotectin, a heterodimer of MRP8 and MRP14 with antimicrobial activity against C. albicans. The up-regulated expression of calprotectin by oral keratinocytes in response to infection has been investigated in vitro and appears to be independent of IL-1ß (370). Finally, keratinocytes express on their cell membranes BPI, which is also an abundant constituent of PMNs (61, 170). BPI on keratinocytes contributes to the killing of gram-negative bacteria that become closely adherent to epithelial cells (61, 170). The role, if any, of BPI in limiting C. albicans colonization or infection of the oral mucosa remains to be determined. (iii) Human oral keratinocytes directly inhibit the growth of blastoconidia and/or hyphae of Candida species in vitro, with a strict requirement for cell contact (411). Growth inhibition appears to involve a carbohydrate moiety on the surface of the keratinocytes but is not mediated by phagocytosis, nitric oxide, superoxide-hydrogen peroxide pathways, or defensin and calprotectin antimicrobial peptides (412). Direct growth inhibition of Candida by oral keratinocytes appears to occur through a novel and distinct mechanism, complementary to other components of the antimicrobial armamentarium of oral keratinocytes. Oral epithelial keratinocytes are thus equipped with numerous redundant defense mechanisms, acting either directly or indirectly against the continuous microbial challenge at the oral mucosal surface. The role of keratinocytes in host protection against Candida at mucosal surfaces appears likely, since C. albicans hyphae are restricted to the upper layers of the oral epithelium in OPC and are some distance away from lymphocytes and Langerhans' cells located in deeper layers.

Macrophages and PMNs. Macrophages and PMNs originate from monoblasts and myeloblasts, distinct populations of myeloid stem cells which differentiate into monocytes and neutrophils in the bloodstream. In the normal uninfected host, circulating monocytes differentiate into resident tissue macrophages, in contrast to PMNs, which are retained almost exclusively within the circulation. Because of their key role in the innate immune response (289), these two cell populations are critical effectors in the first line of defense against oral microbial pathogens. In the normal human oral mucosa, macrophages are located mainly in the lamina propria (86) while PMNs appear in the lamina propria and epithelium only in response to inflammation (268). Macrophages are not a homogeneous cell population but can be separated into biologically active subpopulations which appear at early, intermediate, or late stages of inflammation (185).

Oral mucosal resident macrophages express MHC class II molecules and CD11b, as well as Fc receptors that bind IgG (FcR) (31). Like Langerhans' cells, macrophages present antigenic peptides to CD4⁺ T cells after induction of CD86 costimulatory molecules (112, 113). Th1 CD4⁺ T cells secrete IFN- and IL-2, which activate both macrophages (97) and CD8⁺ cytotoxic T cells to kill intracellular pathogens (113). After activation, macrophages produce TNF-, which activates PMNs, further amplifying the innate immune response (19). For this reason, macrophages play a critical dual role at the crossroads of innate and acquired cell-mediated immunity. Indeed, activation of a specific T-cell response to C. albicans antigens in vitro has been found to require macrophages expressing MHC class II molecules (314).

To date, Langerhans' cells, monocytes, macrophages, and PMNs are the only cells that have been reported to be candidacidal (132a, 213, 310). Macrophages and PMNs have the ability to kill both C. albicans blastoconidia and hyphae by both oxygen-dependent and -independent mechanisms (446). Oxygen-dependent killing by PMNs is mediated by superoxide anion and the myeloperoxidase-hydrogen peroxide-halide system, with the added participation of reactive nitrogen intermediates including NO and peroxynitrite in the candidacidal activity of macrophages which lack myeloperoxidase (446). Production of IFN- by T cells augments NO production by macrophages and enhances resistance to orogastric candidiasis, indicating that T cells indirectly contribute to macrophage killing of C. albicans (213). Macrophages and PMNs are also equipped with oxygen-independent mechanisms including the cationic protein defensins (446) and calprotectin (54, 73, 403), demonstrating the use of an extensive array of oxidative and nonoxidative mechanisms to kill C. albicans blastoconidia and hyphae (446).

In experimental OPC in the mouse model, the early inflammatory response 24 to 48 h postinfection is composed of large numbers of PMNs migrating from the lamina propria to accumulate in the superficial epithelial layers (242). During recovery from primary infection, at 5 to 6 days postinfection, the initial influx of PMNs is replaced by a massive recruitment of macrophages in the lamina propria (71). The presence of both macrophages and PMNs in experimental candidiasis concurs with similar histologic findings in HIV-infected patients with OPC, suggesting a major role for these professional phagocytes in mucosal containment of C. albicans.

In contrast to gastrointestinal and vaginal candidiasis, relatively few hypothesis-driven, cause-and-effect investigations have been conducted to specifically elucidate the mechanisms of host defense against C. albicans in the oral mucosa (157). The accumulated evidence indicates that normal host defense against OPC is the sum of individual redundant mechanisms which include several salivary anticandidal proteins, growth inhibition of C. albicans by oral keratinocytes, and the presence of T-cell-mediated delayed-type hypersensitivity to C. albicans. The evidence implicating anticandidal proteins and oral keratinocytes, described in previous sections of this review, has so far been derived solely from observations of in vitro activity against C. albicans. Although their role in host defense appears likely, no direct demonstration has been presented using compelling approaches such as their depletion, augmentation, or transfer in an experimental model of OPC. Consequently, mechanistic investigations of host defense in experimental OPC have been focused almost entirely on dissecting the precise role of an acquired cell-mediated immune response to C. albicans.

Although this has not yet been directly studied in experimental OPC, oral mucosal Langerhans' cells are most probably involved in the initiation of an acquired cell-mediated immune response to C. albicans. Both human (310) and murine (132a) DCs recognize C. albicans by the mannose-fucose receptor, can phagocytose and degrade Candida, and can subsequently present Candida antigens to T cells. Interestingly, the yeast and hyphal forms of Candida are ingested by different mechanisms and receptors. Phagocytosis of the yeast cells by DCs occurs by coiling phagocytosis, characterized by the presence of overlapping bilateral pseudopods, whereas ingestion of hyphae occurs through a more conventional zipper-type phagocytosis (132a). Human DCs kill Candida as efficiently as human monocyte-derived macrophages do, and killing appears to be mainly oxygen independent, possibly via lysosomal hydrolases (310). In contrast, killing of C. albicans yeast cells or hyphae by murine DCs is correlated with the production of NO (132a). The T-cell proliferation observed with a mixture of human DCs, Candida, and T cells most probably represents a secondary immune response, since C. albicans is a commensal in humans (310). However, analogous experiments conducted using murine DCs required the presence of IL-2 to elicit a priming response since C. albicans is not a commensal organism in mice (132a). In vitro, ingestion of the yeast form of C. albicans activated DCs for IL-12 production and priming of Th1 cells whereas ingestion of hyphae inhibited IL-12 and Th1 priming and induced IL-4 production (132a). The pivotal role of DCs in initiating the immune response to C. albicans was elegantly demonstrated by the generation of protective immunity against intravenous infection after injection of DCs ex vivo pulsed with C. albicans yeasts but not hyphae (132a). Yeast-pulsed DCs from IL-12 knockout mice primed lymphocytes for IL-4 production in vitro and were unable to confer resistance to candidiasis (132a), consistent with the lack of Th1 response development (272). Finally, work from the same group showed that murine DCs pulsed with yeast but not hyphal RNA induce protective immunity to C. albicans in allogeneic bone marrow-transplanted mice (24).

In addition to Langerhans' cells, macrophages and keratinocytes could be potentially involved in the processing and presentation of Candida antigens to CD4⁺ cells and could therefore also participate in the induction of an adaptive immune response to C. albicans in the oral cavity. Keratinocytes of the reproductive tract express MHC class II molecules and can function as APCs (459). In addition, expression of MHC class II molecules by epithelial keratinocytes is enhanced in patients with angular cheilitis (320) or OPC (214), possibly in response to IFN- produced by infiltrating T lymphocytes (18, 448). However, the ability of oral keratinocytes to engage in presentation of Candida antigens is uncertain, since these cells do not appear to have the capacity to engulf C. albicans (412), and the epithelial CD4⁺ cells are located above the basement membrane and therefore not in proximity to the superficial keratinocyte layer where C. albicans is localized. Although not formally demonstrated in OPC, the participation of macrophages in Candida antigen presentation is more likely, since these cells are a prominent component of the innate immune response to C. albicans and fulfill all the requirements for engulfment, killing, and presentation of C. albicans antigens to CD4⁺ cells (18, 173, 314). Of direct relevance to this process, the ability of human monocytes to phagocytose the yeast but not the hyphal form of C. albicans is correlated with enhanced induction of IL-12, again indicating that C. albicans yeasts are specifically involved in promoting Th1 protective immunity