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Surface Glycans of Candida albicans and Other Pathogenic Fungi: Physiological Roles, Clinical Uses, and Experimental Challenges

Department of Pathology, University of Virginia, Charlottesville, Virginia 22908-0904

SUMMARY

INTRODUCTION

GLYCANS IN THE CANDIDA CELL WALL

Basic Components

Glucan.

Chitin.

Mannan.

Sialic acids.

Combination To Form the Cell Wall

FUNGAL GLYCANS IN PATHOGENESIS AND HOST RESPONSE

Protein Folding and Tertiary Structure

Adhesion

Adhesion to host components.

(i) ß-1,2-Mannan.

(ii) Acid-stable mannan.

(iii) O-linked glycans.

Adhesion to other members of the microflora.

Immune Cell Receptors and Interactions

Glycan binding to and activation of immune cells. (i) Mannan.

(ii) Chitin.

(iii) Glucan.

Glycan influence on cytokine production. (i) Mannan.

(ii) Glucan.

Recognition by antibodies.

DIAGNOSTICS AND THERAPEUTICS

Diagnostics

Mannan assays.

Glucan assays.

Therapeutics

ISSUES IN CARBOHYDRATE CHEMISTRY

Detection and Separations

Complexity and Characterization

CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Infections by pathogenic fungi, particularly Candida species, are both widespread and increasing in frequency (68, 276). Oral colonization by Candida albicans has been reported at 17.7% in the healthy population (40). Among hospitalized patients, oral carriage of Candida albicans rises to 40.6%. Healthy, asymptomatic women demonstrate an incidence of vaginal colonization by Candida of 15 to 20%. This percentage rises to 20 to 40% in healthy pregnant women and to 40 to 60% in human immunodeficiency virus-infected pregnant women (26, 209, 254, 273). Hospitals in the United States participating in the National Nosocomial Infection Survey System reported a nosocomial fungal infection frequency of 3.8 per 1,000 discharges in 1990, an increase from 2.0 per 1,000 discharges in 1980 (17). Candida species accounted for 78.3% of all such nosocomial fungal infections, followed by Torulopsis (now Candida) glabrata and Aspergillus species. C. albicans was the most frequently isolated of all the Candida species (17, 219). Furthermore, a European study reported that fungal species in general accounted for 17.1% of intensive care unit-acquired infections (306).

Using the National Nosocomial Infection Survey (1980 to 1990) data, Jarvis and Martone (131) reported that Candida species accounted for 9.4% of nosocomial urinary tract infections, 10.1% of nosocomial infections in adult and pediatric intensive care units, and 7.8% of nosocomial bloodstream infections. In 1990, fungi as a whole accounted for 10% of all nosocomial bloodstream infections (17). Ten years later, the rates of bloodstream infections due to Candida are still rising, with half of these candidemias due to C. albicans (230). More recently, other Candida species—such as C. glabrata, C. tropicalis, and C. dubliniensis—have been isolated with increasing frequency (220). A study carried out by five university hospitals in the Netherlands also reported increased fungemia rates. Between 1987 and 1995, episodes of fungal bloodstream infections rose from 0.37 to 0.76 per 10,000 patient days. Of these infections, 93% were due to Candida species while 7% were due to Cryptococcus species (307).

In addition to the observed increases in incidence, fungal infections continue to be a serious clinical problem with respect to increased morbidity and mortality (194, 314; see also reference 219 and references therein). An analysis of records from the National Center of Health Statistics indicated that mycoses ranked 10th among the underlying causes of death in the United States in 1980 but that by 1997 this rank had risen to 7th (193). Increased morbidity due to nosocomial fungal infections also takes a heavy toll, resulting in longer hospital stays (49, 178, 314) and higher patient care costs (91, 234, 285).

The increasing incidence of fungal infections has, to a certain degree, coincided with the incredible medical advances of the last few years. Life-prolonging technologies, although welcomed by medicine and those it cares for, have unfortunately created numerous opportunities for emerging fungal infections. The rising number of immunocompromised patients, whether through immunosuppressive therapy, AIDS, or other ailments, the prophylactic use of antimicrobics, and the increasing number of indwelling catheters and prosthetic devices seen among patients all unwittingly subject patients to the threat of invasive disease (68). Patients with central intravenous IV catheters were over three times more likely to have bloodstream infections than patients without such catheters (17). Patients undergoing orthotopic liver transplantation demonstrated an incidence of invasive fungal infection of 5 to 42%, with Candida species being the most commonly isolated organism, followed by Aspergillus species (54). A study of heart transplant patients reported that while fungal infections accounted for only 7% of all infections, they were associated with the highest (36%) mortality (194). Taken together, these clinical observations strongly reinforce the necessity for understanding the relationship between fungus and host.

The interaction between fungus and patient occurs first at the level of the cell wall (35). The cell wall of most fungi is composed of glycoproteins embedded within a polysaccharide matrix or scaffolding. Additionally, some fungal species produce a polysaccharide capsule that surrounds the cell wall (e.g. the glucuronoxylomannan capsule produced by Cryptococcus neoformans [221]). It is therefore possible, and even likely, that carbohydrate, not DNA or RNA and perhaps not even protein, is the first component to contact host tissue. Thus, characterization of these exterior carbohydrate groups could lead to (i) a better understanding of fungal adhesion and mechanisms by which fungi avoid the host immune system, (ii) proposals for countering this avoidance so as to enhance the immune response, and (iii) the development of diagnostic tests based on identification of carbohydrate components. The utility of cell wall and cell surface carbohydrate groups has been shown in other areas of clinical microbiology (e.g., Mycobacterium) (23). Fungal cell surface carbohydrates also play an important role in industry (132, 135) and in plant and nonhuman animal fungal pathogenesis.

The purpose of this review is to bridge the areas of fungal pathogenesis and glycobiology. To this end, the manuscript is divided into three major sections. First, a brief introduction to the carbohydrate components of the fungal cell wall and how they are assembled is provided. Second, relevant studies where carbohydrates have been shown to play a role in infection, function in diagnostic assays, and act as therapeutic agents are discussed. The final section is a discussion of the practical issues and difficulties that arise when working with microbial carbohydrates.

This review is limited in scope in two ways. First, although there is an extensive body of literature describing the various structures and pathways by which microorganisms interact with host species, the focus of this review is fungal glycobiology. Therefore, discussion is limited to the polysaccharide components of the fungal cell wall and does not include more general fungal cell fractions, fungal proteins and lipids, or elements of the host cells. In addition, examination of the C. albicans literature illustrates the need for both careful identification of fraction components and strategies for demonstrating that carbohydrate, rather than protein or lipid, is responsible for the observed effect. For example, while C. albicans mannan (defined below) is found only as a glycoconjugate with protein or lipid, many pathogenesis studies use the term "mannan" to refer globally to mannoprotein or similar glycoconjugate fractions.

Second, C. albicans will serve as the example organism for relating glycobiology to clinical microbiology. Studies describing the role of carbohydrate groups in pathogenesis have been carried out mainly with C. albicans; therefore, the review likewise focuses on this organism. A review by Nelson et al. (202) focused on Candida mannan and issues of mannan chemistry, immune suppressive effects, and mechanisms of action. This review expands on their discussion to include glucans and relevant mannan results reported since the publication of the previous review. Although the focus is C. albicans, it should be remembered that carbohydrates, as polysaccharides or components of glycoproteins, play a role in the pathogenesis and diagnosis of other fungal species such as Cryptococcus neoformans, Aspergillus fumigatus, Kluyveromyces lactis, and Paracoccidioides brasilienis. Where appropriate, selected results from studies of these species are included in the discussion.

GLYCANS IN THE CANDIDA CELL WALL

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View larger version (14K): [in this window] [in a new window]   FIG. 1. Monosaccharides found in the C. albicans cell wall.

Chitin. Linear polymers of ß-1,4-D-GlcNAc, called chitin, provide cross-linking and strength to the glucan scaffolding. The amount of chitin found in the cell walls of C. albicans hyphae is three times that found in yeasts (50). In contrast, Kanetsuna et al. (149) found that the GlcNAc content of the mycelial forms of Paracoccidioides brasiliensis and Blastomyces dermatitidis was one-quarter to one-third that of the yeast form. Lipke and Ovalle, based on findings of Kapteyn et al., have suggested that chitin plays a role in a cellular integrity rescue mechanism (153, 181). That is, when the cell wall is weakened by decreased amounts or quality of glucan, the cells compensate by increasing chitin production. Bahmed et al. (12) noted an increase in cell wall chitin in Kluyveromyces lactis and K. bulgaricus strains resistant to amphotericin B. Their results indicate that the net increase in chitin is due to a decrease in chitinase activity rather than an increased chitin synthase activity. However, the specific link between antifungal resistance and chitin content remains to be identified. Chitin also forms the core of the septum during bud growth and separation (see reference 29 for a review of the process in S. cerevisiae). GlcNAc also appears in the fungal cell wall as a component of glycoproteins. A chitobiose [D-GlcNAcß-(14)-D-GlcNAc] forms the link between protein asparagine residue and the inner core of N-linked glycans (Fig. 2).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Glycan structures in C. albicans glycoproteins. The processed oligosaccharide core is extended from the -1,3-linked mannose at its C-6 position (R). R structures represent possible side branch structures that can be incorporated into this outer-chain elongation. X can be up to 60 to 70. The acid-labile phosphooligomannosides are added to a side branch mannotetraose. Circled mannose units are potentially phosphorylated. Inset: Map key for linkage points. All bonds are -1,n, unless otherwise indicated. Structure scheme adapted from reference 88 with permission. Structural information based on work in references 13, 167 to 169, 211, 264, 265, and 293 and R. C. Montijn (1998), Ph.D. thesis, University of Amsterdam. GlcNAc, N-acetylglucosamine; M, mannose.

As alluded to above, mannose can be linked through either - or ß-anomeric bonds. O-linked glycans and the outer chain side branches of N-linked glycans are composed mainly of -1,2- or -1,3-oligomannosides. Three types of ß-1,2-mannose additions have been reported by Suzuki and coworkers (266). The first type forms the acid-labile mannan in strains of C. albicans (both serotype A and serotype B), C. glabrata, and C. tropicalis (164-167, 263) The second is found on the nonreducing terminal of -1,2-oligomannoside outer chain side branches of serotype A C. albicans strains and of C. glabrata and C. tropicalis strains (164-166, 263). The third type is connected to an -1,3-linked mannose in the acid-stable mannan of C. guilliermondii and C. saitoana (261, 266). In addition, the first mannose in the N-linked glycan core is linked ß-1,4 to the second GlcNAc (Fig. 2). The structural models of Rees and Scott (231) predicted that -1,2-mannan forms type B helices like ß-1,3-glucan, -1,3-mannan forms extended ribbon chains (type A), and -1,6-mannan forms type D chains.

ß-1,2-Mannan was predicted to form crumpled or contorted chains (type C). Further, all 1,2-linked glycans, with the exception of -1,2-mannan, were predicted to form these distorted chains (231). Because of steric clashes between nonadjacent residues, the authors expected such structures to be found only rarely in nature. This expectation is supported empirically by the determined structures of commonly occurring polysaccharides. For example, neither starch (-1,4-D-Glc; type B), cellulose (ß-1,4-D-Glc; type A), nor glycogen (-1,4-D-Glc with -1,6-D-Glc branches; types B and D) forms type C chains. Likewise, none of the commonly occurring fungal polysaccharides (Table 1) are predicted to form these crumpled, contorted chains. Nonetheless, examples can be found of 1,2-linked sugars, in addition to C. albicans mannan. Some these examples are Cryptococcus neoformans capsule glucuronoxylomannan (ß-1,2-D-xylose and ß-1,2-D-glucuronic acid), C. neoformans capsule galactoxylomannan (ß-1,2-D-xylose), gum ghatti (ß-1,2-D-glucuronic acid), and gum tragacanth (-1,2-L-glucose) (9, 10, 22, 52, 157). In these cases, however, the 1,2-linked sugars occur either as a terminal group of a side chain or discontinuously as a component of a heteropolymer. In either case, the potential steric conflicts resulting from chain formation are avoided. The two instances of 1,2-linked sugars forming oligomers are a ß-1,2-D-glucan from Agrobacterium species (94, 226) and the ß-1,2-D-oligomannosides from Candida species.

Sialic acids. With only a few exceptions, sialic acids have been found only in higher eukaryotes (i.e., vertebrates and some invertebrates such as Echinoderms) (300). Besides these organisms, sialic acids have been found in Drosophila embryos and the capsule material of some bacteria species. Benhamou and Ouellette (19) also found sialic acid in the cell wall of a fungal plant pathogen, Ascocalyx abietina. Where they are typically found, sialic acids are components of O-linked glycans or complex-type N-linked glycans (Fig. 3). As mentioned above, several groups have presented evidence for the presence of sialic acids in the cell wall of C. albicans (2, 140, 272, 308). Until these reports, there had been no indication that yeast (C. albicans or S. cerevisiae) produce sialic acid in any capacity.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Representative glycans from higher eukaryotic organisms. Inset: Key for linkage positions as in Fig. 2. Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Glc, glucose; M, mannose; SA, sialic acid.

Several aspects of these results are puzzling. First, the C. albicans cells were disrupted by shearing with glass beads. The cell debris, containing the cell wall, was removed by centrifugation, and the supernatant fluid, containing mostly cytoplasmic proteins, was used as the source material for a C3 binding protein. Such a protein would be expected to be membrane or cell wall associated and would thus be more likely to be found with the cell pellet. Second, the removal of terminal sialic acids by neuraminidase seems insufficient to account for a 20% decrease in apparent molecular mass, observed as an increase in relative electrophoretic mobility. Relative electrophoretic mobility is also unlikely to be affected by the presence or absence of negative charge from sialic acids because such charge effects are overwhelmed by the sodium dodecyl sulfate present during electrophoresis. Thus, any change in relative electrophoretic mobility must be due to changes in apparent molecular mass. The neuraminidase preparation used (Sigma; type V) reports potential protease contamination. The presence of proteases could be an alternative explanation for the observed decrease in molecular mass. However, because the 42-kDa protein was unaffected by neuraminidase treatment, the presence of generally acting proteases seems unlikely. Third, Endo F treatment did not deglycosylate the C3d binding proteins. This enzyme cleaves N-linked glycans between the two GlcNAc residues of the oligosaccharide core. It is effective against high-mannose or hybrid glycans but not against complex glycans. While this is consistent with the types of glycans typically containing sialic acids, it is not consistent with the types of glycans made by C. albicans.

Similar evidence was presented by Wadsworth et al. (308) in their characterization of a type 2 complement receptor (CR2). Proteins were extracted from the C. albicans cell wall with dithiothreitol. Glycans were labeled with biotin-hydrazide either before or after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and subsequent transfer to nitrocellulose. Sodium periodate oxidizes cis-diols (e.g., between C-2 and C-3 of mannose), cleaving the ring and producing two aldehydes. These aldehydes can then react with the hydrazide group, resulting in covalent linkage of the attached biotin to the glycan. Wadsworth et al. showed that a 50-kDa protein from C. albicans blastoconidia and a 60-kDa protein from hyphae were recognized by anti-CR2 antisera. Initial results indicated that both proteins were glycosylated by biotin-hydrazide labeling. A 50-kDa protein also bound concanavalin A (Con A) and, to a lesser extent, wheat germ agglutinin (which normally binds GlcNAc but has been reported to also bind sialic acids). However, treatment with N-glycanase (which cleaves between the first GlcNAc and the Asn) or Endo H (which acts in a similar manner to Endo F) did not significantly reduce lectin binding, as would be expected. Furthermore, N-glycanase, Endo F, or neuraminidase treatment had no effect in immunoblots using the anti-CR2 antisera as probes. Based on these results, and considering that the protein separations were one-dimensional PAGE, it seems more likely that the 50-kDa CR2 protein is the deglycosylated form and that the biotin-labeled protein was a comigrant.

The 60-kDa protein also appeared to bind Con A, although this binding also was unaffected by Endo H treatment. This protein gave a stronger signal with regard to wheat germ agglutinin binding, which was markedly reduced on digestion with N-linked glycanase. The remaining results, however, led to some conflicting conclusions. Neuraminidase treatment of the 60-kDa protein led to loss of signal in immunoblots, which might be expected if the epitope included the removed sialic acid. In addition, neuraminidase treatment, either before or after biotinylation, eliminated detection of the biotin signal. This result is consistent with the terminal sialic acid being linked to a galactose (Gal) residue, as is seen in complex-type N-linked glycans. Although galactose can be labeled with biotin-hydrazide, a preliminary reaction with galactose oxidase is required to generate the required hydrazide-reactive aldehydes (248). N-Glycanase and Endo F deglycosylated the 60-kDa protein, as indicated by a decrease in apparent molecular mass in immunoblots. N-Glycanase cleaves both complex and high-mannose N-linked glycans, but Endo F is effective only against high-mannose glycans. This result, in contrast to the neuraminidase result, suggests that the glycans on the 60-kDa protein are high mannose rather than complex.

Jones et al. (140) examined the electrostatic properties of the C. albicans cell surface by using a cationic fluorophore, 9-aminoacridone. The electronegative C. albicans surface attracts the fluorophore, leading to self-quenching and a decrease in detectable fluorescence. Treatment of the cells with neuraminidase resulted in a time-dependent decrease in fluorescence intensity, leading these authors to conclude that the C. albicans surface contained sialic acid groups. However, the neuraminidase used in this study was from the same source as that used by Alaei et al., so that protease contamination is a potential confounding factor for concluding that the observed loss of electronegativity was due specifically to loss of sialic acids.

More direct evidence was presented by Soares et al. (272), who used fluorophore-labeled lectins to detect sialic acids on the C. albicans cell surface. Fluorescein isothiocyanate-conjugated Sambucus niger agglutinin (SNA) strongly labeled the entire surface of blastoconidia but did not appear to label hyphae. This lectin is highly specific for the sialic acid N-acetylneuraminic acid linked -2,6 to galactose or GalNAc. Labeling by SNA was completely eliminated by pretreatment of C. albicans cells with Clostridium perfringens sialidase. Fluorescein-isothiocyanate-conjugated agglutinins from Limax flavus and Limulus polyphemus also bound to the C. albicans cell surface. These lectins are not specific for sialic acid linkage position and do not require a specific subterminal sugar group. As with the SNA result, L. flavus and L. polyphemus agglutinin binding was significantly reduced by pretreatment of the Candida cells with sialidase. In addition, sialidase treatment allowed the binding of peanut agglutinin to occur. This lectin recognizes terminal ß-galactose units, groups that would be exposed from complex-type N-linked glycans following removal of the terminal sialic acids (Fig. 3).

Using similar methods, sialic acids have been found to be also associated with the cell surface or exogenous glycoproteins of Cryptococcus neoformans, Fonsecaea pedrosoi, Paracoccidioides brasiliensis, Pneumocystis carinii, and Sporothrix schenckii (237, 270, 271; reviewed in reference 6). In addition, Rodrigues et al. (236) have recently shown the presence of sialyltransferase activity in C. neoformans. Taken together, these results not only demonstrate the presence of sialic acids on pathogenic fungi but also show that these sugars are probably the termini for complex-type N-linked glycans. This observation is significant because these glycans had not previously been found on these organisms. In mammals, sialylated glycans are often a component of glycoconjugates that modulate intercellular interactions, such as the sialyl Lewis x selectin ligand (155, 252).

A similar role for sialylated glycans has been demonstrated for C. neoformans and S. schenckii, where treatment of fungal cells with neuraminidase significantly increased phagocytosis by murine peritineal macrophages (5, 6, 237). S. schenckii or C. neoformans yeast cells were treated with neuraminidase from Clostridium perfringens or Vibrio cholerae for 1.5 to 2 h and then washed. Treated cells and untreated control cells were then added to adherent peritoneal macrophages. Neuraminidase treatment of C. neoformans cells led to a twofold increase in the phagocytic index (the percentage of macrophages phagocytizing at least one yeast multiplied by the average number of phagocytized yeast cells) (237). For S. schenckii cells, the increase was almost eightfold (207). In the studies above, neuraminidase was specifically chosen to complement the other work demonstrating the presence of sialic acids. It is unknown what effect, if any, treatment with other glycosidases, such as mannosidase, would have on phagocytosis. However, the neuramindase results are consistent with a model, proposed by Schauer and coworkers, that terminal sialic acid groups mask ligands for macrophage-bounds receptors (155, 156, 177). This raises the possibility that sialylated glycans on fungi and other microorganisms can assist in adaptation to life in the host by avoiding recognition or killing by the immune system.

Previous studies of mannoprotein fractions from the cell walls of S. cerevisiae or C. albicans gave no indication of the presence of sialic acids or complex-type N-linked glycans. There are two potential explanations for this (until recently) lack of evidence. First, the usual methods of mannoprotein preparation (hot water or citrate extraction followed by precipitation with Cetavlon or Fehling's reagent [see below]) may be harsh enough to degrade the sialic acid groups, which are known to be sensitive to chemical conditions (301). Second, the N-linked glycans on mannoproteins are strictly high mannose, as described in all previous reports, but the sialic acids are components of other glycoproteins as complex-type glycans. Such glycoproteins would not be expected to precipitate with Cetavlon because they lack the long mannan chains needed to interact with the Cetavlon (discussed in reference 191) and the mannose cis-diols needed to interact with borate (in the Cetavlon precipitation) or copper (in Fehling's reagent).

Visualization of the cell wall structure began with electron microscopy, which showed layers of differing electrodensity (42, 43, 260). These electron micrographs, combined with biochemical data, led to the development of a cell wall schematic (Fig. 4) (35, 232, 260) that has been further refined as the cell wall components have been studied more closely (161, 213). Although these schematics are often depicted as having a layered structure, it has become increasingly apparent that the cell wall is actually a dynamic structure and that any given layer should be more properly referred to as a zone of enrichment (260).

View larger version (33K): [in this window] [in a new window]   FIG. 4. Schematic of the C. albicans wall structure. The wall structural cartoon is based on information from references 150, 152, 213, and 269. Electron micrographs are reprinted from reference 108 with permission. GPI, glycosylphosphatidylinositol-linked glycoproteins; ManPr, mannoprotein; PIR, PIR-family glycoproteins; PM, plasma membrane.

The detailed structure of the fibrils is still unclear. One possibility is that the protein component is globular and lies close to the glucan scaffolding, with the observed fibril being entirely glycan. Alternatively, the fibril may contain a linear or tightly helical protein core decorated with glycan. Some cell wall proteins, which are anchored into the plasma membrane, possess a "lollipop on a stick" structure (134). A long stretch of polypeptide (the stick) extends from the membrane and passes through the wall. Short O-linked glycans are attached along the length of the stick, presumably to help maintain a linear structure. A globular domain (the lollipop) is then stuck out into the extracellular space. However, it is unknown if the fibrils are anchored in such a way into the membrane, covalently attached to glucan or chitin, or simply trapped within the glucan matrix. It is also not known if the fibril is composed of subunits, but some initial experiments suggest that a fibril subunit exists (319).

Some organisms produce extensive carbohydrate structures at the cell wall exterior. For example, C. parapsilosis has been reported to produce a slime layer similar to those produced by Staphylococcus epidermidis and other bacterial pathogens (186, 187). Although the composition of this slime layer has not been characterized in detail, Kuhn et al. (171) have shown that cell wall-like polysaccharides comprise the matrix. Their conclusions were based on staining with a fluorescent Con A conjugate, which binds to terminal mannose and glucose units, and staining with Calcofluor White, which binds to ß-linked polysaccharides such as chitin and ß-glucan. These authors also demonstrated that C. albicans, in addition to C. parapsilosis, was able to construct this slime layer biofilm. As mentioned above, Cryptococcus neoformans constructs a polysaccharide capsule composed mainly of glucuronoxylomannan (52, 297) on the cell wall exterior. Galactoxylomannan and mannoproteins are also elements comprising the capsule. The genetic and temporal regulation of capsule synthesis has recently been reviewed by Bose et al. (22).

FUNGAL GLYCANS IN PATHOGENESIS AND HOST RESPONSE

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Adhesion to host components. The presence of polysaccharide in the intercellular junction of C. albicans cells bound to epithelial or endothelial cells has been known for some time. Marrie and Costerton (188) described a ruthenium red-stainable matrix surrounding the C. albicans cell. Ruthenium red is a cationic dye with affinity for anionic polymers such as certain polysaccharides. The observed matrix was shown to exist between fungal cells and buccal tissue as well as between fungal cells and bacterial cells. Barnes et al. (16) demonstrated fibril connections between the yeast cell surface and the endothelium of renal peritubular and glomerular capillaries. Although the specific composition of the fibrils (i.e., carbohydrate versus protein) was not investigated, the authors speculated that they were the same as, or similar to, polysaccharide fibrils found on the S. cerevisiae cell surface as described by Johnston and Latta (136). Tronchin et al. (296) also reported that fibrillar structures and polysaccharide granules on the yeast cell surface appeared to mediate adhesion to epithelial cells. Some of this material was released from the cell surface into the surrounding medium under certain conditions (109, 183).

Other work focused on specific glycans that might be involved in adhesion events. The majority of evidence pointed to mannan as the active glycan. The studies discussed immediately below established the role of mannan in adhesion. Subsequent work extended these results to show which specific mannan components (ß-1,2-oligomannosides, acid-stable side branches, or O-linked glycans) were the critical ones.

Maisch and Calderone (184) found that the mannoprotein fraction from C. albicans, when conjugated to sheep red blood cells (SRBC), was sufficient to cause the conjugated SRBCs to adhere to a fibrin-platelet matrix. Mannosidase treatment eliminated the adherence, indicating that the mannan was the adhering component. Subsequent results indicated that the matrix component involved in binding to mannan was fibronectin (253).

Sandin et al. (245) showed that pretreatment of yeast cells with Con A inhibited the binding to buccal epithelial cells (BEC), from 100 to 18% adherence. Con A pretreatment of the BECs also reduced binding. Con A is a lectin that binds -linked terminal mannose or glucose residues of an oligosaccharide (93). Of the carbohydrates in the Candida cell wall (mannan, glucan, and chitin) only mannan is -linked. Furthermore, Con A-mediated inhibition of binding was partially reversible with increasing amounts of -methylmannoside added as a mannan competitor. Lectins that bind N-acetylgalactosamine (GalNAc), GlcNAc, fucose, or galactose did not significantly alter percent adhesion. Sandin et al. also reported that -methylmannoside itself inhibited the binding to BEC while GlcNAc, ribose, -methylglucoside, galactose, and xylose did not. This is in partial disagreement with the results of Sobel et al. (274), who reported that treatment of C. albicans with fucose inhibited adhesion but treatment with mannose, -methylmannoside, or galactose did not.

Centeno et al. (47) investigated the possible adhesion interactions between epithelial cells, bacteria, and fungi. Earlier observations had been made that bacterial binding to epithelial cells blocked similar binding by fungi. In addition, bacterial structures (e.g., pili) can bind to fungal cells, leading to fungal agglutination. Centeno et al. found that binding of yeast to BEC was enhanced by pretreatment of the BEC with piliated, but not nonpiliated, strains of Klebsiella pneumoniae or E. coli. The enhanced binding was reversed by the addition of mannose. Further, mannose, but not glucose, inhibited C. albicans binding to urothelial cells and BEC in the absence of bacterial cells. The authors hypothesized that the bacterial pili added mannose-recognizing lectin-like proteins that provided extra attachment sites for the fungal cells.

Sawyer et al. (250) examined a liver perfusion model for studying yeast adhesion. Mouse liver was isolated (in an ex vivo manner) into a perfusion system. C. albicans cells were then infused into the liver, and the system was switched back to perfusion medium. Clearance of cells from the perfusate exiting the system was due either to endothelial trapping or, to a lesser extent, to adherence to and subsequent phagocytosis and killing by Kupffer cells. Treatment of the yeast cells with -mannosidase (from Jack Bean), subtilisin, -chymotrypsin, and papain increased the hepatic killing of yeast cells but did not affect trapping. Mannoprotein, isolated from either C. albicans or S. cerevisiae, blocked fungal cell trapping in the liver and was also able to elute previously trapped cells. These results, combined with previous results from this group showing that mannose or -methylmannoside (but no other sugar) was able to block liver trapping, suggest that it is the carbohydrate portion of the mannoprotein fraction that is involved in hepatic endothelium adhesion.

The use of monosaccharides (e.g., mannose or -methylmannoside) to inhibit binding is commonly applied to demonstrate the recognition of glycans. In the case of competition for the Con A site, this practice seems reasonable since it is known that Con A binds to terminal saccharides. However, other lectins or receptors may recognize oligosaccharides or even oligosaccharides in the context of the polypeptide or lipid to which they are conjugated. Further, these binding interactions can be specific for not only chain length and saccharide composition but also anomericity and linkage. Some of these instances are mentioned in the discussion below. Thus, it seems that the use of bioses or longer oligosaccharides as competitors would be a more physiologically relevant choice. There are a couple of issues, however, that make this choice problematic. First, the specific configuration (i.e., saccharide, anomericity, linkage) needed for a competitor often cannot be known a priori. Therefore, several oligosaccharides may have to be tried before a suitable one is identified. Second, in cases such as the ß-1,2-oligomannoside adhesin described below, the oligosaccharides are not commercially available and must be fractionated and purified in-house, which can be time-consuming. The problem of oligosaccharide availability is discussed in more detail in the last section of this review.

(i) ß-1,2-Mannan. Miyakawa et al. (195) developed C. albicans mutants that lack a ß-1,2-oligomannoside sequence found in the acid-stable mannan of serotype A strains. They observed a decrease in adhesion to BEC and to a mouth squamous carcinoma cell line by the mutant compared to the parent. Adherence of the mutant was comparable to that seen for wild-type serotype B cells that lack the ß-1,2-oligomannosides in the acid-stable region. Pretreatment of the epithelial cells with mannoprotein from the parent strain decreased adhesion of parent cells, but mannoprotein from the mutant did not.

A series of papers from Cutler and coworkers identified specific mannan groups involved in C. albicans binding to macrophages in the splenic marginal zone. Kanbe et al. (147) first demonstrated this binding in an ex vivo assay. Subsequent studies (146, 179) investigated the possible cell wall groups that might be involved in the observed adhesion. C. albicans cells were extracted with ß-mercaptoethanol, and the released material was fractionated using Con A affinity and ion-exchange chromatography (146). The crude ß-mercaptoethanol extract inhibited yeast cell binding to the splenic marginal zone and lymph node epithelial cells in a dose-related manner, and this inhibition activity could be isolated to one particular fraction. Heat and protease treatment of the fraction did not affect inhibition, but periodate and mannosidase treatment did, indicating that the active moiety was carbohydrate. This conclusion was supported by experiments using a monoclonal antibody (10G) that recognizes a ß-1,2-mannotetraose (179). The antibody was able to inhibit C. albicans attachment to murine splenic marginal-zone macrophages in the ex vivo assay. Recently, Fradin et al. (83) identified the receptor molecule on the macrophage for ß-1,2-oligomannosides to be a 32-kDa protein with homology to galectin 3. Since the strain that was used for these studies, A9, is serotype B, the tetraose epitope must be part of the acid-labile fraction of N-linked glycans (Fig. 2).

In a recent study using an in vivo model, Dromer et al. (71) supported the importance of ß-1,2-oligomannosides in adhesion. Intestinal colonization in mice was assayed by both survival and clearance (measuring CFU in fecal pellets). Expression of, or presence of, ß-1,2-oligomannosides, as measured by agglutination with an anti-ß-1,2-oligomannoside monoclonal antibody, positively correlated with virulence as assayed in mouse and rat systemic models. In the developed infant-mouse gastrointestinal (GI) colonization model, the investigators tested virulent (10261, high ß-1,2) and avirulent (10231, low ß-1,2) strains and measured the CFU/fecal pellet. The median score was always lower for 10231, and colonization was more protracted/prolonged with 10261 (which contained more ß-1,2). Synthetic ß-1,2-oligomannosides, but not -1,2-oligomannosides, administered by gavage 1 h preinoculation, decreased or eliminated the CFU/fecal pellet (day 7 postinfection) in a dose-related manner. The authors therefore concluded that ß-1,2-oligomannosides play a role in colonization, presumably at the adhesion step.

(ii) Acid-stable mannan. Other work by Cutler's group (144) showed that the acid-stable mannan inhibited C. albicans binding to splenic marginal zone epithelial cells, demonstrating that this mannan is also involved in attachment. Inhibition was not affected by treatment of mannoprotein with pronase, confirming the importance of the carbohydrate portion. Although the exact structure of the mannan group involved is not yet known, certain characteristics have been determined. Evidence suggests that both the -1,2-linked side branches and the -1,6-linked backbone are involved (145). Mannoproteins from S. cerevisiae MNN2 deletion mutants, which do not attach side branches to the outer chain backbone, did not inhibit binding, showing that the -1,6-linked backbone alone is not sufficient. Acetolysis of the acid-stable mannan cleaves the -1,6-linked backbone bonds and releases the side branches. Acetolysates of acid stable mannan also had no effect on binding, indicating that the side branches alone are not sufficient to produce the observed acid stable mannan-dependent adhesion. Partial sulfuric acid digestion, which sequentially releases the terminal sugar groups from the nonreducing ends, was carried out on acid-stable mannan. As the digestion progressed, producing shorter side branch chains, the inhibition of C. albicans binding decreased. Taken together, these results indicate that the acid-stable mannan mediating C. albicans binding to splenic marginal zone macrophages comprises two or more side branches, joined through the outer chain backbone and of a certain minimum length. Based on their results, Kanbe and Cutler (145) further concluded that chain length was more important than a specific side branch conformation. Other specifics of the macrophage mannose receptor and mannose-binding protein have been recently reviewed (84).

(iii) O-linked glycans. Buurman et al. (27) suggested that O-linked glycosylation plays a role in adhesion. Mutant strains with defects in O-linked glycosylation (leading to truncated glycans) showed decreased adhesion to human buccal epithelial cells in vitro and decreased adhesion to rat vaginal epithelial cells in vivo. They also observed that guinea pigs and mice had increased survival when challenged with the mutant strains. This increased survival was coincident with a decreased organ burden. These results, combined with the in vitro adhesion experiments, led the authors to conclude that decreased adhesion resulted in decreased organ burden and increased survival.

Herrero et al. (110) looked at the issue of glycosylation from the supply side. GDP-mannose is brought in from the cytosol to the Golgi lumen, where mannose is transferred to the growing mannan chain. The other product, GDP, is then hydrolyzed by a GDPase to give GMP and inorganic phosphate. GMP is then transported to the cytosol through an antiporter that brings in the GDP-mannose. Thus, a defect in the GDPase should have an effect on protein glycosylation. Previously, deletion of the S. cerevisiae GDPase gene (20) decreased glycosylation in the Golgi. The authors cloned the C. albicans GDPase gene, designated CaGDA1, and used the URA-blaster (80) procedure to knock out both alleles in C. albicans. Unexpectedly, they observed no change in N-linked glycans. However, a significant change in O-linked mannan was observed. There was a loss of the mannobiose and mannotriose groups and a corresponding increase in the monomer. This suggested a loss of ability to extend the O-linked chain. However, this loss of O-mannosylation did not affect measured adherence to HeLa cells.

These O-linked glycan results present a few possibilities. (i) The defect in the Herrero strain was not severe enough to alter adhesion. The authors themselves speculate that there might be backup GDPases in C. albicans. (ii) the residual O-linked M₁ glycans (from Buurman et al. [27]) or the residual M₄-M₅ O-linked chains observed by Herrero et al. (110) were sufficient to maintain whatever structural requirement was necessary for glycoprotein stability and adhesin function. (iii) Some of the results observed by Buurman et al. (e.g., increased survival, increased vaginal clearing, and decreased organ load) were due to processes other than adhesion per se, although this later case is harder to reconcile with the observed decrease in adhesion to BEC in vitro. (iv) If O-linked glycans play a structural role (as in the lollipop-on-a-stick model), the effect of disrupting O-linked glycosylation may have more to do with protein structure than the glycans acting directly as an adhesin.

There is some evidence for the involvement of glycans other than mannan in adhesion. Reversing the typical manner in which adhesion studies are done, Forsyth and Matthews (81) investigated carbohydrate inhibition of lymphocyte adhesion to C. albicans hyphal cells. While mannose and -methlymannoside both inhibited interleukin-2 (IL-2)-activated mouse lymphocyte adhesion to C. albicans hyphal cells by approximately 40%, GlcNAc inhibited adhesion by activated mouse and human lymphocytes by 80%. No effect on mouse lymphocytes was observed when glucose or sucrose was used. As with the mouse lymphocytes, glucose had no effect on adhesion of IL-2-activated human lymphocytes, but small amounts of ß-glucan (4 mg ml^–1, or approximately 40 µM) inhibited adhesion by 70%. From these results, the authors concluded that lymphocyte binding occurs through Mac-1, which has a lectin-like domain, known ligands for which include ß-glucan and chitin. Placing their observations in context, Forsyth and Matthews concluded that interactions between fungal cells (C. albicans) and lymphocytes are multidimensional but involve lectin-like binding sites on the lymphocytes and glycans on the fungal cell surface.

The inhibition of adhesion by GlcNAc and the chitin binding domain of Mac-1 echoed an earlier result of Segal et al. (255), who found that, of the cell wall polymers, chitin (although not mannoprotein or glucan) inhibited the binding of C. albicans to vaginal epithelial cells. Chitin hydrolysate, GlcNAc, glucosamine, and mannosamine inhibited adhesion to a similar extent (255). On first inspection, these results would seem to be in conflict with the current model of the cell wall that has the chitin component in the inner layers of the cell wall, near the plasmalemma (Fig. 4). It has been shown, however, that the hyphal cell wall is thinner than that of the blastoconidia (43). Further, there is evidence for a small amount of chitin being present on the cell surface (295). These observations serve as illustrations of the dynamic nature of the cell wall and support the evidence by Forsyth and Matthews and Segal et al. for a role of chitin in adhesion, but they do not explain the ineffectiveness of mannose or mannoprotein as an adhesion inhibitor observed by Segal et al.

Another possible explanation is that the experimental conditions are playing a role. Researchers have used various strains, growth conditions, and substrates in adhesion experiments (Table 2). A comparison of additional experimental conditions used in adhesion studies has been done recently by Calderone and Gow (32). The multidimensional aspect of adhesion noted by Forsyth and Matthews may indicate that the relative importance of a specific adhesin depends on the combination of fungal strain and tissue substrate, as well as experimental design.

Hsu et al. (117) looked at coaggregation of C. albicans and oral bacteria isolated from bone marrow transplant patients. They reported that in contrast to previous results (11), sugars did not inhibit coaggregation of C. albicans and A. viscosus. Neither did the sugars inhibit coaggregation with S. sanguis or S. epidermidis, in agreement with the other results. Coaggregation of C. albicans with S. mitis and Lactobacillus salivarius was inhibited by -methylglucoside. Glucosamine inhibited coaggregation with S. salivarius, while glucose inhibited coaggregation with Bacteroides gingivalis. Mannose, -methylmannoside, glucose, and -methylglucoside all inhibited coaggregation with Lactobacillus amylovorus. Coaggregation with F. nucleatum was inhibited by the addition of mannose or -methlymannoside. More recently, Jabra-Rizk et al. (121) showed that mannose inhibited the coaggregation of F. nucleatum with C. dubliniensis as well as C. albicans.

The summary of these results seems to be that the strains of A. viscosus (one strain, tested twice) and F. nucleatum (three strains) tested were positive for coaggregation and that the evidence is that a glycan on the fungal cell surface is involved in that coaggregation. Because the fungal cells were cultured and prepared for assay under different conditions, it is difficult to predict how other fungal phenotypes are involved in coaggregation. For example, results by Jabra-Rizk et al. (121, 122) suggest that cell surface hydrophobicity plays a role in coaggregation of C. albicans and C. dubliniensis with F. nucleatum. This conclusion is supported, to a certain extent, by the observation of Jenkinson et al. (133) that Streptococcus gordonii, S. sanguis, and S. anginosis adhered best to C. albicans germ tubes, structures that are highly hydrophobic, especially at the distal end (106, 107). These authors, however, concluded that cell surface hydrophobicity was not involved since starvation of fungal cells increased coaggregation but had no effect on cell surface hydrophobicity.

In contrast to F. nucleatum and A. viscosus, no strain of Klebsiella pneumonia, Lactobacillus casei, Peptostreptococcus anaerobius, P. magnus, P. micros, Porphyromonas gingivalis, Prevotella intermedia, Pseudomonas maltophila, P. stutzeri (2 strains), P. aeruginosa, or Staphylococcus aureus formed coaggregates. The Streptococcus species present more variable results that seem to be species and strain dependent. For example, S. sanguis appears to form some kind of coaggregate, but the evidence points to the relevant glycan being on the bacterial, not the fungal, cell surface.

Because zymosan phagocytosis was inhibited by a mannoprotein fraction, Speert and Silverstein (275) concluded that phagocytosis occurs after recognition by a mannose receptor. Similarly, Cross and Bancroft (56) reported that phagocytosis of an acapsular Cryptococcus neoformans mutant was inhibited by the addition of mannoprotein from S. cerevisiae. Scaringi et al. (251) reported that injection of mice with C. albicans mannoprotein stimulated the generation of natural killer cells and activated macrophages. Podzorski et al. (224) reported an increase in lymphoproliferation due to mannoprotein. The preceding results, however, lack the evidence necessary to prove that the effect was due to the carbohydrate portion of the mannoprotein fraction rather than the protein.

Several recent studies have linked glycan specifically to effects on the immune system. Podzorski et al. (224) refined their results mentioned above and examined the effect of O-linked mannan on lymphoproliferation. These oligosaccharides, separated from the mannoprotein fraction by beta elimination, inhibited lymphoproliferation in response to Candida antigen, tetanus toxoid, and herpes simplex virus type 1 (224). Fradin et al. (82) showed that pretreatment of macrophages (either peritoneal macrophages or the J774 macrophage cell line) with ß-1,2-linked, acid-labile oligomannosides decreased the macrophage binding of both live and heat-killed C. albicans cells. The extent of inhibition was the same when a synthetic ß-1,2-mannotetraose was used as the inhibitor. Another study from the same group showed that the ß-1,2-oligomannosides can also be released from C. albicans cells during C. albicans-macrophage coculture as a component of a glycolipid fraction (141). A recent paper by Cambi et al. (39) reported that along with the mannose receptor, dendritic cells (DC) bind C. albicans cells through a DC-specific intercellular cell adhesion molecule (ICAM)-grabbing nonintegrin (DC-SIGN) protein. Binding of fungal cells was inhibited in the presence of mannose, but only by 40%. Even with the addition of an anti-DC-SIGN antibody, binding was still 20% of control levels. In addition, when DC-SIGN was transfected into K562 cells (a moderately undifferentiated leukemic derivative cell line), mannose had no effect on C. albicans binding. These results suggest that the interaction of C. albicans cells and DC-SIGN is not mediated solely by lectin-like activity.

Watanabe et al. demonstrated that the glycan component of C. albicans mannoprotein acts as a hemolytic factor (311). RBC were mixed 1:1 (vol/vol) with cell fraction samples, and hemolysis was monitored by measuring the absorbance of the suspension at 405 nm. Hemolysis was associated with a sugar-rich, high-molecular-weight chromatographic fraction characterized as having low protein and high sugar content. Treatment of this fraction with periodate, which cleaves the glycan chains, eliminated hemolytic activity, demonstrating that carbohydrate was the active group.

Cross and Bancroft (56) extended their inhibition experiment to show that mannose binding protein bound to the acapsular Cryptococcus neoformans mutant, but not to encapsulated wild-type cells. These results indicated that the glucuronoxylomannan capsule produced by C. neoformans could mask potential glycan ligands recognized by immune cell receptors. Subsequent work by Mansour et al. (185) showed that mannoprotein released by C. neoformans into the culture supernatant fluid inhibited, in a dose-related manner, the binding of the macrophage mannose receptor to one of its natural ligands, horseradish peroxidase type II. They further showed that deglycosylation drastically reduced the ability of C. neoformans mannoprotein to activate primary T cells. In addition, binding of C. neoformans to DC is mediated, at least in part, by a mannose receptor (283). There is also evidence that peritoneal macrophages bind and phagocytose Paracoccidioides brasiliensis cells via a mannose receptor. Phagocytosis of P. brasiliensis cells was significantly inhibited by the addition of mannose or, to a lesser extent, fucose (3). Mannose reduced the phagocytic index to between one-third and one-half that of the control. This result again raises the issue of using monosaccharides as substitutes for oligomers or polymers. An oligomannoside, used as a competitor, may have completely inhibited phagocytosis because of more complete blockage of the macrophage receptor.

(ii) Chitin. Suzuki et al. (279) first reported the immunological effects of chitin in a murine model of candidiasis. Mice receiving pretreatment with chitin (50 mg kg^–1) were better able to survive a lethal challenge with C. albicans than were mice receiving no treatment. In addition, polymorphonuclear leukocytes (PMN) from circulating blood and peritoneal exudate cells from treated animals showed a higher level of reactive oxygen species (O₂^– and H₂O₂) generation than did those isolated from control animals. However, when macrophages were separated from peritoneal exudates, candidacidal activity was not dependent on reactive oxygen species (280) but was due to a serine protease released by the chitin-treated macrophages. Candidacidal activity was present in macrophage culture supernatant fluid and was inhibited by actinomycin D and diisopropylfluorophosphate, which inhibit protein synthesis and serine protease activity, respectively. One potential confounding factor of these studies is that the authors imply that the control animals were simply untreated rather than receiving sham injections. Thus, it is possible that some of the response is due to the act of injection rather than to receiving chitin specifically. However, chitosan, a deacetylated chitin derivative, was also tested for immunological effects (279, 280), and although chitosan-treated mice also had increased levels of PMN and macrophages, the distribution of these cells (circulation versus peritoneal exudate) was different. Thus, it seems less likely that the immunopotentiating effects of chitin were due entirely to the injection itself.

The effect of fungal chitin (as opposed to chitin extracted from crustacean shells) on immune system components is described in a single study. Rementería et al. (233) extended the earlier studies to examine chitin purified from the C. albicans cell wall. They reported that, as observed by Suzuki et al. (279), injection of mice with purified C. albicans chitin (30 mg kg^–1) increased the survival time in response to a C. albicans challenge of 10⁵ cells. In these experiments, it was explicitly stated that control mice were given an injection of saline to control for the stress of the injection itself. As a further refinement, the authors quantified the chitin present in the organs of experimentally infected animals. Chitin treatments were then based on these measured values.

To begin to look at more specific reasons for this increased survival, peripheral macrophages were isolated over the course of a 20-day study period (233). Macrophages isolated from mice treated with chitin and macrophages isolated from mice treated and then infected with C. albicans both showed increases in phagocytosis and candidacidal activity over those for macrophages isolated from control animals. However, the peak of phagocytosis, measured as (number of macrophages ingesting at least one yeast cell/total number of macrophages) x 100, did not coincide with the peak of cytotoxicity, measured as percentage of killed yeast cells in samples – percentage of yeast cells killed in controls lacking macrophages. Nitric oxide production was increased only in macrophages from mice receiving both chitin treatment and C. albicans infection. Because peak nitric oxide production preceded peak cytotoxicity, the authors concluded, as did Suzuki et al. above (280), that macrophage cytotoxicity in these animals was not occurring through an oxidative burst. In their study, Rementería et al. suggested that a lower dose of chitin (10 mg kg^–1) also produced immunosuppressive effects. However, these data, while suggestive, are not as convincing as those from the experiment with the higher dose.

(iii) Glucan. Studies that examine the interaction between fungal glucan and immune cells can be separated into two groups: those that use soluble glucan and those that use insoluble or particulate glucan. Szabó et al. used soluble glucan (prepared by sonication of S. cerevisiae glucan particles) to characterize the ligand binding domain of a previously identified ß-glucan receptor on mononuclear phagocytes (61, 130, 284). These receptors were shown to also bind C. neoformans glucan (56). In a study mentioned previously, Fradin et al. (82) found that laminarin, a polymer of ß-1,3-linked glucose units with ß-1,6-glucan branches, inhibited the binding of heat-killed, but not live, C. albicans cells to macrophage cells. The authors speculated that exposing the cells to heat released cell surface components and exposed the deeper glucan layers. This supports the evidence that macrophages contain proteins that bind glucan in addition to the mannose receptor. It further suggests that the mannose receptor is not sterically blocked when glucan is bound or that binding of mannan is able to displace bound glucan.

Tokunaka et al. (290) examined the immunopharmacologic activity of a soluble glucan preparation from the C. albicans cell wall (Candida soluble beta glucan [CSBG]). They observed that CSBG increased the hematopoietic response to cyclophosphamide-induced leukopenia, increased vascular permeability, and acted as an adjuvant for the purposes of antibody production. They also observed several effects with respect to cytokine production (discussed below). Kataoka et al. (154) looked at macrophage activation by several types of soluble ß-1,3-glucans by using an NF-B-luciferase reporter assay. Activation culminated in the induction of proinflammatory mediators, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-), and macrophage inflammatory protein 2 (MIP-2). The authors found that a linear ß-1,3-glucan, such as curdlan, gave a stronger response than branched glucans, such as laminarin. They also reported that ß-1,3-oligoglucosides were ineffective at macrophage activational, although a ß-1,3-glucoheptaose was able to inhibit curdlan activation of macrophages. Kikuchi et al. (159), also using the CSBG preparation, showed that ß-glucan augmented or induced maturation of DC. Xia et al. (315) demonstrated that, as with human leukocytes, soluble and particulate ß-glucan binds to mouse leukocytes via CR3 (CD11b/CD18, _Mß₂-integrin). Binding of ß-glucan to natural killer (NK) cells, macrophages and the monocytic cell line P388D₁ was inhibited by other ß-glucans but not yeast -mannan. Further, glucan binding was inhibited by anti-CR3 antibody and did not occur on cells from CR3-deficient mice. Earlier work by this group demonstrated that binding of soluble glucan to CR3 enabled NK cells and neutrophils to lyse iC3b-opsonized SRBC (303). The authors proposed that binding of soluble ß-glucan to these cells primed them for cytotoxicity against iC3b-opsonized cells, even when the opsonized cells are normally resistant to CR3-dependent killing.

Particulate glucan (e.g., in the form of zymosan or unopsonized yeast cells) binds to and is phagocytosed by neutrophils and monocytes in the absence of opsonins (60, 238). Ross and coworkers further demonstrated that binding of particulate, although not soluble, ß-glucan induced superoxide bursts from neutrophils (239, 303). Binding of particulate ß-glucan also led to upregulation of CR3 expression on the surface of NK and cytotoxic T cells (198). Czop and Austen (60) showed that binding of particulate ß-glucan to monocytes induced leukotriene production. This action is inhibited by pretreatment of the monocytes with soluble ß-glucan but not -mannan. In an earlier paper, Glovsky et al. (90) reported that particulate glucan affected human serum complement activity, mainly by decreasing the C2 and C3 activity. Lesser effects on C1 and C5 activity were also seen. This coincided with a release of C3a from normal human serum. In vivo experiments showed that particulate glucan activates guinea pig complement via the alternative pathway. Ishibashi et al. (120) compared soluble and particulate forms of ß-glucan having the same primary structure. The particulate glucan had a significantly greater effect than did the solubilized glucan on the activation of various cells, as measured by production of IL-8 by human peripheral blood mononuclear cells (PBMC), TNF- production by a murine macrophage cell line (RAW 264.7), and peroxide production in ICR mouse peritoneal exudate. These results indicate that immune cell activation is a function of both solubility and particle size (termed degree of assembly) (120). Vassallo et al. (302), working with particulate ß-glucan