Marta Feldmesser¹ and Arturo Casadevall^1,2

1Departments of Medicine (Division of Infectious Diseases) and Microbiology and Immunology², Albert Einstein College of Medicine, Golding Building Room 701, 1300 Morris Park Ave., Bronx, NY 10461

Received 1/5/98 Accepted 1/9/98

8. POTENTIAL MECHANISMS OF AB ACTION

The mechanism by which mAb administration prolongs survival in animal models is unknown at the present time. Although mAb binding to GXM produces structural alterations in the cryptococcal capsule (111) (illustrated in figure 3), several groups have shown no effect of Ab on fungal growth or viability (100,107,112). Agglutination, a classically described Ab property, may contribute to protection in some infections by preventing adhesion or by mechanically clumping organisms and preventing their dissemination. As noted above, the role of agglutination in cryptococcal infection is uncertain, as protective and nonprotective mAbs are agglutinins (84). It is likely that the effects of mAb are multifactorial, and involve actions on macrophages as effector cells, macrophages as antigen presenting cells, altered inflammatory cell recruitment, and differences in cytokine expression. The removal of the toxic and immunomodulatory effects of GXM could provide a common pathway for the mechanism of Ab action. Potential mechanisms of mAb action are summarized in figure 4.

Figure 3. Top: Scanning electron micrograph of encapsulated C. neoformans shows fibrillar appearance of the polysaccharide capsule; X 9,000. Bottom: Incubation of yeast in mAb to GXM demonstrates alteration in structural appearance of the polysaccharide capsule; X 8,000. Micrographs were provided by W. Cleare.

Figure 4. Summary of potential mechanisms of action of mAb in C. neoformans infection.

8.1 Opsonization/phagocytosis

The potential mechanism of Ab action in cryptococcal disease that has received the most attention is opsonization for enhanced phagocytosis by macrophages, with the expectation that enhanced phagocytosis results in stimulation of effector cell fungicidal activity. While the requirements for phagocytosis vary based upon the species and tissue from which macrophages are derived and the state of macrophage activation, most commonly, acapsular strains of cryptococcus are readily phagocytosed (113), while encapsulated strains are not efficiently phagocytosed in the absence of opsonins (114-116). In the absence of opsonins, acapsular strains are ingested following binding to mannose and beta-glucan receptors in the yeast cell wall that are blocked by the presence of the capsule (113). While unencapsulated cryptococci are readily ingested by macrophages in the presence of complement, much less ingestion of encapsulated organisms occurs (117). For both encapsulated and acapsular strains, C3b is the major serum opsonin involved in phagocytosis (118), and phagocytosis follows binding to CR1, CR3 and CR4 in human cultured macrophages (55). The cytokines TNF-alpha and GM-CSF enhance phagocytosis by increasing the affinity of CR3 for the yeast (119), and interferon (IFN)-gamma produced by endogenous T cells is required for maximal degrees of phagocytosis and expression of markers of macrophage activation in mice (120).

The ability of polyclonal anticapsular Ab and mAbs to GXM to induce FcR-mediated phagocytosis of encapsulated cryptococci by human and murine macrophages from a variety of tissues and by macrophage-like cell lines is well described, though with some variation in efficacy (55,83,100,107,114,117). However, neither complement receptor- nor Ab-mediated ingestion can be equated with fungal killing, as has been shown in a variety of systems. Absence of fungal killing has been described in guinea pig and human alveolar macrophages where complement was the opsonin (21,114). Intracellular cryptococcal growth is described in vitro in human peripheral blood mononuclear cells (121). mAbs to GXM result in Fc-mediated ingestion of cryptococci by microglia, and phagolysosomal fusion is seen. While transient fungistasis follows, some internalized yeast proliferate intracellularly, initiating a chain of events that culminate in cell death (122).

Nonetheless, the ability of Ab to function as an opsonin in the absence of complement is potentially of clinical importance, as complement depletion has been reported in patients and guinea pigs with disseminated cryptococcal disease, but not in patients who are not fungemic (123). Evidence that mAb is beneficial in the role of opsonin in this setting is provided by studies of an i.v. infection model in complement deficient mice. In this model, C5 deficiency results in increased susceptibility to C. neoformans (124). Complement deficient (C5^-) mice die rapidly from pneumonia, while complement sufficient strains (C5⁺) die from subacute meningoencephalitis. Soon after infection, PMNs are recruited to the lung in C5⁺ but not in C5^- mouse strains (125). The IgG1 mAb E1 prolongs survival of C5^- mice when given prior to infection without restoring the protective PMN infiltration seen in C5⁺ strains. In the absence of altered inflammatory cell recruitment, survival prolongation and reduction in lung CFU is attributed to enhanced opsonization of cryptococci, with resulting enhancement of macrophage effector function (100).

While increased macrophage killing of cryptococci is likely to result from enhanced phagocytosis, several lines of evidence suggest that this is not the sole mechanism of Ab action. As noted above, the ability of different isotypes to mediate phagocytosis does not completely correlate with enhancement of killing (107). In i.t. models, C5 deficiency is not associated with altered numbers of PMNs recruited to the lung or with decreased fungal clearance (126). Further, in an i.t. murine infection model, cryptococci are phagocytosed rapidly by alveolar macrophages without administration of exogenous Ab (93). While mAb administration prolongs survival in both C5- and C5+ mouse strains, small reductions in lung CFU are seen at most (61,93). However, it is possible that mAb serves different functions in disseminated disease than in initial infection. Further, it is unknown whether the outcomes of FcR- and CR-mediated phagocytosis are the same in cryptococcal infection and this remains an area for further study. Thus, while it is likely that opsonization of cryptococci with increased phagocytosis is one effect of Ab, is unlikely that this is the sole mechanism by which mAb exerts its protective effect.

8.2 Macrophage effector functions (killing)

In many microbial infections, phagocytosis allows more effective killing of microorganisms by the production of antimicrobial peptides, including lysozyme, defensins, cryptidins and histone proteins (127) as well as the production of toxic oxygen and nitrogen radicals (128). Several of these mechanisms may be operative in extracellular killing. Knowledge of the role of these mechanisms in the host response to cryptococcal infection is incomplete, and their relative roles may vary between species and populations of cells. However, it is known that killing of acapsular and encapsulated cryptococci by murine resident peritoneal macrophages is enhanced by stimulation with IFN-gamma (129) and that killing by bone marrow macrophages may occur extracellularly (130).

The effect of Ab to GXM on the ability of macrophages to kill C. neoformans has varied in in vitro systems. As noted above, in in vitro studies, mAb to GXM increases phagocytosis and killing of fungi by the murine macrophage-like cell line J774 (107) and by murine peritoneal and alveolar macrophages (93,100). Diamond reported that in the presence of polyclonal anticapsular Ab, human peripheral blood mononuclear cells can kill C. neoformans extracellularly, by a non-phagocytic mechanism (131). Thus, Ab may mediate anti-cryptococcal activity by effector mechanisms that do not require phagocytosis. However, Levitz found no effect of Ab opsonization on human peripheral monocyte/macrophages, with no killing of an encapsulated strain or an isogenic acapsular mutant (132).

Anticapsular IgG activates murine peritoneal macrophages to synthesize nitrite and to kill cryptococci. In the absence of Ab, stimulation with IFN-gamma is required for fungal killing and both IFN-gamma and serum opsonization are necessary for nitrite production (129). Both anticapsular IgG1 mAb and IFN-gamma are required for stimulation of the respiratory burst of encapsulated yeast, while acapsular organisms stimulate the respiratory burst without a requirement for opsonins. Nitrite synthesis, but not phagocytosis, respiratory burst stimulation or lysosomal enzyme release, correlate with fungal killing in this system (129). Opsonization of encapsulated C. neoformans with mAb increases peroxynitrite production and fungicidal activity of IFN-gamma-activated resident peritoneal macrophages and stimulates O₂^- production from unstimulated cells (133).

Naslund found that while acapsular strains induce NOS production by J774 cells as measured by nitrite production, conditions which promote attachment and/or phagocytosis do not lead to NOS induction, including opsonization with specific polyclonal rabbit serum (134). Fc receptor activation by GXM-mAb complexes enhances production of nitrogen related oxidants by IFN-gamma-stimulated J774 cells (135). However, IgG1-mediated killing of C. neoformans by J774 cells occurs despite inhibition of NOS and ROI scavengers (136). The specific mediators of this fungal killing are unknown. Similarly, the transient fungistasis following incubation of C. neoformans with fetal human microglial cells in the presence of an IgG1 mAb is not inhibited by NOS inhibitors and ROI scavengers (137).

In vivo, administration of mAb to the polysaccharide capsule reduces fungal burden in a variety of murine models of infection. It reduces lung CFU following i.v. infection with a variety of C. neoformans strains and reduces brain CFU following i.c. but not i.v. infection (90-92). In i.t. infection, despite marked prolongation of survival, CFU reductions in the lung are small and there is no reduction in extrapulmonary sites (93). Increased fungal killing by macrophages may contribute to the beneficial effect of Ab, but the lack of association of survival prolongation with reductions in fungal burden suggest that this is not the major mechanism of Ab action in i.t. infection.

8.3 Granulocytes as effector cells

Comparatively little attention has focused on the role of neutrophils and eosinophils in Ab-mediated protection against C. neoformans. Both cell populations are present transiently after infection in animal models, though their importance in the immune response in human infection is unknown. In i.v. infection, neutrophils are present in the pulmonary vasculature of murine lung, where ingestion is dependent on the presence of C5 and terminal complement components. Though prominent among intravascular inflammatory cells at 30 min after infection, they are rare by 24 h. While after i.t. infection no role for these mechanisms has been seen (126), neutrophils are present in pulmonary infiltrates during the first 7 d, but not subsequently (138). Neutrophils are the first inflammatory cells to migrate toward cryptococci in the peritonea of rabbits infected i.p., and are then replaced by monocytes (65). In vitro phagocytosis of cryptococci by human peripheral blood PMNs is dependent mainly on the presence of components of the alternate complement pathway (112). Neutrophil activation enhances phagocytosis of cryptococci opsonized with normal human serum, and this enhancement may be related to increased CR3 expression (139). Once ingestion occurs, killing of yeast is relatively efficient, and myeloperoxidase and H₂O₂ are necessary for killing (112). Incubation of human peripheral blood neutrophils with C. neoformans or with GXM results in production of the proinflammatory cytokines TNF-alpha, IL-8, IL-1beta and IL-6 (140).

Results of in vitro studies using polyclonal sera vary. While one study concluded that Ab to C. neoformans does not significantly increase ingestion or killing by neutrophils in vitro (112), another showed that anticapsular Ab is required for maximal phagocytosis by these cells (141). A third demonstrated that Ab is required for neutrophil-mediated killing, and that in the presence of antiserum, neutrophil killing of C. neoformans is more efficient than that by monocytes (142). A recent study showed that a human anti-GXM IgM increases neutrophil phagocytosis and growth inhibition of cryptococci compared to complement alone, suggesting that Ab-mediated deposition of complement components on the capsule can enhance neutrophil complement receptor-mediated antifungal activity (143).

Eosinophils are prominant in pulmonary inflammatory cells 14 d after i.t. infection in some mouse strains, but then gradually decrease in number (144). Eosinophils are uncommonly reported in inflammatory responses to human infection. Phagocytosis of cryptococci by rat peritoneal eosinophils is induced by IgG1 and IgE to GXM in vitro, and degranulation is seen. Minimal phagocytosis occurs in the absence of Ab (144). In vivo, administration of IgG1 results in reduction in the number of granules per eosinophil in murine lung, suggesting Fc-mediated degranulation, and in occasional eosinophil phagocytosis of cryptococci (61). The effect of eosinophil phagocytosis or degranulation on cryptococcal killing by yeast is unknown, though the lack of reduction in lung CFU seen in mAb-treated mice in this model suggests that this mechanism is unlikely to be a major contributor to Ab-mediated effects in mouse lungs.

Both neutrophils and eosinophils may have important roles in the host immune response to this pathogen. Their transient appearance after initial infection may account for their lack of prominence in human pathology specimens of C. neoformans infection, which are usually from patients with longstanding infection. Intravascular neutrophil phagocytosis of cryptococci suggests that these cells may be important for prevention of intravascular dissemination. Both cells can function as effector cells through multiple mechanisms, including target killing and cytokine secretion. Ab may potentially act by enhancing these roles. Further study is required for determination of the roles of these cells both in the immune response to infection as well as in Ab-mediated protection.

8.4 Antigen presentation

A mechanism of Ab action in cryptococcal disease currently under investigation is enhancement of antigen presentation. To date, all studies of this effect have been done in vitro. In cryptococcal infection, lymph node cells primed with acapsular organisms proliferate in response to acapsular organisms but not to encapsulated yeast. Thirty fold more encapsulated yeast are required to induce such a response. Therefore, inhibition of antigen presentation is another deleterious effect of cryptococcal capsular polysaccharide. However, once ingestion of organisms occurs, the capsule has no effect on processing or presentation of cryptococcal antigens and subsequent T cell activation (145). Vecchiarelli has shown that normal human serum allows phagocytosis of thinly encapsulated cryptococci by human alveolar macrophages, but that phagolysosomal fusion is inhibited. Co-culture of alveolar macrophages with autologous T cells produces a massive blastogenic response of alpha/beta TCR-bearing T cells that is regulated by IL-1 produced by the macrophages in response to cryptococci (146). Thus, macrophages may be important Ag presenters in cryptococcal disease.

Addition of the IgG1 mAb 2H1 to cocultures of cryptococcal-laden monocytes plus autologous T cells increases lymphoproliferation, and the magnitude of effect is dependent on mAb concentration (147). This alteration is associated with a reduction of IL-10 found in the culture supernatant, and removal of IL-10-induced downregulation of MHC class II expression may be responsible for the increased lymphoproliferation (147). mAb 2H1 increases B7-1 expression on peripheral blood mononuclear cells in the presence of encapsulated strains of C. neoformans, but not in the presence of acapsular strains or in the absence of C. neoformans (35). Thus, the capsular polysaccharide may interfere with antigen presentation by downregulation of MHC class II expression in an IL-10 dependent process, and may prevent the upregulation of expression of co-stimulatory molecules. In vitro studies suggest that Ab may reverse these deleterious effects and allow more effective inflammation. In vivo studies will further define this possible mechanism.

8.5 Antibody-dependent cell-mediated cytotoxicity (ADCC)

NK cells are the principal mediators of this nonspecific arm of the immune system, in which NK cells develop cytolytic capacities without need for prior contact with Ag through binding of aggregated IgG to FcgammaRIII. In the absence of exogenous Ab, NK cells are responsible for growth inhibition of C. neoformans in nonadherent spleen cells in vitro (148) but treatment with anti-asialoGM-1 Ab to decrease NK activity does not affect survival in a murine i.v. model of infection (6). The importance of this mechanism in native infection is therefore unknown.

However, human NK cells function as effectors in ADCC only in the presence of rabbit anticryptococcal antiserum (149). In vitro, polyclonal rabbit anti-cryptococcal IgG accelerates the anticryptococcal effects of NK cells, which mediate their activity by extracellular killing through binding to the cryptococcal cell wall (150). Thus, a potential mechanism of Ab action is the enhancement of NK cell effector activity. In vivo, beige mice, which have defective NK cells that are unable to lyse NK-sensitive targets, have increased susceptibility to C. neoformans infection (151). In this mouse strain, the effects of mAb are similar to those seen in immunocompetent mice after i.v. infection, raising question as to the role of NK cells in Ab-mediated protection (106). Demonstration of the role of Ab in this regard requires further study.

8.6 Removal of toxin (GXM)

As noted in section 4.1 above, GXM has many immunomodulatory effects. Hence, the reversal of GXM-induced immunosuppression is likely to contribute to the action of mAb in cryptococcal infection. Removal of GXM by Ag-Ab complex formation potentially reverses these deleterious actions on the immune system and may also reverse the direct toxicity of polysaccharide in tissue. In human autopsy specimens from patients who died from cryptococcal meningoencephalitis, CNPS is detectable by immunohistochemistry in the brain parenchyma and meninges. Higher percentages of tissue cross sectional area are involved in specimens from patients with AIDS than from those without AIDS (8). Capsular polysaccharide spreads through CNS tissue after intracranial inoculation in rats, resulting in cell swelling (152,153). In murine i.v. infection models, CNPS is shed into tissue, and administration of mAb results in removal of immunohistochemical staining for GXM in areas away from fungi in the brain. In the lung, polysaccharide lines the alveolar epithelium and bronchial lumen, while in mAb-treated mice, staining is limited to granulomas (90). mAb administration results in reduction in serum GXM in mice infected by i.p., i.v., and i.t. routes (84,90,93) and mAb effectively removes GXM from rats inoculated i.v. (154). mAb-treated mice have lower brain weights despite no reduction in CFUs. This finding may reflect reduced brain edema and suggests a "mechanical" protective mechanism (90,92). Protective efficacy of mAbs administered after established infection does not correlate with reduction in serum GXM (94). Results of studies with polyclonal IgG show that cryptococcal Ag-Ab complexes may exert detrimental effects by preventing macrophages from ingesting immunologically coated cryptococci via their FcRs (155). Nonetheless, the reversal of GXM-induced immunosuppression and the removal of direct toxic effects are likely to be important components of the action of mAb in cryptococcal disease.

8.7 Altered pathology

Containment of cryptococci is associated with the development of granuloma formation while in disseminated disease, absence of inflammation is characteristic. Though the mechanism of granuloma formation in cryptococcal infection is not well understood, one potential mechanism of Ab action is the enablement of a more "effective" pattern of inflammation. Early ultrastructural studies describe granuloma initiation in the peritonea of rabbits and in vitro with rabbit and guinea pig peritoneal cells. While small yeast are phagocytosed by PMNs or monocytes, larger fully encapsulated organisms are first surrounded by rings of PMNs that are later substituted by monocytic rings. These rings then fuse into giant cells, with release of hydrolytic enzymes (156,157). The formation of multinucleated giant cells is dependent on the presence of CD4⁺ cells (16).

Early studies by Aronson, et al., and Schneerson-Porat et al, show differences in the inflammatory response of mice infected i.p. when cryptococci are incubated in immune rabbit serum. Mouse monocytes require addition of immune serum for phagocytosis of cryptococci and ring formation. When yeast are incubated in Ab, mononuclear cells adhere to cryptococci and cryptococci agglutinate. Ab opsonization results in the formation of three dimensional structures in which yeast are surrounded by mononuclear cells which then fuse (158,159). In rabbits and guinea pigs, two species that are less susceptible to infection, immune rabbit serum is not required for ring formation, though organisms are only completely enclosed in the presence of Ab opsonins. In rabbits, serum is not required for ring formation, but plasma cells are seen in the inflammatory infiltrate (65). These studies suggest that Ab to the capsular polysaccharide alters the inflammatory response that mononuclear cells can form.

In mice infected i.t., prolongation of survival by administration of an IgG1 mAb to GXM is associated with alteration in lung pathology such that yeast are contained within foci of inflammation, while in control mice cryptococci spread through the alveolar spaces (93). These studies suggest that Ab can result in more effective containment of cryptococci within granulomas. Since granuloma formation, which is analogous to chronic DTH, results from products of macrophage activation, such as cytokines, a possible mechanism for the pathology differences seen in the presence of Ab is alteration of cytokine production by host inflammatory cells.

8.8 Inconsistencies

While some or all of the mechanisms described above may contribute to the mechanism of mAb action against C. neoformans, inconsistencies in the effects of mAbs in in vitro and in vivo systems demonstrate that our knowledge of mAb action is far from complete. For example, IgG3 are non-protective, yet are opsonic, promote fungal killing in vitro by macrophages and clear serum polysaccharide (94,107). IgG1 are protective in beige mice that have no functional NK cells (106). These results suggest that care must be taken in the extrapolation of in vitro results. Further, the discrepancies indicate that the in vivo effects may be the cumulative result of multiple mechanisms, all of which are not presently known.

Furthermore, it is uncertain why Ab administration fails to clear the infection. Possibilities include a problem with the models used, an inherent limitation to Ab-mediated protective mechanisms, active interference with Ab-mediated protection by the fungus and/or a combination of the above.

8.9 New directions: Altered cytokine expression/cellular recruitment

Recent study has shown that resistance to cryptococcal infection and development of protective inflammatory responses are associated with the production of the T_H1-associated cytokines (14), as is typical for diseases in which granulomatous inflammation is responsible for containment. In this regard, IFN-gamma and IL-12 are required for inflammatory cell recruitment in the lung following murine i.t. cryptococcal infection. IFN-gamma is required for containment of yeast within inflammatory foci (160), while IL-12 suppresses dissemination (161). Administration of anti-TNF-alpha mAb reduces inflammatory cell recruitment, increases CFU and prevents the development of DTH (162). The inflammatory changes produced by these cytokines parallel the histopathological changes seen following mAb administration in susceptible mice, as described above. Limited studies of the ability of mAb administration to alter cytokine production in vivo have been performed to date. However, preliminary data suggests that an effect of mAb in murine pulmonary infection may be to alter cytokine production, possibly through reduction of T_H2-associated cytokines (163).

In in vitro studies, during coculture of human alveolar macrophages with T cells and cryptococci, following a lymphoblastogenic response, high levels of IFN-gamma and IL-2 are found in culture supernatants, while IL-4 is undetectable. Levels of IFN-gamma and IL-2 are higher in response to incubation with acapsular than with encapsulated strains (164). IL-10 production causes dose-dependent inhibition of TNF-alpha and IL-1beta release by peripheral blood mononuclear cells in response to C. neoformans and reduces mRNA expression for TNF-alpha (165). PBMs produce higher levels of IL-10 in response to encapsulated cryptococcal strains than acapsular strains (166). A mechanism of Ab action in cryptococcal infection may be reduction of IL-10 production, removing the inhibitory effect on proinflammatory cytokine production (147).

Alteration in cellular recruitment at the chemokine level as opposed to at the effector cytokine level is another possible pathway for the histopathological changes that accompany mAb administration. Additional pathways for cellular recruitment in cryptococcal infection are newly being studied. Recently, Huffnagle et al reported that MIP-1alpha, which is chemotactic for a variety of inflammatory cells, selectively recruits macrophage/monocytes and PMNs to the lungs of mice infected with C. neoformans and that depletion of this chemokine is associated with reduced fungal clearance. Induction of MIP-1alpha secretion is dependent on MCP-1 production (167). There is no data on the ability of mAb to GXM to produce changes in these chemokines. However, this is a potential area for further exploration.