The regulation by Cryptococcus neoformans encapsulation of interleukin 1␤ (IL-1␤) and tumor necrosis factor alpha (TNF-␣) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-␣ secretion induced by LPS or acapsular C. neoformans. In contrast, no regulatory effect on IL-1␤ was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-␣ does not affect IL-1␤ production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococcal polysaccharide could be devised.

The contribution of human alveolar macrophages (AM) from normal subjects in Cryptococcus neoformans infection was investigated. AM were able to efficiently phagocytize the fungus after opsonization, but killing activity did not occur at an effector-to-target ratio of 10:1 in a 6-h incubation since there was an inhibition of phagosome-lysosome fusion. Moreover, the role of AM as antigen-presenting cells was investigated. Cryptococcus-laden AM were co-cultured with autologous T lymphocytes and lymphoproliferation was determined; a massive blastogenic response of alpha/beta TCR-bearing T lymphocytes was observed. The response started after 1 day of co-culture and was triggered and regulated by IL-1 produced by AM in response to C. neoformans. Finally, the antigen-presentation process was associated with HLA class II DR molecules. This finding suggests that AM play a key role in the lung as antigen-presenting cells and, through the secretion of IL-1, regulate proliferation and activation of T lymphocytes, which are important in mediating pulmonary clearance. We speculate that in immunodepressive conditions, the impairment of AM functions could contribute to the spread of C. neoformans infection from the lung.

In this study, we demonstrated that purified capsular polysaccharide of Cryptococcus neoformans is a potent inducer of interleukin-10 (IL-10) secretion by human monocytes. Endogenous IL-10 was involved in regulating tumor necrosis factor alpha and IL-1␤ secretion by human monocytes in response to encapsulated C. neoformans strains. Our results suggest a new immunosuppressive effect exerted by glucuronoxylomannan through the induction of IL-10, a potent downregulator of proinflammatory cytokines. Cryptococcus neoformans is a potentially pathogenic yeast that is ubiquitous in our environment. Cryptococcosis is unusual among healthy individuals, but encapsulated forms of this organism may produce disseminated disease in individuals with weakened immune systems (9). The capsule is an important virulence factor because it has tolerogenic and antiphagocytic properties (2, 3, 7, 8) which are related to one or more of the structural features of the major polysaccharide antigen of the capsule, glucuronoxylomannan (GXM). Recently, we reported that the secretion of cytokines by human monocytes is markedly influenced by encapsulation of C. neoformans as well as by the presence of purified capsular polysaccharide (14). Of particular interest was the ability of GXM to suppress lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-␣). To date, little is known about the mechanism(s) by which C. neoformans influences cytokine secretion by monocytes. Since interleukin-10 (IL-10) has been reported to be a potent downregulator of TNF-␣ and IL-1␤ (4, 15), we hypothesized that inhibition of proinflammatory cytokine secretion could be mediated by endogenously produced IL-10 by human peripheral blood monocytes (PBM) secreted in response to C. neoformans and its capsular material. Therefore, the focus of this research was to examine the ability of capsular material to regulate IL-10 production by monocytes by using an acapsular (7698), thinly encapsulated (6995), or heavily encapsulated (3168) strain of C. neoformans and purified GXM alone or in combination with the acapsular strain. The following strains of C. neoformans were obtained from J. Orendi: a thinly encapsulated strain of C. neoformans var. neoformans serotype A (CBS 6995 ϭ NIH 37; National Institutes of Health, Bethesda, Md.), a heavily encapsulated serotype A strain (NCPF 3168; National Collection of Pathogenic Fungi, London, United Kingdom), and an acapsular mutant of C. neoformans var. neoformans (CBS 7698 ϭ NIH B-4131). The cultures were maintained by serial passage on Sabouraud agar (Bio Merieux, Lyon, France), harvested by suspending a single colony in RPMI 1640 medium (Eurobio Laboratories,

Disseminated infections by the opportunistic yeast Cryptococcus neoformans are characterized by accumulation in tissues of glucuronoxylomannan (GXM), the major component of the capsular polysaccharide. We investigated binding, uptake, and disposal of GXM by peripheral blood neutrophils and monocytes, and the effect of GXM uptake on phagocytic cell function. GXM was efficiently bound and internalized by both types of phagocytic cells, with maximal loading at 50 ? g/ml, a GXM concentration found in serum and cerebrospinal fluid of some cryptococcosis patients. However, substantial differences were noted in the kinetics for uptake by macrophages and neutrophils. Whereas neutrophils rapidly ingested limited amounts of GXM and then expelled or degraded it after 1 h of incubation, macrophages demonstrated continuous intracellular accumulation for up to 1 week of incubation. Accumulation of GXM by neutrophils was accompanied by reduced anticryptococcal activity, suggesting one more mechanism for virulence enhancement by the major capsular component of C. neoformans.

We previously demonstrated that the principal component of capsular material of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces interleukin-10 (IL-10) secretion from human monocytes. Here we report that encapsulation of the yeast with GXM is able to down-regulate interleukin-12 (IL-12) production by monocytes that would normally occur in the absence of encapsulation. This phenomenon appeared to be the result of inhibition of the phagocytic process by encapsulation with GXM as well as of negative signals such as IL-10 secretion produced by interaction of GXM with leukocytes. Decreased secretion of IL-12 correlated with decreased release of gamma interferon (IFN-␥) from T cells, suggesting a role for encapsulation with GXM in hindering a T helper type 1 (Th1) response. This is supported by the ability of encapsulation with GXM to limit increased expression of B7-1 costimulatory molecules that otherwise might limit IL-10 secretion. Endogenous IL-10 played a critical role in modulatory activity associated with encapsulation with GXM. Blocking IL-10 with monoclonal antibody to IL-10 resulted in increased (i) IL-12 secretion, (ii) IFN-␥ release from T cells, and (iii) killing of C. neoformans by monocytes. These results suggest that encapsulation with GXM limits development of a protective Th1-type response, an inhibitory process in which IL-10 plays a critical role. Scavengers of GXM and/or IL-10 could be useful in a protective Th1-type response in patients with cryptococcosis.