Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by ironindependent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis. (

We studied the receptors on human cultured macrophages (MO-MO)

Tumor necrosis factor alpha (TNF-ox) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-a when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-ot following stimulation with C. neofonnans and lipopolysaccharide. Detectable TNF-ot release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-ot. TNF-a gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-o from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-a following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-ox release from human leukocytes. However, several variables profoundly affected the amount of TNF-a released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins.

Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-␣) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-␣ release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1␤ from PBMC stimulated by all three stimuli. TNF-␣ mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1␤ mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-B in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus-and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-␣ and both transcriptionally and posttranscriptionally for IL-1␤.

Cryptococcus neoformans infections are a major cause of morbidity and mortality for HIV-infected persons. Containment of the initial respiratory inoculation to the lung appears defective in patients with AIDS despite the low burden of HIV in bronchoalveolar macrophages. We have studied the fungistatic activity of human bronchoalveolar macrophages (BAM) cultured with an encapsulated strain of C. neoformans in the presence of pooled human serum. We observed 51.6% fungistasis after 24 h of culture. Fungistasis was diminished if the pooled human serum was heat-inactivated but was not affected by anticryptococcal capsular IgG. HIV envelope protein (gp120) has been shown to interfere with lymphocyte activation in vitro. We studied the effects of gp120 on BAM function and found that fungistatic activity was inhibited 25% (p < 0.001). Although binding of yeasts was not affected, gp120 inhibited the internalization of bound yeasts by 46% (p = 0.025). These experiments indicate that gp120 decreases the internalization and fungistasis of C. neoformans by human BAM, and they suggest a mechanism to explain how a small number of HIV-1-infected cells in the lung could impair the containment of C. neoformans.

The capacity of human peripheral blood monocytes, neutrophils, and monocyte-derived macrophages to bind the yeast Cryptococcus neoformans was increased when the phagocytes were cultured on surfaces containing recombinant human mannose-binding protein. In contrast, soluble mannose-binding protein had no effect on cryptococcal binding by phagocytes.

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C. neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per l of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.