Recently we demonstrated that the nonadherent (to plastic) fraction of human PBMC could be activated by
Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth 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.
The chemokine monocyte chemoattractant protein 1 (MCP-1) is produced predominantly by mononuclear phagocytes and stimulates recruitment into infected tissues of blood monocytes and T cells. These cell types are thought to be critical to host defenses against infections due to Cryptococcus neoformans, a major cause of disease in persons with AIDS and other disorders of cell-mediated immunity. Accordingly, in the present study, we examined the conditions under which human monocytes and bronchoalveolar macrophages (BAM) are stimulated by C. neoformans to produce MCP-1. C. neoformans was a potent inducer of MCP-1 release from monocytes, with levels of chemokine secreted similar to that seen following stimulation with lipopolysaccharide (LPS). BAM, in contrast, were stimulated by LPS, but not by C. neoformans, to secrete MCP-1. A peak in MCP-1 mRNA was seen 8 h following cryptococcal stimulation of monocytes. Nine strains of C. neoformans stimulated monocytes to release MCP-1, and there was only modest variation between strains. However, when an individual strain was used, the capacity of C. neoformans to stimulate monocyte MCP-1 release did vary, depending upon the conditions used to grow the fungal stimuli. Finally, C. neoformans stimulated comparable quantities of MCP-1 release in monocytes from donors with and without human immunodeficiency virus infection. These data establish C. neoformans as a potent stimulator of MCP-1 in monocytes, but not in BAM. The failure of C. neoformans to stimulate MCP-1 in BAM, if occurring in vivo, could result in a diminished cell-mediated inflammatory response following inhalation of airborne fungi.
Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.
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