Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2-7 1-bis(2carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% ؎ 2%, 35% ؎ 14%, 50% ؎ 11%, 51% ؎ 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% ؎ 17%, 22% ؎ 9%, and 40% ؎ 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

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Tumor necrosis factor alpha (TNF-␣) release by peripheral blood mononuclear cells (PBMC) during disseminated infection by Cryptococcus neoformans may initiate and amplify the immune response of the host, leading to elimination of the fungus. The ability to induce TNF-␣ in PBMC by four clinical strains of C. neoformans, a laboratory strain (NIH 37), and the purified cryptococcal components glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MP1 and MP2) were investigated under different opsonic conditions. In the absence of serum, the levels of TNF-␣ induced by all strains and cryptococcal components were not above background levels. Normal human serum (NHS) enhanced TNF-␣ induction by whole cryptococci and the different cryptococcal components, with MP2 being the most potent TNF-␣ inducer. Inactivation of complement (HI NHS) almost abrogated the ability of whole cryptococci and the GXMs to induce TNF-␣. In contrast, when MP1, MP2, and GalXM were incubated with HI NHS, 48, 71, and 44%, respectively, of the original TNF-␣ levels remained. MPs incubated with heat-inactivated immunoglobulin G (IgG)-depleted serum still induced 50% of the levels of TNF-␣ induced by components incubated with HI NHS. Both these sera contained the same very low levels of anti-MP IgG antibodies, indicating the opsonic effect of a heat-stable factor other than antibody. Two anti-CD14 monoclonal antibodies (60BCA and 3C10) inhibited the production of TNF-␣ induced by MP2. The results indicate that (i) induction of TNF-␣ by C. neoformans and GXMs strongly depends on complement, (ii) MP1 and MP2 induction of TNF-␣ is facilitated by a heat-stable serum factor other than Ig, and (iii) CD14 may be involved in the induction of TNF-␣ by MP2.

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