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.

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans‐induced enhancement of HIV replication in HIV‐1‐infected PBMC. Similar to whole C. neoformans, cell‐wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV‐infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II‐mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule‐1 (ICAM‐1) and lymphocyte function‐associated antigen‐1 (LFA‐1) also inhibited C. neoformans‐induced cell proliferation. In addition, anti‐MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II‐mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast‐induced enhancement of HIV replication in PBMC.

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The influence of subinhibitory concentrations of clindamycin on opsonization and phagocytosis of Staphylococcus aureus was studied. S.aureus was grown overnight in the presence or absence of one half or one quarter of the minimal inhibitory concentration (MIC) of clindamycin. Radioactively labeled S. aureus was opsonized for various periods of time in different concentrations of normal serum, heated antiserum and serum of patients with agammaglobulinaemia or C3 deficiency. Complement-as well as antibody-dependent phagocytosis of the antibiotic treated S. aureus was significantly enhanced, compared to phagocytosis of the untreated control. Killing experiments showed that clindamycin-treated S. aureus was also better killed by the granulocytes than untreated S. aureus. The mechanism of action is likely to be an increased susceptibility of clindamycin-treated bacteria to antibody- and complement-dependent phagocytosis.

Polymorphonuclear leukocytes (PMN) are able to exert cell-mediated cytotoxic reactions in order to eliminate tumor cells and virus-infected cells. Appropriate stimulation is needed to activate the potential cytotoxic arsenal of the PMN in contrast to the spontaneous cytotoxicity mediated by natural killer cells. Stimulation with phorbol esters induces an oxygen-dependent killing mechanism which results in highly efficient lysis of red blood cell targets. Tumor target cells are more resistant to oxygen-dependent killing mechanisms due to effective antioxidant capacities. Antibody-coated tumor target cells are easily recognized and bound by PMN via a cooperative action of Fc receptors and adhesion molecules. This firm contact and receptor occupation result in efficient killing of the tumor cells which does not require production of oxygen radicals. The mechanisms of PMN-mediated cytotoxicity are discussed and compared with data known from natural killer cells and monocytes.