Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans.

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.     

Production of tumor necrosis factors alpha and beta by human mononuclear leukocytes stimulated with mitogens, bacteria, and malarial parasites.

Tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta) are multifaceted polypeptide cytokines which may mediate some of the significant changes in cellular homeostasis which accompany the invasion of the mammalian host by viruses, bacteria, and parasites. Although it is well established that bacterial lipopolysaccharide is a potent inducer of TNF-alpha, there is still very little known of the types of agents which can trigger the production of TNFs in mononuclear leukocytes. Using an enzyme-linked immunosorbent assay for measuring TNF-alpha and TNF-beta, we examined the capacity of various T-lymphocyte and beta-lymphocyte mitogens as well as microbial components to stimulate production of these cytokines in culture. The mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen induced production of both TNF-alpha and TNF-beta, while whole-killed Staphylococcus aureus and Bordetella pertussis, like lipopolysaccharide, were potent inducers of TNF-alpha but failed to stimulate TNF-beta production. TNF-alpha production was detectable within 1 h after stimulation, while TNF-beta production was not detected until after 8 h of culture. The bacterial products tetanus toxoid, purified protein derivative, pertussis filamentous hemagglutinin, and pertussis toxin were all able to induce TNF-alpha and TNF-beta production. Disrupted (frozen-thawed) Plasmodium falciparum-infected erythrocytes were also potent inducers of TNF-alpha and TNF-beta. The results demonstrated that a wide variety of microbial components are inducers of TNF-alpha. Some may not only be more effective than lipopolysaccharide but can also induce TNF-beta production. Furthermore, evidence is presented showing that TNF-beta but not TNF-alpha production correlates with lymphoproliferation.     

Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.     

Porins of Pseudomonas aeruginosa induce release of tumor necrosis factor alpha and interleukin-6 by human leukocytes.

The aim of this study was to examine the ability of Pseudomonas aeruginosa components to induce release of cytokines from human leukocytes. Human whole-blood cultures were incubated with several concentrations of purified P. aeruginosa products, including porins, exomucopolysaccharide, lipopolysaccharide, and toxin A. Supernatants were assayed for tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) activities. All of the P. aeruginosa components except toxin A were able to stimulate the release of both cytokines. On a weight basis, porins were as effective as lipopolysaccharide and significantly more effective than exomucopolysaccharide in inducing IL-6 release (P < 0.05). Moreover, porins were more potent than either exomucopolysaccharide or lipopolysaccharide in inducing TNF-alpha release (P < 0.05). Further experiments using isolated leukocytes suggested that monocytes were the cell population predominantly responsible for the production of both cytokines. These data indicate that P. aeruginosa porins are able to induce significant cytokine production. These components may be responsible for the chronically overactive inflammatory response associated with persistent lung infection in cystic fibrosis patients.     

Cryptococcus neoformans and cryptococcal glucuronoxylomannan, galactoxylomannan, and mannoprotein induce different levels of tumor necrosis factor alpha in human peripheral blood mononuclear cells.

Tumor necrosis factor alpha (TNF-alpha) 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-alpha 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-alpha induced by all strains and cryptococcal components were not above background levels. Normal human serum (NHS) enhanced TNF-alpha induction by whole cryptococci and the different cryptococcal components, with MP2 being the most potent TNF-alpha inducer. Inactivation of complement (HI NHS) almost abrogated the ability of whole cryptococci and the GXMs to induce TNF-alpha. In contrast, when MP1, MP2, and GalXM were incubated with HI NHS, 48, 71, and 44%, respectively, of the original TNF-alpha levels remained. MPs incubated with heat-inactivated immunoglobulin G (IgG)-depleted serum still induced 50% of the levels of TNF-alpha 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-alpha induced by MP2. The results indicate that (i) induction of TNF-alpha by C. neoformans and GXMs strongly depends on complement, (ii) MP1 and MP2 induction of TNF-alpha is facilitated by a heat-stable serum factor other than Ig, and (iii) CD14 may be involved in the induction of TNF-alpha by MP2.     

Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus.

Cryptococcus neoformans and Aspergillus fumigatus are airborne fungi and the alveolar macrophages (AM) constitute a first line of host defence against both pathogens. We investigated the ability of rat AM to produce nitric oxide (NO) when challenged in vitro with C. neoformans, A. fumigatus conidia or inert silica particles alone and together with interferon gamma (IFN-Gamma). The role of NO in the killing of C. neoformans as well as the relationship between phagocytosis of the yeast or A. fumigatus conidia and NO production by AM were studied. Both fungi, but not the inert particles induced a small but significant increase in NO production by AM. A synergistically enhanced NO production by AM was observed when each fungus, but not silica particles, were incubated together with IFN-Gamma. AM treated with IFN-Gamma and challenged with C. neoformans showed higher killing activity than untreated AM, a finding that correlated with increased NO production by AM. Both effects were reduced by an inhibitor of NO synthesis. Increased NO production by IFN-Gamma activated AM was found together with an increased accumulated attachment of A. fumigatus conidia and serum opsonized, but not unopsonized C. neoformans. The IFN-Gamma dependent increase in accumulated attachment of the fungi might be responsible for the synergistic effect of the fungi and IFN-Gamma on the NO production. Our data suggest that activated rat AM might efficiently use the antimicrobial nitric oxide system in the defence against these pathogens in the normal host.     

Bacterial-lipopolysaccharide-induced release of lactoferrin from human polymorphonuclear leukocytes: role of monocyte-derived tumor necrosis factor alpha.

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.     

Production of tumor necrosis factor alpha by resident and activated murine macrophages.

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.     

Simultaneous induction of interferon gamma and tumor necrosis factor alpha by different seleno-organic compounds in human peripheral blood leukocytes.

Ebselen is known as anti-inflammatory and anti-oxidant selenium containing drug. We have synthetized 13 seleno-organic compounds, analogs of ebselen. Seven of them were found to be inducers of interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) in human peripheral blood leukocytes (PBL) cultures. The most active cytokine inducers were: 2-phenyl-1,2-benzisoselenazol-3(2H)-one (1, ebselen), bis [2-(N-phenylcarbamoyl)]phenyl diselenide (7) and bis (2-[N-(2-pyridyl)carbamoyl])phenyl diselenide (8). The amounts of IFN and TNF produced by PBL cultures in response to the seleno-organic compounds were found to be similar to that induced by phytohemagglutinin (PHA). The activities of the seleno-organic compounds were dose-dependent and related to the chemical structure of the drugs suggesting involvement of the specific cytokine-inducer receptor. The simultaneous inductions of IFN-gamma and TNF-alpha were highly correlated, but independent on each other.     

Production of interleukin-6 by human and murine mononuclear leukocytes stimulated with Plasmodium antigens is enhanced by pentoxifylline, and tumor necrosis factor secretion is reduced.

When pentoxifylline was present during stimulation of human mononuclear leukocytes with Plasmodium falciparum exogenous antigens, an increase in interleukin-6 production was observed simultaneously with a reduction of tumor necrosis factor secretion. Similar results were obtained in murine macrophages stimulated with P. vinckei antigens. This indicates the independence of interleukin-6 and tumor necrosis factor secretion in response to malaria antigens.     

Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components.

While there is considerable evidence that both interleukin-1 (IL-1) and tumor necrosis factor (TNF) are central mediators of inflammation caused by gram-negative bacteria and endotoxin, the roles of these two mediators in gram-positive infection are unknown. Pneumococcal infections are characterized by an intense inflammatory reaction in infected tissues. Current evidence suggests that the component of the pneumococcus which causes this inflammation in many body sites is the cell wall. We determined the ability of native pneumococcal cell wall, lipoteichoic acid, and cell wall subcomponents to stimulate secretion of IL-1 and TNF from human monocytes. Each pneumococcal cell surface component was found to have a different specific activity for induction of IL-1. Teichoication was an important determinant of this activity: teichoicated species were at least 10,000-fold more potent than endotoxin and 100-fold more potent than teichoic acid-free peptidoglycan. IL-1-inducing activity was greatly reduced by chemical alteration of the teichoic acid. In contrast to endotoxin, cell wall did not induce production of TNF. This dissociation of the production of IL-1 and TNF during the response of the human monocyte to pneumococcal surface components suggests that, in at least some circumstances, the mechanisms for generation of an inflammatory response to infection may be fundamentally different between gram-positive and gram-negative disease.     

Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis.

We studied the production of tumor necrosis factor alpha (TNF-alpha) by peripheral blood monocytes taken from patients with pulmonary tuberculosis and from healthy controls. It was found that the monocytes from patients with newly diagnosed tuberculosis released significantly greater amounts of TNF-alpha in vitro in response to lipopolysaccharide than did those from healthy controls (P less than 0.05). However, the monocytes from patients with chronic refractory tuberculosis released significantly lower amounts of TNF-alpha than did those from patients with newly diagnosed tuberculosis (P less than 0.005). Even when the cells were primed for 24 h with 500 U of recombinant interferon gamma per ml, the same pattern of results was observed. The depressed TNF-alpha production by the monocytes from patients with chronic refractory tuberculosis was also shown in response to Mycobacterium bovis BCG. This depressed TNF-alpha production did not recover, even when cultured for 1 to 7 days in the sera of healthy individuals. The sera from patients with chronic refractory tuberculosis did not have any suppressive effect on the lipopolysaccharide-induced TNF-alpha production. Thus, it was demonstrated that the levels of TNF-alpha produced by monocytes were related to the disease states of pulmonary tuberculosis and that the depressed TNF-alpha production by monocytes in patients with chronic refractory tuberculosis might not be acquired.     

Modulation of C5a-mediated effector functions of human polymorphonuclear leukocytes by tumor necrosis factor alpha and granulocyte macrophage colony-stimulating factor

At the site of acute inflammation, leukocytes are confronted with multiple mediators which are expected to modulate each other with respect to cell responses to the individual ligand. In the present study, we compared the effects of the classical chemoattractants FMLP, PAF and LTB4, of the chemokine IL-8 and of TNFalpha, GM-CSF, IFN-gamma and IL-1beta on C5a-induced chemotaxis, degranulation, oxidative burst and expression of adhesion molecules of human neutrophils in vitro. Upon preincubation, TNFalpha as well as GM-CSF dose-dependently inhibited C5a-mediated chemotaxis, but augmented the release of elastase as well as respiratory burst activity. The effects of the two cytokines were accompanied by a downregulation of C5a receptors as determined by Scatchard analysis using (125)I-labeled C5a. Compared on a molar basis, TNFalpha was more effective than GM-CSF. C5a-induced expression of beta(2)-integrins was only moderately influenced by TNFalpha and GM-CSF. C5a itself diminished chemotaxis as well as degranulation and oxidative burst in response to a second dose of the same ligand (homologous desensitization), whereas heterologous desensitization by FMLP and IL-8 was restricted to C5a-induced degranulation or not observed (PAF, LTB4]. The cytokine effects are likely to be a consequence of altered C5a receptor expression as well as of postreceptor events. In concert with C5a, certain cytokines may shift neutrophil effector functions from migration to exocytosis, an essential step within the sequence of events in a coordinated inflammatory response. Copyright Copyright 1999 S. Karger AG, Basel     

Tumor necrosis factor-inducing activities of Cryptococcus neoformans components.

Cryptococcus neoformans-induced tumor necrosis factor alpha (TNF-alpha) production may lead to increased human immunodeficiency virus replication in patients with AIDS. In order to identify cryptococcal components that are predominantly responsible for stimulating TNF production, various concentrations of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), mannoproteins (MP), and alpha(1-3) [corrected] glucan were added to whole-blood cultures. All of the cryptococcal components tested, as well as whole heat-killed cryptococci, were capable of inducing TNF-alpha release in a dose-dependent manner. MP were significantly more potent than any of the other cryptococcal components tested or heat-killed cryptococci in stimulating TNF-alpha production (P < 0.05). GXM, in contrast, was significantly less potent in this activity than either GalXM or MP (P < 0.05). As little as 0.5 microg of MP per ml was sufficient to produce moderate but significant elevations of TNF-alpha release. Maximal MP-induced TNF-alpha levels were similar to those induced by Salmonella enteritidis lipopolysaccharide, our positive control. Further experiments using isolated leukocytes suggested that monocytes were the cell population mainly responsible for TNF-alpha production, although the participation of other cell types could not be excluded. The presence of complement-sufficient plasma was a necessary requirement for TNF-alpha induction by GXM, GalXM, and low doses of MP. High MP concentrations (100 microg/ml) were also capable of stimulating TNF-alpha production in the absence of plasma. These data indicate that soluble products released by C. neoformans are capable of inducing TNF-alpha secretion in human leukocytes. This may be clinically relevant, since high concentrations of such products are frequently found in the body fluids of AIDS patients infected with C. neoformans.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes.

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.     

Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na125I by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interleukin-1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte-macrophage-colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml). The mean concentration of TNF in 24-h culture supernatants of human MNC exposed to different concentrations of lipopolysaccharide (LPS) was found to increase in dose-dependent fashion and then level off between 50 and 100 ng/ml. The concentrations of IL-1 beta and alpha detected by specific RIAs in these supernatants were between 0.2 and 19 ng/ml and 0.04 and 1 ng/ml, respectively. The amount of TNF produced by human MNC in vitro was determined in a cohort of 50 normal volunteers. Without exogenous stimuli, TNF concentrations were almost always below the detection limit; with 0.5 ng/ml LPS, the median concentration of TNF was 2 ng/ml, and with PHA the median was 3.8 ng/ml. In cultures performed in the presence of indomethacin significantly (p less than 0.005) more TNF was produced. Using this RIA, we could detect TNF in the circulation of mice injected with Hu rTNF. When plasma samples of patients with febrile illnesses were added directly to the RIA, TNF was not detectable, with the exception of patients with malaria. These studies demonstrate the range and sensitivity of LPS-induced and mitogen-induced production of immunoreactive TNF by human MNC in vitro without interference of similar cytokines in bioassays.     

Cytokine enhancement of complement-dependent phagocytosis by macrophages: synergy of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor for phagocytosis of Cryptococcus neoformans.

We have examined the regulation of complement dependent phagocytosis by macrophage-activating cytokines. Tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not interferon-gamma, interleukin-4 or macrophage-CSF, stimulated ingestion of the encapsulated fungal pathogen Cryptococcus neoformans by resident peritoneal macrophages in vitro. This was dependent upon opsonization of the yeasts with complement, 72 h of incubation with the cytokines for maximum effect, and the obligate involvement of the macrophage CR3 receptor. TNF-alpha and GM-CSF synergized at low concentrations, resulting in dramatic up-regulation of phagocytosis when compared to either cytokine alone. Supernatants from C. neoformans-specific T cells also increased macrophage phagocytic efficiency. Finally, the administration of neutralizing mAb specific for TNF-alpha and GM-CSF increased mortality in C. neoformans-infected mice, and induced the rapid progression of disease with involvement of the brain and meninges. We conclude that TNF-alpha and GM-CSF are potent regulators of complement-dependent phagocytosis by murine macrophages. Macrophage activation with these two cytokines can completely overcome the anti-phagocytic properties of the virulent yeasts. Our results, therefore, implicate TNF-alpha and GM-CSF as important mediators of resistance to encapsulated pathogens such as C. neoformans where ingestion of the organism is a critical process in host resistance.