Recombinant and natural gamma-interferon activation of macrophages in vitro: different dose requirements for induction of killing activity against phagocytizable and nonphagocytizable fungi.

Recombinant murine gamma-interferon (IFN) and supernatants from concanavalin A (ConA)-stimulated spleen cells were tested for their ability to activate resident peritoneal macrophages (M phi) for fungicidal activity. M phi monolayers pulsed overnight with IFN exhibited significantly enhanced fungicidal activity against Candida albicans (44 +/- 12 versus 0.0%) and Blastomyces dermatitidis (34 +/- 1 versus 3 +/- 3%). The effect of IFN was dose dependent; however, less IFN (10 U/ml) was required to activate M phi to kill phagocytizable C. albicans than to kill nonphagocytizable B. dermatitidis (1,000 U/ml). ConA-stimulated spleen cell supernatants were also able to activate M phi for fungicidal activity against both fungi. The capacity of ConA-stimulated spleen cell supernatants to activate M phi for fungicidal activity was neutralized in the presence of antibody to murine IFN. ConA-treated monolayers acquired the ability to kill C. albicans, but not B. dermatitidis, which was shown to be associated with residual (10%) lymphocytes in the monolayers. Lipopolysaccharide (0.001 to 10 micrograms/ml) failed to consistently activate M phi for fungicidal activity. These data show that IFN can exert an immunoregulatory role on M phi defense against these fungal pathogens.     

Gamma-interferon activation of macrophages for killing of Paracoccidioides brasiliensis and evidence for nonoxidative mechanisms

Fungicidal activity of murine peritoneal macrophages for the yeast form of the dimorphic fungal pathogen P. brasiliensis was studied. Killing was assessed by reduction of colony forming units (CFU) using a new medium which has a good plating efficiency. Resident peritoneal macrophages phagocytosed but did not kill P. brasiliensis. Macrophages treated overnight with recombinant gamma-interferon (IFN), lymph node cells plus concanavalin A (Con A) or Con A-stimulated spleen cell culture supernatants (Con A Sup) reproducibly killed three different isolates of P. brasiliensis (35 - 55%, P less than 0.05 - P less than 0.001). This is the first demonstration of killing of this organism by macrophages. Activated macrophages did not show enhanced phagocytosis of P. brasiliensis. Activation of macrophages for killing by IFN was dose-dependent and, varying with the isolate, 100 - 10,000 U/ml was required for inducing significant fungicidal effects against P. brasiliensis. Activation of macrophages by IFN or Con A Sup was abrogated by anti-IFN antibody. These results suggest that immune modulation may be an approach to therapy of paracoccidioidomycosis. Killing was not significantly inhibited in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethylsulfoxide (300 mM) or azide (1 mM). This indicated that killing mechanism(s) did not depend upon products of the oxidative burst. These results show that P. brasiliensis can be significantly killed by activated macrophages without products of the oxidative burst.     

Fungicidal activity of murine inflammatory polymorphonuclear neutrophils: comparison with murine peripheral blood PMN.

A characteristic of inflammation is the influx of polymorphonuclear neutrophils (PMN) from peripheral blood to the inflammatory reaction. We report on metabolic alterations and alterations in fungicidal activity in PMN elicited intraperitoneally with different inflammatory agents. The fungicidal activity of murine peripheral blood PMN (PB-PMN) against phagocytosable Candida albicans and nonphagocytosable Blastomyces dermatitidis was compared to that of murine inflammatory PMN. PMN elicited with sodium caseinate exhibited enhanced killing of B. dermatitidis (93 +/- 3%) compared to PB-PMN (38 +/- 11.7%). In contrast, thioglycollate medium elicited PMN had significantly less ability to kill B. dermatitidis (3 +/- 5%) than PB-PMN. Incubation of caseinate PMN with thioglycollate medium for 1 h significantly reduced their ability to kill B. dermatitidis (95% vs 25%). This effect was not due to cytotoxicity of thioglycollate medium for PMN. Candidacidal activity of inflammatory PMN (caseinate or proteose peptone-elicited) was not significantly greater than that of peripheral blood PMN. However, inflammatory PMN had significantly greater candidacidal activity than thioglycollate-elicited PMN.     

Activation of murine polymorphonuclear neutrophils for fungicidal activity by recombinant gamma interferon

Recombinant murine gamma-interferon (IFN) was tested for its ability to enhance murine polymorphonuclear neutrophil (PMN) fungicidal activity in vitro. PMNs, elicited by intraperitoneal injection of thioglycollate 4 hr prior to collection, were treated with 0.00003-300,000 units of IFN per milliliter for 1 hr prior to challenge with yeast form Blastomyces dermatitidis. These PMNs were not fungicidal for Blastomyces in the absence of IFN; significant enhancement of PMN fungicidal activity by IFN treatment occurred in a dose-dependent manner with maximal enhancement observed at 30,000 U/ml (21% killing). Pretreatment of IFN with rabbit anti-IFN antiserum before addition to PMNs eliminated the enhancement of fungicidal activity by effective doses of IFN. PMN fungicidal activity against phagocytizable Candida albicans was significantly (P less than .001) higher (71.3 +/- 17.4%) than against B. dermatitidis. Candidacidal activity was not significantly enhanced by IFN treatment of PMNs. Exogenously added lipopolysaccharide, at levels corresponding to those found in this preparation of IFN, did not activate PMNs for enhanced fungicidal capacity. These data indicate a stimulatory role for IFN in the killing of B. dermatitidis by PMNs, suggesting that IFN is an active component of the communication between T lymphocytes and PMNs with respect to antimicrobial resistance. They suggest a natural role for IFN in host defense against blastomycosis and other fungal infections, and a possible therapeutic use for exogenous IFN in fungal disease.     

Fungicidal activity of rabbit alveolar and peritoneal macrophages against Candida albicans.

We tested the ability of rabbit macrophages to kill Candida albicans in vitro. Resident (unstimulated) alveolar macrophages killed 28.1 +/- 1.9% of ingested organisms in 4 h, whereas resident peritoneal macrophages killed only 15.2 +/- 1.3% (mean +/- standard error of the mean, P < 0.01). Peritoneal macrophages obtained from rabbits treated 3 weeks earlier with complete Freund adjuvant showed enhanced candidacidal activity relative to normally resident peritoneal cells (28.2 +/- 3.1%, P < 0.01). Candidacidal activity by alveolar macrophages recovered from such treated animals was slightly enhanced relative to untreated alveolar macrophages (32.9 +/- 2.3%). Candidacidal activity by peritoneal and alveolar macrophages was not decreased by several agents (cyanide, azide, sulfadiazine, and phenylbutazone) that inhibit the ability of human blood monocytes to kill C. albicans. In contrast, candidacidal activity by alveolar macrophages was greatly diminished by iodoacetate, an ineffective inhibitor of this function in human monocytes. We conclude that rabbit macrophages kill C. albicans by a fungicidal mechanism distinct from the peroxidase-H2O2 mechanism of human granulocytes and monocytes, and that the fungicidal properties of peritoneal and alveolar macrophage populations are enhanced after nonspecific stimulation with complete Freund adjuvant.     

Enhanced killing of Blastomyces dermatitidis by gamma interferon-activated murine peripheral blood polymorphonuclear neutrophils

Normal peripheral blood polymorphonuclear neutrophils (PB-PMNs), challenged in vitro with yeast form Blastomyces dermatitidis, reduced inoculum colony-forming units of a virulent strain by 37.5 +/- 9.5%. Pre-incubation of PB-PMNs with 10-100,000 U/ml of purified recombinant murine gamma-interferon (IFN) for 1 h prior to challenge with fungi resulted in significant enhancement of PB-PMN fungicidal activity. No direct fungicidal activity by IFN alone was observed. Pretreatment of selected concentrations of IFN shown to have PMN-enhancing activity (100 or 1000 U/ml) with rabbit hyperimmune anti-IFN antiserum for 1 h before addition to PB-PMNs abrogated the enhancement of fungicidal activity. Isolated peripheral blood mononuclear cells failed to kill B. dermatitidis, even when mononuclear cells were present at a concentration ten times greater than that normally used in killing assays, and failed to be activated by IFN. Treatment of unstimulated or IFN-activated PB-PMNs with complement and hybridoma-derived monoclonal antibody specific for PMNs eliminated PB-PMN fungicidal activity. Exogenously added lipopolysaccharide (0.0005-50,000 ng/ml) did not activate PB-PMNs, whether added alone or in conjunction with IFN. The PB-PMN activating capacity of IFN could be destroyed by heat treatment (100 degrees C, 15 min) or by acid treatment with HCl (pH 2). These results demonstrate that recombinant gamma-interferon can stimulate PB-PMNs to kill B. dermatitidis, that the PB-PMN activating moiety is IFN and that PB-PMNs are responsible for fungal killing in this assay system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fungicidal mechanisms of activated macrophages: evidence for nonoxidative mechanisms for killing of Blastomyces dermatitidis.

The mechanism(s) by which lymphokine-activated peritoneal macrophages kill Blastomyces dermatitidis was studied. Resident peritoneal macrophages from BALB/cByJ mice, when treated overnight with lymph node cells plus concanavalin A, supernatants from concanavalin A-stimulated spleen cells, or recombinant gamma interferon, were then able to kill a virulent B. dermatitidis isolate (ATCC 26199) (at levels of 25% +/- 4%, 28% +/- 8%, and 21% +/- 5%, respectively). Killing was not significantly decreased or enhanced in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethyl sulfoxide (300 mM), or azide (1 mM). Viable B. dermatitidis elicited a brisk oxidative burst and superoxide anion production in activated macrophages as measured by lucigenin-enhanced chemiluminescence, e.g., 10(4) cpm. However, these responses were not significantly different from those of control macrophages. Luminol-enhanced chemiluminescence responses by activated or control macrophages were meager (less than or equal to 10(2) cpm). These results indicate that activated macrophages kill B. dermatitidis by a mechanism(s) independent of products of the oxidative burst.     

Enhanced oxidative burst in immunologically activated but not elicited polymorphonuclear leukocytes correlates with fungicidal activity.

Polymorphonuclear neutrophils (PMN) induced locally in immune mice by intraperitoneal injection of antigen exhibit enhanced fungicidal activity compared with PMN elicited with thioglycolate. The mechanism of the differences in these PMN populations was studied. Sublethal infection was used to produce immunity to Blastomyces dermatitidis. A correlation was sought between the ability of PMN to kill, or not kill, B. dermatitidis and the production of the oxidative burst, as measured by luminol-enhanced chemiluminescence (CL). Although elicited PMN cocultured with Candida albicans produced a burst of CL and were candidacidal, killing did not occur when PMN were cocultured with B. dermatitidis. Lack of killing of B. dermatitidis by elicited PMN correlated with lack of stimulation of a brisk oxidative burst. In contrast to elicited PMN, PMN induced by B. dermatitidis antigen responded to this fungus with a burst of CL and a significant reduction of inoculum CFU (80%). Furthermore, these PMN when cocultured with C. albicans produced an enhanced burst of CL, and killing was enhanced compared with that by elicited PMN, e.g., 86 versus 58%. The CL burst and killing of B. dermatitidis by antigen-induced PMN was abrogated in the presence of catalase, implying a critical role for hydrogen peroxide. Partial but significant depression of CL and killing in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, identified hydroxyl radical, or its metabolites, as a toxic product(s) responsible for a significant fraction of fungicidal activity. These results indicate that the metabolic activity and microbicidal activity of PMN can be altered (enhanced) at the site of an immunological reaction and thus could constitute an important factor in resistance.     

Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

In vivo activation of peripheral blood polymorphonuclear neutrophils by gamma interferon results in enhanced fungal killing.

The effect of in vivo administration of murine recombinant gamma interferon (IFN) on the fungicidal activity of murine peripheral blood polymorphonuclear neutrophils (PB-PMNs) was studied. Mice were injected intramuscularly with 250, 2,500, 25,000 or 250,000 U of IFN 5 h before collection of peripheral blood. Purified PB-PMNs were cocultured in vitro with Blastomyces dermatitidis yeast cells for 2 h. PB-PMNs from untreated mice killed 44.5 +/- 12.5% of the fungal inoculum, whereas PB-PMNs from mice treated with 25,000 or 250,000 U of IFN showed significantly enhanced in vitro killing (68.0 +/- 9.4% [P less than 0.005] and 72.3 +/- 1.1% [P less than 0.001], respectively). Treatment with 250 or 2,500 U of IFN or 25,000 U of heated (100 degrees C, 15 min) IFN had no effect. The IFN-induced activation of PB-PMNs was transitory. Significant enhancement of PB-PMN killing activity occurred 1, 2, or 5 h after in vivo IFN administration, but no enhancement was observed 16 or 24 h after IFN treatment. Enhanced fungicidal activity by PB-PMNs from mice treated for 5 h with 25,000 U of IFN correlated with an increased release of superoxide anion (O2-) in vitro after stimulation of PB-PMNs with phorbol ester; normal PB-PMNs and IFN-activated PB-PMNs, respectively, produced 2.2 +/- 2.5 and 23.5 +/- 4.8 nmol of O2- per 10(6) PB-PMNs per 30 min (P less than 0.005). The exogenous addition of compounds that antagonize or inhibit the formation of oxygen radicals (superoxide dismutase, catalase, dimethyl sulfoxide, or sodium azide) significantly inhibited fungal killing by both normal and IFN-activated PB-PMNs. In addition to the enhanced microbicidal activity and superoxide generation demonstrated in vitro with constant cell numbers, there was a transient leukocytosis (particularly neutrophilia) in peripheral blood at doses of IFN and at times after IFN administration where enhanced activity was also demonstrated. In summary, our results indicate that PB-PMNs can be activated in vivo for enhanced killing of a fungal target. The enhanced killing capacity of IFN-activated PB-PMNs is due at least in part to the enhancement of oxidative killing mechanisms.     

In vitro study of contact-mediated killing of Candida albicans hyphae by activated murine peritoneal macrophages in a serum-free medium.

Activated peritoneal macrophages obtained from Listeria-immune mice were demonstrated to kill nonphagocytosable Candida albicans hyphae by contact-mediated mechanisms in a serum-free synthetic medium. The actual killing of hyphae was confirmed by a microculture technique utilizing the dimorphic nature of the fungus. The most efficient candidacidal activity was demonstrated by the macrophages obtained from mice first immunized with live Listeria monocytogenes and then elicited with heat-killed L. monocytogenes cells. Resident macrophages from control mice showed only low candidacidal activity against C. albicans hyphae and yeast cells. Direct physical contact appeared to be required for macrophages to efficiently kill oversized C. albicans hyphae. Efficient in vitro killing of hyphae also required relatively high effector/target cell ratios (50 or higher). The contact-mediated candidacidal activity of activated macrophages was not significantly abrogated by oxygen-radical scavengers, suggesting the involvement of oxygen-independent mechanisms. These results suggest that the enhanced nonspecific immunity to candidiasis seen in Listeria-immune hosts can be attributed, at least in part, to activated fungicidal macrophages. The ability of macrophages to detect and destroy both yeast and hyphal C. albicans cells is clearly an important element of the host defense against candidiasis.     

Candidacidal factors in murine bronchoalveolar lavage fluid.

Respiratory secretions provide an efficient method for protecting the large surface area of the lower respiratory tract. To determine whether lung secretions contribute to antifungal defenses, we tested bronchoalveolar lavage fluid for fungicidal activity. Candida albicans (blastoconidia) was incubated in unconcentrated cell-free lavage fluid from Swiss Webster mice and then cultured quantitatively to measure residual viability. In control buffer the residual fractions of viable fungi were 1.03 +/- 0.12 at 60 min and 0.84 +/- 0.05 at 120 min, whereas the residual fractions in lavage fluid were 0.64 +/- 0.07 and 0.23 +/- 0.05, respectively (P less than 0.05 by t tests). This activity was trypsin sensitive and heat stable (56 degrees C) and did not require divalent cations. It did not sediment with the surfactant fraction of lung lavage fluid. Unconcentrated lavage fluid reduced the adherence of C. albicans to serum-coated glass tubes to 2.3 +/- 1.5% of that of control Candida suspensions (n = 5, P less than 0.05 by t test). It did not alter Candida ingestion or intracellular processing by alveolar macrophages. Lavage fluid also killed clinical isolates of Candida tropicalis and Torulopsis glabrata but did not kill Candida krusei or Candida parapsilosis. Lavage fluid was concentrated and passed through an acrylamide-agarose gel matrix. The chromatogram indicated that the candidacidal activity eluted in a peak with a molecular weight range of 29,000 to 40,000. After electrophoresis on 15% sodium dodecyl sulfate-polyacrylamide gels, these fractions resolved into three bands. These were transferred to nitrocellulose and then eluted with Triton X-100; this procedure permitted the isolation of a single band of candidacidal activity with a molecular weight of 29,000. In summary, murine lavage fluid contains a heat-stable protein with direct antifungal activity. This soluble factor may contribute to lung defense processes by reducing fungal viability and adherence to tissue surfaces.     

Activation of murine polymorphonuclear neutrophils for fungicidal activity with supernatants from antigen-stimulated immune spleen cell cultures.

An in vitro model of in vivo immunological activation of murine polymorphonuclear neutrophils (PMN) was developed. Culture supernatants of spleen cells from Blastomyces dermatitidis-immunized mice stimulated with B. dermatitidis antigens in vitro were studied. Incubation of the supernatants with thioglycolate-elicited PMN enabled the cells to significantly reduce (31 +/- 6%) B. dermatitidis inoculum CFU. Optimum production of active supernatants occurred after 4 to 6 days of stimulation in vitro and required 200 micrograms of nonviable B. dermatitidis cells per ml. Generation of activity by immune spleen cells was shown to be antigen specific in that stimulation with a heterologous antigen or stimulation of nonimmune spleen cells with B. dermatitidis antigen did not produce active supernatants. The activity in supernatants was dose dependent, nondialyzable (molecular weight greater than or equal to 14,000), and relatively heat labile (80 degrees C, 30 min). Activation of PMN by supernatants for fungicidal activity against B. dermatitidis required only a short incubation period (1 h) followed by a 2-h coculture (challenge) period. Stimulation of normal spleen cells with concanavalin A also resulted in the production of supernatants capable of activating PMN for significant fungicidal activity (31.1 +/- 8.5%). These findings demonstrate for the first time a link between soluble factors produced by antigen stimulation of sensitized lymphoid cells and activation of PMN for enhanced microbicidal activity. Such a process defines an additional immune defense mechanism whereby the immune host may clear specific microorganisms.     

Susceptibility to pulmonary blastomycosis in young compared to adult mice: immune deficiencies in young mice.

The immunological basis for differences in resistance to pulmonary blastomycosis between young (3 to 4-week-old) and adult (7 to 8-week-old) CD-1 mice is unknown. We assessed whether there were differences in fungicidal activity of phagocytes and Th-1 lymphocyte cytokine production. The fungicidal activity of young bronchoalveolar macrophages (BAM) (20%) against Blastomyces dermatitidis (Bd) was comparable to killing by adult BAM (25%). However, IFN-gamma enhanced the killing by adult BAM (from 30 to 69%) to a greater extent than BAM from young animals (from 20 to 30%). Killing of Bd by young peritoneal macrophages (PM) (46%) and adult PM (42%) was similar, and the enhancement of cells of both by IFN-gamma was similar. TNFalpha production by young macrophages (BAM or PM), when cocultured with Bd for 18 h, was half of TNFalpha secreted by adult macrophages. We found that polymorphonuclear neutrophils (PMN) from young mice had deficient fungicidal activity against Bd (37%) compared with adult PMN (80%). Interferon-gamma (IFN-gamma) treatment increased PMN killing of Bd by PMN of young animals from 37 to 80%. In an assessment of innate responses, we found spleen cells from young mice produced three-fold less IFN-gamma and three-fold less IL-2 than adult spleen cells in response to 1 microg/ml concanavalin A (Con A). The young spleen cells also produced more NO, which we demonstrated reduced Con A-induced proliferation. These in vitro results demonstrate several immunological deficiencies in cells from young mice and these deficiencies correlate with susceptibility. In a pilot reconstitution experiment in pulmonary blastomycosis, treatment of infected young mice with IFN-gamma (18.5 x 10(3) U, s.c.) on days 0, 1, and 2 significantly increased survival.     

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.     

Candidacidal mechanisms of peritoneal macrophages activated with lymphokines or gamma-interferon

The mechanisms by which resident peritoneal macrophages, activated in vitro by lymphokines (LK) or recombinant gamma-interferon (IFN), kill Candida parapsilosis or C. albicans were studied. Resident non-activated peritoneal macrophages killed C. parapsilosis (55.5% SD 6.8%), but not C. albicans. This killing was completely inhibited by superoxide dismutase (SOD), partially by dimethyl sulphoxide (DMSO), but not by catalase or azide. Killing correlated with a brisk lucigenin-dependent chemiluminescence (CL) response by macrophages interacting with C. parapsilosis. No enhanced luminol-dependent CL response was observed in this system. This suggests that C. parapsilosis is killed by resident macrophages via a mechanism dependent on the presence of superoxide anion. By contrast, killing of C. parapsilosis by activated macrophages (49.0% SD 5.9%) was not inhibited by SOD or DMSO, suggesting the induction of a non-oxidative candidacidal mechanism. C. albicans was killed only by macrophages activated with IFN (52.0% SD 3.7%) or LK (55.7% SD 2.8%). Inhibition of killing by SOD was greater in IFN- than in LK-activated macrophages. Conversely, killing by LK-, but not IFN-, activated macrophages was significantly inhibited by catalase, DMSO or azide. The killing by LK-activated macrophages, and its inhibition by scavengers, correlated with the luminol-dependent CL response. The non-killing resident macrophages interacting with C. albicans made lucigenin-dependent CL responses similar to those of activated macrophages. The mechanisms enabling killing of C. albicans induced by activation appear to be different for LK and IFN, and appear to depend upon the myeloperoxidase systems and superoxide respectively.     

The involvement of nitric oxide in the anti-Candida albicans activity of rat neutrophils.

Rat peritoneal neutrophils (PMN) spontaneously release nitric oxide (NO) when incubated in vitro. Addition of the NO synthase inhibitor L-monomethylarginine (L-NMMA) to the PMN reduces NO production and impairs the killing of the yeast Candida albicans, both effects being reversed by L-arginine. These data strongly suggest that oxidative metabolism of L-arginine by PMN is involved in the candidacidal activity of these cells. Rat blood PMN, which do not produce significant amounts of NO, exhibit a reduced killing capacity compared with peritoneal cells, except when they are obtained from lipopolysaccharide (LPS)-treated rats. In this case they produce measurable amounts of nitrite and express high fungicidal activity in vitro. Confirming the candidacidal activity of NO, the exposure of the C. albicans cultures to different concentrations of NO donors leads to a reduction in their survival. The candidacidal activity related to the NO pathway in rat PMN is phagocytosis dependent, since the activity can be inhibited by cytochalasin B. However, the oxidative products of oxygen released by rat PMN do not seem to be involved in their candidacidal activity, as incubation of the cells with phorbol myristate acetate (PMA) increases release of superoxide anion but does not affect the pattern of killing. Our results suggest that NO could be an important candidacidal pathway in rat neutrophils.     

A monoclonal antibody directed against a Candida albicans cell wall mannoprotein exerts three anti-C. albicans activities

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.     

Involvement of reactive oxygen metabolites in the candidacidal activity of human neutrophils stimulated by muramyl dipeptide or tumor necrosis factor

In the presence of the adjuvant glycopeptide muramyl dipeptide (MDP), purified human PMN exhibited an enhanced capacity to kill Candida albicans cells at various cell ratios. A significant effect was obtained at 100 ng/ml MDP, and the maximum was reached at 1 micrograms/ml MDP. Recombinant human tumor necrosis factor (rHuTNF), a monokine that enhances host resistance to bacterial and fungal infections, also stimulated the candidacidal potency of PMN with a maximal effect at 10(-2) ng/ml rHuTNF. When MDP- or rHuTNF-stimulated PMN were cultured with yeast cells, the intracellular production of oxygen metabolites was enhanced. Pretreatment with inhibitors of oxidative burst demonstrated that the yeast cell killing by MDP-stimulated PMN was not affected by SOD but was inhibited by sodium azide, indicating the involvement of myeloperoxidase (MPO)-halide system in fungicidal mechanisms induced by MDP. When PMN were stimulated with rHuTNF, the killing of yeast cells was neutralized by iodoacetamide, showing that the candidacidal potency of stimulated-PMN was due to oxygen derivatives. Inhibition by sodium azide and sodium benzoate indicated that these oxygen metabolites could be derived from the MPO-halide system but also from hydroxyl radical production. Moreover, SOD partially inhibited the fungicidal potency of rHuTNF-stimulated PMN, thus indicating a possible reutilization of the released O2- anion for intracellular killing. Cytochalasin B abrogated the PMN fungicidal potency in all cases.     

Modulation of rat lymphocyte transformation by plasma fibronectin

Purified rat plasma fibronectin (Fn) was studied for its ability to influence nonspecific lymphocyte transformation to various mitogens. Fn added to lymph node cells (LNC) stimulated with phytohemagglutinin (PHA), concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) resulted in a noncytotoxic dose-dependent inhibition of the blastogenic response. Effective inhibition (greater than 50%) of LNC responses to PHA and LPS occurred with concentrations of Fn that ranged from 10-50 micrograms/culture. The proliferative response to Con A was less affected by the presence of Fn: 50% inhibition was observed only with the highest concentration of Fn studied. Fn at low concentrations was more effective than Fn-depleted plasma in inhibiting lymphocyte responses to PHA. Optimal expression of the regulatory activity of Fn occurs during and following the peak proliferative period for both PHA and LPS responses. In order to evoke maximum inhibition it is necessary for Fn to be present within 12 hours following stimulation of LNC with mitogen. Inhibition does not appear to be the result of Fn-mitogen complexes which reduce the amount of mitogen available for stimulation, since increasing concentrations of either PHA or LPS did not significantly reduce the inhibitory effect. Furthermore, inhibition was not influenced by the concentration of fetal calf serum present in the culture medium. Thus, Fn may function as an important nonspecific immunoregulatory factor at inflammatory sites and in areas of tissue repair.