Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin.

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.     

Colony-stimulating factors activate human macrophages to inhibit intracellular growth of Histoplasma capsulatum yeasts.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.     

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes.

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.     

Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon.

Recombinant murine gamma interferon as well as lymphokines prepared from immune splenocytes and concanavalin A-stimulated T-cell hybridoma activated normal mouse peritoneal macrophages to inhibit the intracellular growth of Histoplasma capsulatum. The activities of the lymphokine from immune splenocytes and of recombinant murine gamma interferon were neutralized by rabbit anti-murine gamma interferon antibody. The intracellular yeasts were not killed by the interaction even though growth was completely inhibited.     

T-cell hybridoma-produced lymphokine that activates macrophages to suppress intracellular growth of Histoplasma capsulatum.

Supernatants from concanavalin A-stimulated T-cell hybridomas activated macrophages to suppress the intracellular growth of Histoplasma capsulatum and to kill tumor cells. The supernatants had high interferon activity and were pH 2 sensitive and heat resistant. Gamma interferon is suggested to be among the active substances in the supernatants. Neither alpha interferon nor beta interferon activated macrophages for the Histoplasma growth inhibitory activity.     

Recombinant murine gamma interferon stimulates macrophages of the RAW cell line to inhibit intracellular growth of Histoplasma capsulatum.

Macrophages of the RAW 264.7 cell line, activated by pretreatment with recombinant murine gamma interferon, inhibit the intracellular growth of Histoplasma capsulatum. Growth inhibition occurred by a mechanism that was operative only when L-Arg metabolism was allowed to occur. When activated macrophages were cultured in the absence of L-Arg or in the presence of NG-monomethyl-L-Arg, a competitive inhibitor of L-Arg metabolism, activation to the antihistoplasma growth-inhibitory state did not occur. An increase in levels of NO2-, an end product of L-Arg metabolism, was detected only after activation of RAW 264.7 cells to the growth-inhibitory state. In contrast, only baseline levels of NO2- were detected when L-Arg was excluded or when NG-monomethyl-L-Arg was added to the culture medium. Nitric oxide (NO.), a reactive intermediate product of L-Arg metabolism, was implicated as the relevant antihistoplasma effector molecule. When H. capsulatum yeast cells were cultured for 24 to 28 h in a system designed to generate soluble NO., a dose-dependent cytotoxic effect was observed.     

Stimulation of murine lymphocyte blastogenesis by mitogens in heat-killed Histoplasma capsulatum yeast cells.

In vitro blastogenesis by normal murine splenocytes from several mouse strains has been detected after exposure to heat-killed Histoplasma capsulatum yeast cells. Maximal lymphocyte stimulation induced by 10(4) heat-killed cells resulted in 20- to 45-fold increases in [3H]thymidine uptake by splenocytes when compared with responses by normal unstimulated lymphocytes. The kinetics for this response to heat-killed H. capsulatum cells has shown peak mitogenesis 3 days after culture. Examination of the mitogenic potential of soluble antigen preparations from H. capsulatum has revealed stimulation of lymphocyte blastogenesis with yeast cell sonicates and autolysates but not substances from autoclaved yeast cells. The levels of lymphocyte blastogenesis induced by sonicates or autolysates were comparable to mitogen responses stimulated by heat-killed cells. Preliminary biochemical characterization of the mitogenic factor(s) associated with yeast cell sonicates show two peaks of activity, at 178,000 and less than 12,000 Mr, which have a protein or glycoprotein nature. Finally, analysis of lymphocyte blastogenesis in cultures enriched for selected lymphocyte subpopulations has shown that T lymphocytes are preferentially stimulated by yeast cell mitogens.     

Immune responsiveness following intratracheal inoculation with Histoplasma capsulatum yeast cells

Splenic in vitro immune responses from C57B1/6 mice have been evaluated after intratracheal (i.t.) or intravenous (i.v.) inoculation with live Histoplasma capsulatum yeast cells. Significant (P less than 0.005) numbers of cells reactive to heat killed H. capsulatum (HK-Hc) were present 1 week after i.t. inoculation with 5 X 10(5) viable yeast cells. In contrast, blastogenic responses to HK-Hc were not detected until 3 weeks after i.v. H. capsulatum inoculation. Examination of non-specific immunity, i.e. mixed lymphocyte responses to alloantigens and antibody formation to sheep erythrocytes, revealed a significant (P less than 0.005) and transient depression of these responses 1 week following i.t. H. capsulatum inoculation. The non-specific decreases associated with early (1 week) i.t. infection were not mediated by suppressor cells but were related to the number of viable yeast administered.     

Characterization of antigens from type A and B yeast cells of Histoplasma capsulatum.

The antigenic composition of cytoplasmic extract and culture filtrate antigens of type A and B yeast cells of Histoplasma capsulatum grown in a synthetic medium was studied. These preparations from type A and B yeast cells contained varying amounts of protein, carbohydrates, and protein-carbohydrate complexes. The antigenic analysis of these preparations was performed by antigen-antiserum absorption with subsequent immunodiffusion and cross-immunoelectrophoresis with absorption in situ in an intermediate gel. All protein antigens observed in culture filtrate of either type A or B yeast cells were also present in the cytoplasmic extracts of the same type. The cytoplasmic extract of type A and B yeast cells each contained certain characteristic antigens that were not shared by the other type. Rocket immunoelectrophoresis with different molecular weight fractions of the antigenic preparations from both types indicated the polydisperse nature of Histoplasma yeast cell antigens.     

Killing of Histoplasma capsulatum by gamma-interferon-activated human monocyte-derived macrophages: evidence for a superoxide anion-dependent mechanism.

The interaction of human macrophages with the yeast form of the thermally dimorphic fungal pathogen, Histoplasma capsulatum, was studied. Macrophages derived from monocytes by culture in vitro for 3 days ingested H. capsulatum, but were neither fungicidal or fungistatic. In contrast, when monocytes were exposed to human recombinant gamma-interferon (gamma-IFN) during their differentiation into macrophages, those macrophages were able to reduce the number of ingested or adherent cfu of H. capsulatum by 44-75% in 2 h. Activation of macrophages for fungicidal activity by gamma-IFN was dose dependent and 500-1000 units ml were optimal. Antibody to gamma-IFN abrogated the gamma-IFN activation process. Killing of H. capsulatum by activated macrophages in 2-h assays could be inhibited by superoxide dismutase but not by sodium azide.     

Blastogenic responses of lymphocytes from mice immunized by sublethal infection with yeast cells of Histoplasma capsulatum.

Blastogenic responses of spleen cells to histoplasmin and ribosomal antigens and to the mitogens concanavalin A. phytohemagglutinin, and lipopolysaccharide were studied in normal and immunized mice (10(5) live yeast cells of Histoplasma capsulatum given by the subcutaneous route). Cells (10(6) per well) were cultured with and without antigens and mitogens in microtiter plates with RPMI 1640-5% heat-inactivated normal mouse serum for 72 h at 37 degrees C. Cells were harvested after a 16- to 18-h pulse with 1 microCi of [3H]thymidine (6.7 Ci/mol), and thymidine incorporation was measured by scintillation counting. The initial blastogenic response to concanavalin A (54 X 10(3) cpm) was suppressed (P less than 0.001) from 4 to 14 days post-immunization and returned to control levels on day 21. The response to phytohemagglutinin was suppressed up to 21 days. Lipopolysaccharide responses, however, were affected to a lesser degree. Blastogenic responses to histoplasmin and H. capsulatum ribosomes were similar on day 0 in normal and immune lymphocytes, but by day 4 cells from immunized mice were more responsive (P less than 0.01). The maximum response to H. capsulatum antigens was detected on day 42 and was 9- to 16-fold higher than in controls. These results demonstrate in vitro responses of primed lymphocytes on exposure to H. capsulatum antigens and suppressed responses to mitogens during early stages of the immune response.     

Histoplasma capsulatum fails to trigger release of superoxide from macrophages.

The yeast form of the dimorphic fungus Histoplasma capsulatum survives within macrophages after phagocytosis. To do so, it must avoid, inhibit, or resist a variety of toxic oxygen metabolites. Using ferricytochrome c reduction to assay superoxide release, we examined the response of mouse macrophages to the yeast form of various H. capsulatum strains. Doses of zymosan as low as 20 particles per macrophage elicited superoxide, whereas H. capsulatum failed to induce superoxide even at 160 yeast cells per macrophage. This phenomenon was observed with two virulent strains of H. capsulatum (G217B and G186A) and with an avirulent variant of G186A. Over a 15- to 150-min observation period, zymosan stimulated increasing reduction of ferricytochrome c, but H. capsulatum did not. When added concurrently with zymosan, H. capsulatum had no effect on superoxide production. Therefore, H. capsulatum was unable either to inactivate the oxygen radical or inhibit host cell superoxide response to other competent stimuli. Enzymatically generated superoxide reduced ferricytochrome c even in the presence of H. capsulatum, again implying that the organism does not readily inactivate superoxide. This experiment also demonstrated that the yeast did not interfere with the assay used. Thus, rather than inhibiting superoxide generation or inactivating the anion, H. capsulatum yeast cells appear to avoid the toxic effects of superoxide by failing to trigger its release.     

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.     

Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum.

Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's). Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H. capsulatum-containing murine peritoneal M phi's. An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H. capsulatum but not latex spheres. These results suggest that one way in which H. capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.     

Recognition of Histoplasma capsulatum yeast-cell antigens by human lymphocytes and human T-cell clones.

We have prepared a detergent extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells that is recognized by T cells from mice immunized with viable organisms or with the extract. A 62-kd antigen from this extract has been isolated and has been shown to be antigenic and to confer protective immunity in mice. In this study, we examined the in vitro proliferative response by human lymphocytes and human T-cell clones to both the extract from the cell wall and cell membrane and the 62-kd antigen, termed HIS-62. Seven healthy individuals were identified whose peripheral blood mononuclear cells responded to the cell wall and cell membrane extract from H. capsulatum yeast cells. Mononuclear cells from all seven individuals recognized histoplasmin. T-cell clones were generated from peripheral blood mononuclear cells from a single person using the extract from cell wall and cell membrane as the antigenic preparation. Nineteen clones were propagated; all expressed the surface phenotype CD3+, CD4+, T-cell receptor alpha/beta +. The clones were antigen-specific. Of 17 clones studied, none responded to extracts from Blastomyces dermatitidis or Coccidioides immitis or to tetanus toxoid. Sixteen of 19 clones recognized HIS-62 in vitro. Additional analysis revealed that cells from each of the seven individuals who responded to the extract mounted a proliferative response to HIS-62. Thus, the extract from the cell wall and cell membrane and HIS-62 are targets of the human cell-mediated immune response to H. capsulatum. Moreover, HIS-62 appears to be an immunodominant antigen.     

Interactions between Histoplasma capsulatum and macrophages from normal and treated mice: comparison of the mycelial and yeast phases in alveolar and peritoneal macrophages.

Interactions between macrophages (alveolar and peritoneal) from normal, vaccinated (with heat-killed yeast cells), and Mycobacterium bovis BCG-treated mice and the mycelial and yeast phases of Histoplasma capsulatum were observed. Phagocytosis of microconidia, small hyphal fragments, and yeast cells occurred 4 to 6 h after the infection of macrophage cultures. Conversion to the yeast phase began at 6 to 7 h and was complete after a 72-h incubation at 37 degrees C. Macrophages surrounded and adhered to macroconidia and large hyphal elements. More macrophages (65 to 68%) from BCG-treated mice contained fungi at 24 h than did macrophages from normal or vaccinated mice. Although there was no increase in the number of fungi in macrophages from vaccinated mice, only the macrophages from BCG-treated mice contained fewer fungi after 48 h of infection with the mycelial phase of H. capsulatum. Fungal growth was not inhibited in any of the macrophage cultures when infected with the yeast phase. The macrophages infected with yeast cells were destroyed after 48 to 72 h in the culture. Only BCG-treated macrophages survived infection with the mycelial phase, whereas macrophages from normal and vaccinated mice were destroyed by the infection.     

An aberrant variant of Histoplasma capsulatum var. capsulatum.

We studied an aberrant culture of Histoplasma capsulatum var. capsulatum isolated from synovial fluid collected from the right elbow of a patient from Kansas. Colonies on Sabouraud glucose agar and other routine mycological media were glabrous to soft, moist, heaped, deeply folded or convoluted, and orange-brown with a white, irregular margin. Microscopically, hyphae were hyaline, septate, and branched and remained totally devoid of conidiation over a period of 2 years on all mycological media. Conversion to the yeast form was achieved on Pine's medium at 37 degrees C. Colonies at early stages of growth were smooth, moist, pasty, shiny, and orange-brown but soon became wrinkled and slightly raised and produced oval, thin-walled cells measuring 2 to 3 by to 4.5 microns which multiplied by polar budding. The identity of the isolate was further confirmed by utilizing the Accuprobe DNA and the exoantigen test for H. capsulatum var. capsulatum.     

Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum.

The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs. Little is known about the interaction of BAM and Hc, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis. A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-activated BAM. Fungistatic activity of BAM was determined with a 24-h assay. A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used. Activated BAM killed Hc (reduced inoculum CFU by 25 +/- 12%; n = 4). The fungicidal activity of BAM was abrogated by 0.2 mM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase. In fungistatic assays activated BAM inhibited multiplication of Hc by 61 +/- 4% (n = 3) compared with cocultures with control BAM. However, Hc multiplied 100% more in control BAM cocultures than in medium alone. Data indicated that this was due to advantages that Hc has in the intracellular environment. Only NMMA inhibited fungistatic activity of activated BAM. In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained. In conclusion, activated BAM and PM kill yeast cells of Hc by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway.     

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.