Keiko Kagaya

Murine defense mechanism against Candida albicans infection. II. Opsonization, phagocytosis, and intracellular killing of C. albicans.

The phagocytic and intracellular killing activities of normal mouse phagocytes against Candida albicans were studied to elucidate the role of these activities in nonspecific and specific defense mechanisms. In the presence of fresh normal mouse serum, viable C. albicans cells were ingested by mouse peripheral blood leukocytes (PBLs) and peritoneal macrophages (PMPs) at the same rate. Serum‐chelation experiments indicated that the factors involved in the alternative complement pathway are opsonins for C. albicans. PBLs killed intracellular C. albicans more effectively than PMPs. Lymphokine‐activated PMPs manifested marked intracellular killing activity and the occurrence of increased superoxide anion‐ and singlet oxygen production, in the absence of increased myeloperoxidase (MPO) production, suggests that the enhanced, MPO‐independent, oxidative mechanism may play an important role in the candidacidal activity. Specific rabbit antibodies played no role in the phagocytosis and intracellular killing of C. albicans. These results suggest that PMNs and factors involved in the alternative complement pathway, and lymphokine‐activated macrophages play major roles in the protection of mice against C. albicans infection.

Murine defense mechanism against Candida albicans infection. I. Collaboration of cell-mediated and humoral immunities in protection against systemic C. albicans infection.

Mice immunized with viable C. albicans cells demonstrated a high incidence of cell‐mediated and a low incidence of humoral immune response. There was good agreement between the final survival rate of C. albicans infected mice and the rate of simultaneous cell‐mediated and humoral immune response acquisition. Immunized mice with positive delayed hypersensitivity (DTH) against C. albicans crude antigen showed significant protection against intravenous challenge with C. albicans. Furthermore, the transfer of immunoglobulins from rabbit anti‐C. albicans serum to DTH‐positive mice enhanced protection, while it did not protect control mice against a subsequent challenge with C. albicans. These results suggest that cell‐mediated immunity plays a major role and humoral immunity a side role in the defense mechanism(s) of C. albicans infected mice.

Characterization of Pathogenic Constituents of Cryptococcus neoformans Strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 × 105 or 1 × 107 viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: (i) a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; (ii) a cell surface structure for responsible for resistance to intra‐ or extracellular killing and induction of the granulomatous lesion; (iii) a growth rate suitable for interacting with phagocytic elimination.

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes.

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.

Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Role of Mannopyranosyl Residue of the MAF Molecule in Macrophage Activation

Activation of macrophages with macrophage activating factor (MAF) was evaluated by measuring the intracellular killing activity of murine macrophages against Salmonella typhimurium. Concanavalin A (Con A)‐induced MAF‐rich fraction was obtained by a Sephadex G‐100 column, which contained molecules ranging from 25,000 to 67,000 daltons. The intracellular killing ability of mouse peritoneal macrophages against S. typhimurium was found to be increased by 0.1 m d‐mannose as well as by Con A‐induced MAF‐rich fraction. Both 0.1 m d‐mannose and MAF exhibited a similar timing pattern for macrophage activation. The same concentration of d‐glucose or l‐rhamnose did not change bacterial uptake and intracellular killing by macrophages. Moreover, when MAF‐rich fraction was applied to a Con A‐Sepharose column, a fraction that was adsorbed on Con A and eluted with 0.1 m α‐methyl d‐mannoside exhibited MAF activity. These results suggest the possibility that mannopyranosyl residues in the MAF molecules play an important role as a ligand in macrophage activation.

Anti‐Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti‐chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)‐ and weakly (W)‐encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H‐ < W‐strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan‐activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan‐activated serum was incubated with heat‐killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O‐acetyl groups in their molecules by 1H‐nuclear magnetic resonance spectroscopy. The de‐O‐acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de‐O‐acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti‐chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O‐acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.

Biological and biochemical characterization of macrophage activating factor (MAF) in murine lymphocytes: physicochemical similarity of MAF to gamma interferon (IFN-γ)

During the course of an investigation designed to separate macrophage activating factor (MAF) activity from interferon (IFN) antiviral activity in the lymphokine‐rich fraction (LKF) produced by stimulation of murine splenic cells with concanavalin A (Con A), we found molecular evidence for the similarity of the two activities. MAF activity was expressed as the rate of inhibition of intracellular growth of Salmonella typhimurium in macrophages based on the linear correlation between relative MAF activity and LKF concentration. The antiviral substance in LKF was identified as IFN‐γ based on the observation that its activity was inactivated at pH 2 and neutralized with anti‐mouse IFN‐γ serum but not with anti‐mouse IFN‐α/β serum. MAF and IFN antiviral activities displayed identical sensitivity to pH 2 and temperature. Further, neither activity was affected by β‐mercapto‐ethanol, but both were inactivated by guanidine hydrochloride and by sodium dodecyl sulfate, suggesting that the structures related to conformation of the protein of the two molecules may be similar. In affinity chromatography of the LKF on a Con A‐Sepharose column, MAF and IFN activities were found in both the nonadsorbing (F I) and adsorbing (F II) fractions. However, the rates of F II of MAF and IFN activities increased proportionally when the sample was applied on a column of higher capacity, suggesting that the molecular structure of the mannose‐containing glycosyl moiety of the two molecules may also be similar. Moreover, the intact or modified form of MAF and IFN activities of different LKF preparations showed a strong correlation, indicating that the production and denaturation of MAF activity were proportional to those of IFN antiviral activity. The results of this study provide strong evidence that MAF and IFN antiviral activities may reside in virtually the same molecular species.

Enhancement of phagocytosis and bactericidal activity of neutrophils in miniature pigs by dihydroheptaprenol, a synthetic polyprenol derivative

Dihydroheptaprenol (DHP), a synthetic polyprenol derivative, markedly stimulated the generation of peripheral blood neutrophils after intramuscular injection in miniature pigs. The generated neutrophils exhibited enhanced phagocytic activity against latex particles and also enhanced killing activity against Escherichia coli. The effective dose in miniature pigs (1.4 mg/kg) was markedly less than that required in mice (100 mg/kg). These results indicate that DHP induces resistance to some bacterial infections in pigs, suggesting the applicability of DHP for humans.

Antigen-specific suppression of antibody responses by T lymphocytes cytotoxic for antigen-presenting cells

In this study we demonstrated an alternative model of the antigen (Ag)‐specific suppression of antibody response in mice. Splenocytes that were taken from BALB/c mice immunized by i.v. injection of soluble human serum albumin (HSA) or ovalbumin exhibited MHC‐restricted Ag‐specific cytotoxicity for the respective antigen‐presenting cells (APC). When HSA‐primed splenocytes cultured with Ag and interleukin‐2 (IL‐2) were treated with anti‐CD4 or anti‐CD8 monoclonal antibody (mAb) plus complement, CD8+ and CD4+ T cells exhibited nearly the same level of cytotoxicity against APC. Furthermore, HSA‐primed CD4+ and CD8+ T cells released the same amount of interferon‐γ (IFN‐γ) when stimulated with Ag and IL‐2. Recombinant IFN‐γ was shown to suppress the in vitro plaque‐forming cell (PFC) response to sheep red blood cells (SRBC) only when it was added within 24 h after addition of Ag. The supernatants from both HSA‐primed CD4+ and CD8+ T cells suppressed the PFC response to SRBC in vitro, and the suppressive activity was abrogated by anti‐IFN‐γ mAb, but increased by anti‐IL 4 mAb. These results suggest that in our system the effector cells for Ag‐specific suppression of the antibody response in mice are both the cytotoxic type 1 clones (IFN‐γ‐producing) of CD4+ and CD8+ T cells for APC, and that IFN‐γ is a major extracellular effector molecule for such suppression.

Characteristics of Macrophage Activation by Gamma Interferon for Tumor Cytotoxicity in Peritoneal Macrophages and Macrophage Cell Line J774.1

We investigated the characteristics of macrophage‐mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN‐γ). In PMP, MTC was demonstrated when they were stimulated with IFN‐γ for 12 hr (short‐term stimulation) and was abrogated when they were stimulated for 48 hr (long‐term stimulation). Enhanced H2O2 generation was observed in PMP activated by long‐term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non‐treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short‐ and long‐term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN‐γ stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN‐γ for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN‐γ. The results suggest that TNF‐like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.