Lucia Scaringi

Group B Streptococcus Induces Apoptosis in Macrophages

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS β-hemolytic activity, suggesting that GBS β-hemolysin could be involved in apoptosis. β-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.

Cell Wall Components of Candida albicans as Immunomodulators: Induction of Natural Killer and Macrophage-mediated Peritoneal Cell Cytotoxicity in Mice by Mannoprotein and Glucan Fractions

Cell wall components from Candida albicans were compared to intact cells for their ability to induce natural cytotoxic immunoeffectors in the peritoneal cavity of mice. A soluble mannoprotein extract (MP) and an insoluble glucan fraction (GG) strongly stimulated the generation of peritoneal effectors capable of lysing YAC-1 and P-815 tumour cell lines in vitro. The anti-YAC-1 effectors were characterized as natural killer (NK) lymphocytes while the anti-P-815 effectors appeared to be activated macrophages. The activity of each fraction was typically dose-dependent and both fractions differed from whole cells in the kinetics of induction of cytotoxicity. However, the NK and macrophage effectors generated by these materials had similar functional and phenotypic properties, irrespective of the material used as inducer. No mannoprotein was detected in the insoluble glucan fraction GG. Hence, the immunoenhancing activity of GG could not be attributed to the presence of some MP or MP-like component. Mannan-rich fractions with low (less than 3%) protein content (M) or extracted by hot alkaline reagent (M-alk) were inactive as NK and macrophage inducers. Thus, the cell wall of C. albicans contains at least two distinct macromolecular complexes which mediate the induction in murine peritoneal exudates of cytotoxic effectors active against tumour cell lines.

Omeprazole Induces Apoptosis in Jurkat Cells

We report for the first time a potent apoptotic effect of omeprazole (OM). Apoptosis was induced in Jurkat cells in a time and concentration-dependent mode. Caspase 3 and PARP were rapidly cleaved in response to OM, but apoptosis was only partially inhibited by the caspase 3 inhibitor DEVD-CHO. OM also induced an early lysosomal destabilization which increased progressively and was correlated with a parallel increase in apoptotic cells. The cysteine protease inhibitor E64d gave strong protection against apoptosis thus proving the involvement of lysosomal enzymes in OM-induced apoptosis whereas, it did not impede the caspase 3 cleavage. Instead ZVAD-fmk, a general caspase inhibitor, also able to inhibit cathepsin activity, protected cells completely from OM-induced apoptosis. It therefore seems that both caspases and cysteine cathepsins are involved in the execution stage of OM-induced apoptosis.

Omeprazole induces apoptosis in normal human polymorphonuclear leucocytes.

We investigated in vitro apoptosis in human polymorphonuclear neutrophils (PMN) induced by omeprazole. This drug, both in the native (OM) and acidified (OM-HCl) form, is a potent inducer of PMN apoptosis. The effect is time- and dose-dependent. OM-HCl is more efficient than OM in inducing PMN apoptosis. In fact, after 24 h incubation in vitro at 1×10 −4M OM-HCl induces apoptosis in 70% of the cell population compared to 37% induced by OM. Apoptosis induced by both forms of the drug is caspase dependent being significantly reduced by pretreating cells with the caspase 3 inhibitor (DEVDH-CHO). However, some differences in the apoptosis mechanisms between the two forms of the drug seem to exist because PMN treatment with the specific caspase 8 inhibitor (Z-IETD-FMK) only blocks OM-HCl mediated apoptosis. We observed cleavage of caspase 8 only in the cells incubated with OM-HCl while the executioner caspase 3 was activated with both forms of the drug. Furthermore, pretreatment with GM-CSF, a known activator of intracellular survival pathways in PMN, partially protected cells from OM-HCl induced apoptosis but did not contrast the apoptotic effect of OM. Cysteine cathepsin proteases also seem involved in the apoptotic mechanism of both drug forms since the specific inhibitor E64d gave a significant protection. To verify if OM-HCl induced apoptosis was dependent on the sulfenamide bound with the cell sulfhydryl groups we used molecules with thiol groups such as β-mercaptoethanol (β-ME) and reduced glutathione (GSH). Reactions of OM-HCl with cellular sulfhydryl groups are strongly involved in both the triggering and evolving phase of the apoptotic mechanism since significant protection from apoptosis was obtained when PMN were pretreated for 1h with β-ME (lipid-permeable) or GSH (lipid-impermeable). These results show that OM and OM-HCl induce apoptosis in human PMN and suggest that the second binds the sulfhydryl groups, present on the cell membrane, to then penetrate the cell thus causing a further significant increase in apoptosis. OM-induced PMN apoptosis during the treatment of gastric inflammatory disease could be an advantage for the resolution of the phlogosis state. However, this aspect should be further elucidated to assess the optimal therapeutical regimen for gastric diseases which are related to infective agents.

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.

A rapid Candida albicans hyphal‐form growth inhibition assay: determination of myelomonocytic‐mediated antifungal activity

Summary. An in vitro microassay for the measurement of Candida albicans hyphal‐form growth inhibition by myelomonocytic cells is described. The assay is rapid, easy‐to‐perform and objective. A Candida strain capable of in vitro dimorphic transition from yeast to hyphal form has been employed. The assay is based on the incorporation of 3H‐glu‐cose by the fungus, the effect being dependent upon the time of pulse, size of the inoculum and concentration of radiolabelled metabolite. In particular, C. albicans hyphal form, obtained by a 3 h incubation in vitro in the presence of 10% fetal calf serum, is co‐incubated with the effector cells. A pulse with 3H‐glucose in water is then performed and the radioactivity incorporated by the residual Candida is taken as an indication of hyphal growth. We found that polymorphonuclear cells, peritoneal macrophages and the cloned GG2EE macrophage cell line significantly inhibited hyphal growth, the effects being time and effector‐to‐target cell ratio dependent.

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.

Learning from other diseases: protection and pathology in chronic fungal infections

Fungal commensals coexist in a complex milieu of bacteria within the human body. An increased understanding of the importance of microbiota in shaping the host’s immune and metabolic activities has rendered fungal interactions with their hosts more complex than previously appreciated. Metagenomics has revealed the complex interactions between fungal and bacterial commensals that, either directly or through the participation of the host immune system, impact on immune homeostasis at mucosal surfaces that, in turn, lead to secondary fungal infections. Metabolomics has captured the dialogue between the mammalian host and its microbiota. It appears that the host tryptophan catabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) plays a dominant role in the interplay between tryptophan catabolism by microbial communities, the host’s own pathway of metabolite production, and the activation of the aryl hydrocarbon receptor (AhR)/IL-22 axis, eventually impacting on mucosal immune homeostasis and host/fungal symbiosis. Thus, the regulatory loop involving AhR and IDO1 may be exploited for the development of multi-pronged host- and microbiota-directed therapeutic approaches for mucosal and systemic fungal diseases.

Activation of cytokine genes during primary and anamnestic immune response to inactivated c. albicans.

Recent evidence suggests that after repeated stimulations with inactivated C. albicans (CA) cells, CD2F1 mice respond with a cytokine pattern typical of T‐helper 1 (Th1) subset development. The purpose of this study was to analyse the sequence of immunological events which, soon after priming mice with CA, lead to the development of primary and anamnestic response. A comprehensive kinetics analysis of cytokine mRNA expression was performed by Northern blot assay, in peritoneal exudate cells (PEC), at different phases of immune response to CA: after priming (one i.p. injection of 2×107 CA cells/mouse), during development of the primary immune response (five progressive CA i.p. injections over a 2‐week period) and in the anamnestic response (CA booster 30days after the primary response). In vitro assays were performed 2 and 24hr after every CA stimulation. The response to CA priming was characterized by an early and high expression of interleukin‐2 (IL‐2) and IL‐1β mRNAs. At 24hr, IL‐2 mRNA was still at a high level, while IL‐1β had greatly decreased. A weak expression of IL‐10 was only induced at 2hr, whereas IL‐12 p40 subunit, interferon‐γ (IFN‐γ), IL‐4 and IL‐5 mRNAs were undetectable. In this phase no in vitro proliferative response of PEC to CA was observed, whereas a significant natural killer (NK) activity was induced. From the second CA injection, the IFN‐γ mRNA was already induced at 2hr. Its expression level increased progressively with the number of CA injections persisting up to 24hr after the fifth stimulation. A progressive increase of IL‐2 mRNA expression was also induced whereas IL‐1β and IL‐10 mRNAs were always transiently expressed at 2hr at levels similar to those observed after the priming. IL‐12 p40 subunit, IL‐4 and IL‐5 mRNAs were never detectable. The expression of this selected cytokine pattern typical of Th1 response was correlated with the development of CA‐specific T lymphocytes as confirmed by the in vitro proliferative response of CA‐5d‐induced PEC to CA. NK activity also increased progressively with the number of CA injections and after the fifth stimulation lymphokine‐activated killer (LAK) activity was also induced. The anamnestic response to CA was characterized by a very quick induction of high levels of IL‐2, IFN‐γ and IL‐1β mRNAs. IL‐2 and IFN‐γ mRNAs remained high up to 24hr while IL‐1β mRNA decreased strongly. A weak, transient expression of IL‐10 mRNA was induced at 2hr whereas the IL‐12 p40 subunit, IL‐4 and IL‐5 mRNAs were not detectable. The presence of CA‐specific memory lymphocytes was confirmed by the in vitro specific proliferative response of PEC to CA. CA booster caused also a very rapid and high level of NK/LAK activation. In conclusion, these results indicate that CA is able to progressively trigger differentiation of the Th1 subset which develops in the absence of IL‐12, and that Th memory cells retain the same selected Th1 cytokine profile developed in the primary immune response.

In vivo modulation of lymphokine-activated killer cell activity by cell wall components of Candida albicans

We have previously reported that inoculating CD2F1 mice intraperitoneally with five doses of 2 x 10(7) inactivated Candida albicans (CA) cells was associated with the induction of lymphokine-activated killer (LAK)-like effectors. In this study we investigated the ability of some purified cell wall components of CA (CA-CW) to induce LAK-like cells in vivo. Multiple administrations of glucan ghost (GG), a mannoprotein mixture (MP) and a low-protein mannan fraction (M) at variance with whole CA did not induce LAK-like cells in the peritoneal cavity. However, the broad-spectrum antitumor cytotoxicity induced by CA could be recalled to a high level by a booster dose of MP and M, but not GG, given up to 70 days after the multiple CA-treatment. This induced cytotoxicity was maximum when the booster was given on Day +14 after CA-treatment and minimum on Day +70. In CA-treated mice, inoculated on Day +30 with CA or MP, LAK-like cytotoxicity was already significantly increased 4 hr after the booster, but the maximum value was reached at 24 hr. Anti-mannan antibodies did not interfere with LAK-like cells induction by CA because splenectomy before CA-treatment or passive administration of anti-mannan antibodies had no effect on the rapid activation of cytotoxicity by CA or a booster dose of MP. Administration of recombinant human interleukin-2 (rhIL-2) to CA-treated mice induced a higher level of NK activity than that induced by the same dose in untreated control mice, but did not activate LAK-like effectors. The results indicate that LAK-like effectors are easily generated in the peritoneal cavity by a booster with a defined antigenic constituent of CA cell wall for a long period in CA-sensitized mice.