Mechanisms of killing of Toxoplasma gondii by rat peritoneal macrophages.

Rats are resistant to Toxoplasma infection, and macrophages are thought to mediate this resistance. We performed a series of experiments to investigate the mechanism of the anti-Toxoplasma activity of resident rat peritoneal macrophages. Resident rat peritoneal macrophages killed more than 90% of ingested Toxoplasma gondii in vitro. This capacity was reduced progressively with the prolongation of culturing of macrophages in vitro before challenge with T. gondii. Exhaustion of the respiratory burst of macrophages with phorbol myristate acetate impaired their ability to kill and limit the replication of T. gondii. Histidine and diazabicyclooctane, presumed scavengers of singlet oxygen, were the only members of a battery of scavengers of metabolites of the respiratory burst that impaired the anti-Toxoplasma activity of macrophages. Ingestion of heat-killed Candida albicans by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium dye, whereas little dye was reduced by the ingestion of T. gondii. Challenge of macrophages with T. gondii released no detectable superoxide anion, as measured by the reduction of ferricytochrome c, whereas stimulation of macrophages with phorbol myristate acetate or ingestion of heat-killed Candida by macrophages released abundant superoxide anion. These data are consistent with the contributions of oxygen-dependent and oxygen-independent mechanisms to the anti-Toxoplasma activity of rat peritoneal macrophages. In addition, neonatal rats are known to be susceptible to Toxoplasma infection in vivo. However, resident neonatal rat peritoneal macrophages ingested and killed T. gondii to the same extent as did adult macrophages. Thus, the susceptibility of neonatal rats to Toxoplasma infection probably resides in other aspects of macrophage function or the immune response.     

Inhibition of Chlamydia psittaci in oxidatively active thioglycolate-elicited macrophages: distinction between lymphokine-mediated oxygen-dependent and oxygen-independent macrophage activation.

Immune sensitization of spleen cells was required to generate lymphokines (LK) that activated thioglycolate-elicited peritoneal macrophages (thio MACs) to respond via both oxygen-dependent and oxygen-independent systems. LK produced by incubating spleen cells from immunized A/J and LAF mice with concanavalin A stimulated a response by thio MACs to phorbol-12-myristate-13-acetate (PMA)-induced chemiluminescence and activated these cells to inhibit intracellular Chlamydia psittaci replication. Concanavalin A-incubated spleen cell preparations from unimmunized animals stimulated neither PMA-induced chemiluminescence nor antichlamydial activity. Activated thio MACs demonstrated a rapid chemiluminescence response to the intracellular protozoan Toxoplasma gondii, but C. psittaci did not induce chemiluminescence in LK-activated thio MACs, although cells exposed to C. psittaci retained their responsiveness to PMA-induced chemiluminescence. The PMA-induced response was inhibited by the addition of exogenous superoxide dismutase and catalase and was therefore related to the production of superoxide anion (O2 . -) and H2O2 by these cells. LK preparations incubated at 56 degrees C before macrophage treatment retained antichlamydial activity, but heated preparations no longer stimulated thio MACs to respond in the chemiluminescence assay. These data provide evidence that macrophage oxygen-dependent and oxygen-independent systems are simultaneously activated by LK, and these preparations comprise at least two distinct activities. The portion responsible for activating oxygen-dependent systems (PMA-induced chemiluminescence) is heat labile, whereas the portion responsible for activating oxygen-independent systems is heat stable. It is the latter system that results in restriction of chlamydial growth and in vitro parasite persistence.     

Nonoxidative microbicidal activity in normal human alveolar and peritoneal macrophages.

Although Toxoplasma gondii multiplies within normal murine alveolar and peritoneal macrophages, it is killed by normal rat alveolar and peritoneal macrophages. The killing by rat macrophages is by a nonoxidative mechanism. Studies on normal human alveolar macrophages have reported disparate results in regard to their ability to inhibit or kill T. gondii. We considered it of interest to explore further the effect of normal human alveolar and peritoneal macrophages on T. gondii. Unstimulated alveolar macrophages from each of seven individuals demonstrated a marked ability to kill or inhibit multiplication of T. gondii in vitro (e.g., the number of parasites per 100 alveolar macrophages was 31 at time zero and 2 at 18 h, whereas this value increased from 37 at time zero to 183 at 18 h in murine macrophages assayed in parallel). In quantitative assays of superoxide, alveolar macrophages released a substantial amount of superoxide when exposed to phorbol myristate acetate or to candidae. In contrast, alveolar macrophages incubated with T. gondii released no more superoxide than when in medium alone. Scavengers of superoxide anions, hydrogen peroxide, singlet oxygen, and hydroxyl radicals failed to inhibit killing of T. gondii by alveolar macrophages. Peritoneal macrophages from each of six normal women undergoing laparoscopy killed T. gondii in vitro; results of quantitative superoxide assays and scavenger experiments demonstrated that no oxidative burst was triggered in these macrophages by exposure to T. gondii. These data indicate that normal human alveolar and peritoneal macrophages can kill an intracellular parasite by nonoxidative mechanisms and suggest that these mechanisms are important in inhibition or killing of other opportunistic intracellular pathogens.     

Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages.

The Salmonella typhimurium gene for Mn-cofactored superoxide dismutase (sodA) was cloned by complementation of an Escherichia coli sodA sodB mutant for growth on minimal medium. Sequence analysis revealed an open reading frame of 618 bp encoding a polypeptide with 97% identity to E. coli SodA. A S. typhimurium sodA mutant was created by allelic exchange and tested for the ability to survive in the murine macrophage-like cell line J774. Growth of bacteria under iron-limiting conditions, inactivation of the Fur repressor, or expression of sodA from a plasmid resulted in increased resistance to early killing by J774 cells, which was abolished in the sodA mutant. These results suggest that resistance to the early oxygen-dependent microbicidal mechanisms of phagocytes involves the SodA gene product. The S. typhimurium sodA mutant was not significantly attenuated in mice, however, which suggests that resistance to early oxygen-dependent microbicidal mechanisms in vivo may play only a minor role in Salmonella pathogenesis.     

Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro.

After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice. One of the immunodominant antigens of Mycobacterium spp. is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages. After an i.p. injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T. gondii, and killed L. monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative. When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65. In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon. Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p. booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T. gondii, and increased rate of intracellular killing of L. monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65. The rate of phagocytosis of L. monocytogenes was not affected by Hsp 65 treatment. Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L. monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.     

Deficient intracellular killing of bacteria by murine alveolar macrophages

Microbiologic methods were used to assess the in vitro phagocytosis and intracellular killing of various species of bacteria by freshly isolated murine peritoneal and alveolar macrophages. Peritoneal macrophages showed effective phagocytosis of opsonized Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Listeria monocytogenes, and moderate ingestion of Staphylococcus aureus and Escherichia coli. Alveolar macrophages were poor in phagocytosing opsonized S. pyogenes, S. aureus, and E. coli; ingestion of S. pneumoniae, P. aeruginosa, and S. epidermidis was moderate. Peritoneal macrophages killed 40 to 80% of these bacteria intracellularly, but alveolar macrophages showed almost no intracellular killing of bacteria. To find out whether there is a correlation between the poor bactericidal activity of alveolar macrophages and the oxygen-dependent microbicidal mechanisms of these cells, we determined the uptake of oxygen and the release of superoxide anion and hydrogen peroxide by macrophages at rest and after stimulation with phorbol myristate acetate (PMA) or opsonized S. aureus. Upon exposure to these stimuli, peritoneal macrophages, but not alveolar macrophages, showed an increased uptake of oxygen and release of superoxide anion and hydrogen peroxide. Because alveolar macrophages contain surface active material (SAM), we investigated the phagocytosis and intracellular killing of bacteria and the release of hydrogen peroxide by peritoneal macrophages pretreated with SAM. The results showed reduced phagocytosis and impaired intracellular killing of S. epidermidis by these macrophages. The release of hydrogen peroxide by SAM-pretreated peritoneal macrophages upon stimulation with PMA or opsonized S. aureus was equal to that of the control peritoneal macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in microbicidal activities of human macrophages against Toxoplasma gondii and Trypanosoma cruzi.

The microbicidal activities of normal human pelvic macrophages against infection by the intracellular protozoa Toxoplasma gondii and Trypanosoma cruzi were examined. Macrophages allowed infection by T. gondii and yet possessed remarkable microbicidal activity against this organism. In contrast, human macrophages were not microbicidal against T. cruzi.     

Gamma interferon-activated human macrophages and Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani: antimicrobial role of limiting intracellular iron.

Iron-saturated transferrin did not reverse the intracellular killing or inhibition of Toxoplasma gondii, Chlamydia psittaci, or Leishmania donovani by gamma interferon-activated human macrophages. Deferoxamine, an iron chelator, also did not impair replication within unstimulated macrophages. Limiting the availability of intracellular iron is an unlikely mechanism in human macrophage activity against these three diverse pathogens.     

Comparison of cytotoxic and microbicidal function of bronchoalveolar and peritoneal macrophages.

Studies were carried out with mice to explore in vitro the effector function(s) of macrophages from two different anatomical compartments (peritoneal cavity and lungs). The cytotoxic capacity of macrophages was measured by determining their cytostatic and cytocidal effects on EL-4 tumour target cells, and the microbicidal capacity of macrophages was measured by determining their ability to kill or inhibit the intracellular protozoan, Toxoplasma gondii. Neither peritoneal macrophages (PM) nor bronchoalveolar macrophages (BAM) from normal mice were ever microbicidal or cytotoxic. Intravenous treatment with Corynebacterium parvum greatly enhanced (activated) both effector functions of PM but did not activate BAM. Chronic infection with Toxoplasma activated PM throughout the period of observation (greater than 140 days), but the presence of activated BAM was transient and appeared to coincide with the occurrence of an inflammatory response in the lungs.     

Effect of hydrocortisone on macrophage response to lymphokine.

To define the suppressive effects of corticosteroids on mononuclear phagocyte antiprotozoal activity, normal resident peritoneal macrophages were exposed to hydrocortisone (HC) before, during, and after in vitro activation with cell-free supernatants (lymphokines). The presence of pharmacological concentrations of HC before or during lymphokine activation prevented normal macrophages from acquiring the capacity to either respond oxidatively to Toxoplasma gondii ingestion or to inhibit intracellular toxoplasma replication. HC had no effect, however, on the cells fully stimulated by lymphokine or on macrophages previously activated in vivo. These findings indicate that although HC does not impair the ability of activated macrophages to control intracellular protozoan infection, it does compromise the antimicrobial activity of the cell-mediated immune system by rendering normal macrophages unresponsive to lymphokine.     

Macrophage oxidative metabolism and intracellular Toxoplasma gondii

We explored the mechanisms by which Toxoplasma gondii avoids destruction by the oxidative metabolism of normal macrophages. Unelicited murine peritoneal macrophages were infected with T. gondii and exposed to different experimental conditions. As endpoints we used measurement of hydrogen peroxide (H2O2) release and intracellular reduction of nitroblue tetrazolium dye (NBT). Three main observations were made. Firstly, different T. gondii preparations (live or dead, opsonized or not) failed to trigger the respiratory burst. Combined challenges also showed that a primary T. gondii infection was able to block H2O2 release triggered by heat-killed (HK)-Candida albicans. The H2O2 release, however, once triggered by HK-C. albicans, was not inhibited by a subsequent challenge with T. gondii. Secondly, when a respiratory burst was obtained in T. gondii-infected macrophages--for instance by stimulation with phorbol myristate acetate (PMA)--the toxic oxygen metabolites (as determined by the NBT reduction test) did not seem to reach the vacuoles containing the parasite. Thirdly, when a respiratory burst occurred in T. gondii-infected macrophages, the intracellular development of T. gondii did not seem to be affected. In conclusion, we hypothesize that T. gondii is not damaged by the macrophage oxidative metabolism because the parasite fails to encounter toxic oxygen metabolites. The killing of intracellular T. gondii, as it is commonly observed in activated macrophages, does not appear oxygen-dependent.     

Inhibition of interferon-gamma-mediated activation in mouse macrophages treated with lipoarabinomannan.

Lipoarabinomannan (LAM), purified from the cell walls of Mycobacterium leprae and M. tuberculosis, is a potent inhibitor of interferon-gamma (IFN-gamma) mediated activation of macrophages. The capability of LAM to inhibit IFN-gamma activation of macrophages in vitro was dose dependent and required a 24-h pre-exposure. Defective activation was evident as a block in IFN-gamma-induced cytocidal activity for tumour cell targets and microbicidal capacity for intracellular Toxoplasma gondii. Additionally, LAM treatment blocked the induction of surface Ia antigens on peritoneal macrophages by IFN-gamma. The requirement for pretreatment with LAM was further substantiated by the finding that peritoneal macrophages that were activated in vivo were not affected by LAM treatments and retained full microbicidal function. However, once inhibited by LAM treatment in vitro, macrophages remained fully refractory to IFN-gamma activation for up to 5 days in culture. Inhibition of IFN-gamma activation in macrophages treated with LAM was not overcome by 100-fold increases in the dose of IFN-gamma used or by a constant dose of IFN-gamma in combination with 100-fold increases in the level of endotoxin used to trigger cytotoxic activity. The defect in IFN-gamma unresponsiveness was not due to altered receptor function, as control and LAM-treated macrophages showed similar capacity to bind, internalize, and digest radiolabelled IFN-gamma. Based on the in vitro findings reported here, the inhibition of IFN-gamma-mediated macrophage activation by exposure to LAM may contribute to defective macrophage function observed in lepromatous granulomas and thus constitutes an important aspect of pathogenesis in mycobacterioses.     

Specific antibody-dependent killing of Toxoplasma gondii by normal macrophages.

The requirement for specificity of antibody-dependent inhibition or killing of intracellular Toxoplasma gondii trophozoites by normal mouse peritoneal macrophages was evaluated in vitro using light microscopy and autoradiography. Anti-toxoplasma antibody in the presence of 'accessory factor' rendered extracellular T. gondii trophozoites non-viable and non-infectious for cells, whereas exposure of extracellular trophozoites to heat-inactivated immune serum did not appear to damage the parasites. Although pretreatment of extracellular trophozoites with heat-inactivated immune serum neither diminished nor prevented infection of normal mouse peritoneal macrophages, it did confer upon macrophages the ability to inhibit or kill the organisms once they were intracellular. In contrast, pretreatment of trophozoites with either heat-inactivated normal or Besnoitia jellisoni immune serum did not enable normal macrophages to inhibit or kill T. gondii; rather, such organisms multiplied intracellularly in normal macrophages. Thus, pretreatment with specific antibody alone prepared T. gondii trophozoites for intracellular destruction by normal mouse peritoneal macrophages. These results suggest that spesific antibody acting in concert with normal macrophages may play a role in controlling infection with T. gondii.     

Infection of murine macrophages with Toxoplasma gondii is associated with release of transforming growth factor beta and downregulation of expression of tumor necrosis factor receptors.

Toxoplasma gondii is capable of invading and multiplying within murine peritoneal macrophages. Previous studies have shown that treatment of macrophage monolayers with recombinant gamma interferon but not tumor necrosis factor (TNF) is associated with intracellular killing of T. gondii by macrophages. Furthermore, infection of macrophages with T. gondii prevents their stimulation for mycobactericidal activity by TNF. Since transforming growth factor beta (TGF-beta) is known to suppress a number of functions in macrophages, we investigated the influence of infection with T. gondii on macrophage TNF receptors and on production of TGF-beta. Infection with T. gondii was associated with increased production of TGF-beta and downregulation of TNF receptors. This effect was observed early after infection and was partially inhibited by anti-TGF-beta 1 antibody.     

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.     

Prevention of peroral and congenital acquisition of Toxoplasma gondii by antibody and activated macrophages.

Intramuscular administration of Toxoplasma gondii lysate antigens to mice produced titers of T. gondii-specific antibody (measured by Sabin-Feldman dye test) greater than or equal to 1:1,024 in their sera. Intravenous administration of heat-killed Propionibacterium acnes to mice produced peritoneal macrophages with enhanced microbicidal capacity against T. gondii. Mice with high antibody titers or activated peritoneal macrophages or both had reduced numbers of Toxoplasma cysts in their brains 30 days after peroral challenge. Specific antibody and activated macrophages appeared to act together to significantly (P = 0.01) reduce the numbers of Toxoplasma cysts. A reduction in tissue infection as a result of these treatments was also demonstrated by subinoculation of brain tissue. A high antibody titer alone did not protect against congenital infection. Mice treated with P. acnes delivered reduced numbers of T. gondii-infected pups (P greater than 0.05). Treatment that produced high titers of Toxoplasma antibody and activated macrophages provided significant protection against congenital infection (P less than 0.05).     

Role of reactive nitrogen and oxygen intermediates in gamma interferon-stimulated murine macrophage bactericidal activity against Burkholderia pseudomallei.

We examined the contributions of reactive nitrogen and oxygen intermediates (RNI and ROI, respectively) in macrophage bactericidal activity against Burkholderia pseudomallei, the causative agent of melioidosis, in order to understand host defense mechanisms against infection caused by this bacterium. The bacteria multiplied in unstimulated murine macrophage cell line J774.1. However, a strong dose-dependent inhibition of intracellular bacterial growth was observed when gamma interferon (IFN-gamma)-activated macrophages were used. The induction of bactericidal activity correlated well with the production of nitric oxide (NO) by IFN-gamma-activated macrophages and was markedly suppressed by N(G)-monomethyl L-arginine (L-NMMA), a competitive inhibitor of NO synthesis. Superoxide dismutase (SOD) and catalase significantly inhibited macrophage bactericidal activity, and the combined addition of L-NMMA, SOD, and catalase resulted in the complete inhibition of IFN-gamma-stimulated activity. The bacteria were susceptible to the killing effects of chemically generated NO and superoxide anion in a macrophage-free system. Our results indicate that IFN-gamma-induced macrophage bactericidal activity against B. pseudomallei is mediated to a large extent by RNI killing mechanisms and to a lesser extent by ROI-dependent mechanisms.     

A method to evaluate the capacity of monocytes and macrophages to inhibit multiplication of an intracellular pathogen.

A new method to assess intracellular inhibition of multiplication or killing by normal and activated mouse peritoneal macrophages, human peripheral blood monocytes, and human monocyte-derived macrophages is described. This method involves measurement of incorporation of [3H]uracil into nucleic acids of the obligate intracellular parasite Toxoplasma gondii. The method utilizes the observation by Pfefferkorn and Pfefferkorn (1977) that [3H]uracil is incorporated in substantially greater amounts by T. gondii than by certain mammalian cell types. Differential uptake of [3H]uracil by Toxoplasma-infected and uninfected cultures allows for evaluation of the ability of macrophages or monocytes to inhibit or kill this organism. This method has been adapted to microsystem.     

A Role for Activated Macrophages in Resistance to Infection with Toxoplasma

Activated macrophages from mice which were chronically infected with Toxoplasma gondii or Besnoitia jellisoni, or which had received Freund complete adjuvant, had an enhanced capacity to to kill intracellular Toxoplasma. Enhanced killing by activated macrophages was demonstrated by decreased incorporation of isotopically labeled uridine by intracellular Toxoplasma and by inhibition of plaque formation. The latter resulted from lack of proliferation of the intracellular Toxoplasma which is normally followed by destruction of the host cell (macrophage) and secondary invasion and destruction of fibroblast monolayers.     

Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 10(4) to 10(7) bacteria in C3H/HeN and 10(1) to 10(4) bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.