Effect of normal and activated human macrophages on Toxoplasma gondii.

Human macrophages derived from in vitro culture of peripheral blood monocytes were studied under a variety of conditions to determine their microbicidal capacity for the obligate intracellular protozoan, Toxoplasma gondii. The effect of macrophages on intracellular Toxoplasma was evaluated morphologically by light and phase microscopy and by autoradiography. When macrophages from dye test (DT)-negative or DT-positive individuals were infected with Toxoplasma in the presence of normal human serum, the organisms were able to multiply intracellularly with resultant destruction of the monolayer. Once organisms were intracellular, the presence of antibody-containing serum in the medium did not alter this inability of the macrophages to kill Toxoplasma. However, when Toxoplasma were incubated in the presence of heat-inactivated DT-positive serum just before infection of the monolayers, the intracellular organisms were inhibited or killed by normal macrophages. Attempts were made to activate macrophages in vitro to kill Toxoplasma. Macrophages incubated in the presence of sensitized lymphocytes and Streptokinase-Streptodornase (SK-SD) or Toxoplasma lysate antigen (TLA) were found to kill Toxoplasma when compared to macrophages incubated in the presence of lymphocytes from DT-negative individuals and TLA or lymphocytes alone. Thus, in vitro induction of resistance (both specifically and nonspecifically) in human macrophages was accomplished by culturing these cells in the presence of specifically sensitized lymphocytes and antigen. These results suggest that, as in the mouse model, activated human macrophages have the ability to inhibit or kill intracellular Toxoplasma and that these cells may be important as effector cells in cell-mediated immunity (CMI) to toxoplasmosis in man.

Human lymphokine-activated killer cells are cytotoxic against cells infected with Toxoplasma gondii.

Experiments were conducted to determine whether human lymphokine-activated killer (LAK) cells are cytotoxic against cells infected with Toxoplasma gondii. Nylon wool nonadherent (NWNA) peripheral blood lymphocytes, as well as purified natural killer cell (NK) (CD3- CD16+ CD56+) and T (CD3+ CD16- CD56-) cells obtained from five healthy T. gondii seronegative volunteers exhibited minimal cytotoxic activity against T. gondii-infected cells. When standard LAK (S-LAK) cell preparations were induced by incubation of NWNA cells with recombinant interleukin 2, induction of remarkable cytotoxic activity against T. gondii-infected cells. When standard in LAK cell preparations from each of the volunteers. The phenotype of the LAK precursor and effector cells varied depending on the target cell used. Whereas the precursor and the effector cells of most of the LAK activity against K562 and Daudi cells were cells with NK phenotype, when T. gondii-infected cells were used as targets, both cells with NK and T cell phenotypes were precursors and effectors of the lysis. When cytotoxic activity of S-LAK cells was compared with the activity of adherent LAK (A-LAK) cells, A-LAK cells displayed higher cytotoxic activity against T. gondii-infected cells, as well as against K562 and Daudi cells. Cold target inhibition experiments suggested that there is a subset of LAK effector cells capable of lysing both T. gondii-infected cells and Daudi cells, whereas other subsets preferentially or exclusively lyse one of these target cells.

Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens.

As previously reported, normal human monocytes (11) and activated mouse macrophages (9) are able to kill or inhibit intracellular replication of Toxoplasma that are not antibody coated, whereas normal human and mouse macrophages are not (7, 9). Each of these types of mononuclear phagocytes is able to kill antibody-coated Toxoplasma. In our studies, phagocytosis of antibody-coated Toxoplasma stimulated the respiratory burst by each of these types of mononuclear phagocytes, whereas phagocytosis of organisms that were not antibody coated stimulated the respiratory burst only by human monocytes and by activated mouse macrophages. Phagocytosis of Toxoplasma did not inhibit production of reactive oxygen metabolites by normal macrophages; rather, it failed to stimulate their production. Killing of Toxoplasma by monocytes from a child with X-linked chronic granulomatous disease and his heterozygote mother was impaired. Thus, reactive oxygen metabolites, perhaps in conjunction with lysosomal contents, appear to be first-line mechanisms whereby mononuclear phagocytes kill this organism. We were not able to determine the exact mechanisms whereby mononuclear phagocytes inhibit the replication of those Toxoplasma that were not killed, although both oxygen-dependent and other nonlysosomal mechanisms may be involved. The differences we observed in oxidative response to phagocytosis of Toxoplasma appear to be one determinant of the antimicrobial activity of these cells and may account for the ability of some intracellular pathogens to survive within phagocytes. These differences may be membrane related. Further studies of Toxoplasma membranes, phagocyte membrane receptors for Toxoplasma, and membrane-related mechanisms for activation of the respiratory burst are needed to define their true basis.

Oxygen-independent killing by alveolar macrophages.

We have found that normal alveolar macrophages can kill an intracellular parasite by a mechanism that does not involve toxic metabolites of oxygen. We studied the interaction between Toxoplasma gondii and rat alveolar macrophages in vitro. We were interested in Toxoplasma because it causes pneumonia in immunosuppressed patients but not in healthy individuals, and we chose the rat because it resembles immunocompetent human subjects in being resistant to T. gondii. Resident rat alveolar macrophages could kill large numbers of T. gondii. This occurred without a respiratory burst as judged by intracellular reduction of nitroblue tetrazolium and quantitative release of superoxide. Furthermore, scavengers of toxic oxygen metabolites had no effect on the toxoplasmacidal activity of the alveolar macrophages, nor did prior exhaustion of their respiratory burst with PMA. Whereas acid pH (e.g., 4.5-6.0) rapidly kills extracellular T. gondii, raising of the intralysosomal acid pH of rat alveolar macrophages by incubating them with weak bases did not inhibit their ability to kill T. gondii. Killing of Toxoplasma occurred within 1 h of initial exposure to the alveolar macrophages. However, there was no evidence that killing preceded ingestion; Toxoplasma attached to the surface of the cell appeared viable, and when phagocytosis was blocked with sodium fluoride the organisms survived. These results indicate that rat alveolar macrophages possess a powerful nonoxidative microbicidal mechanism, which is distinct from acidification of the phagolysosome but which probably involves phagosome formation. This mechanism may be clinically relevant, for we have recently observed that human alveolar macrophages also kill T. gondii by an oxygen-independent process.

Association of CD4+ T cell-dependent, interferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.

Since there is a remarkable difference in susceptibility to peroral infection with Toxoplasma gondii among inbred strains of mice, we performed studies to examine the mechanism(s) of this difference in susceptibility. After peroral infection with the ME49 strain of T. gondii, C57BL/6 (B6) mice all died whereas BALB/c mice all survived. At day 7 of infection (when B6 mice began dying), massive necrosis of the villi and mucosal cells in the ilea were observed in B6 but not in BALB/c mice. To analyze the role of T cells in resistance against death and development of necrosis in the ilea after infection, studies were performed using athymic nude and euthymic control B6 and BALB/c mice. Athymic B6 mice all died after infection, but surprisingly, they survived significantly longer than control B6 mice, indicating that T cells predispose to early death in these mice. Necrosis in the ilea was observed in control B6 but not in athymic B6 mice; however, significantly less numbers of tachyzoites were observed in the ilea of the former than the latter mice. These results indicate that necrosis in the ilea of the B6 mice was not due to destruction of tissue by tachyzoites but was mediated by T cells. This deleterious effect of T cells appears to contribute to early death in these mice. In contrast, T cells conferred resistance against death in BALB/c mice but did not cause necrosis in their ilea. To analyze the T cell subset(s) that induces necrosis of the ilea in B6 mice, we examined histological changes of the small intestines after infection of mutant mice deficient in different T cell subsets (with the same H-2b haplotype as B6 mice). Mice deficient in alpha/beta or CD4+ T cells did not develop necrosis in the ilea, whereas wild-type control mice and mice deficient in gamma/delta or CD8+ T cells did, suggesting that the cells that induce necrosis in the ilea after infection are CD4+ alpha/beta T cells. Since interferon (IFN)-gamma has been shown to be critical for survival of BALB/c mice after infection with T. gondii, we examined the role of this cytokine in resistance/susceptibility of infected B6 mice. Treatment of B6 mice with anti-IFN-gamma monoclonal antibody shortly before they developed illness prolonged time to death and prevented necrosis in the ilea in these mice. These results indicate that IFN-gamma mediates necrosis in the ilea of B6 mice after infection. This CD4+ T cell-dependent, IFN-gamma-mediated necrosis of the small intestines appears to be a mechanism that underlies the genetic susceptibility of B6 mice to peroral infection with T. gondii, whereas the same cytokine plays a critical role in the resistance of genetically resistant BALB/c mice.

Co-infection of macrophages modulates interferon gamma and tumor necrosis factor-induced activation against intracellular pathogens.

Co-infection of macrophages (M phi) with Toxoplasma gondii and Mycobacterium avium-intracellulare complex (MAC) has been observed in patients with acquired immunodeficiency syndrome (AIDS). In this study we have demonstrated that co-infected murine M phi respond differently to cytokine stimulation than M phi infected with either of the microorganisms alone. Whereas treatment with interferon gamma (IFN-gamma) activated both single and co-infected groups of M phi to kill T. gondii, treatment with TNF did not influence the rate of MAC growth in co-infected M phi, in contrast with the inhibition of growth observed in MAC-infected M phi. These results suggest that in AIDS patients suffering infection with multiple intracellular pathogens, the ability of cytokines to stimulate microbicidal or static activity in mononuclear phagocytes can be impaired by the presence of more than one of the intracellular organisms.

Polymorphisms in the tumor necrosis factor alpha (TNF-alpha) gene correlate with murine resistance to development of toxoplasmic encephalitis and with levels of TNF-alpha mRNA in infected brain tissue.

Murine resistance to development of toxoplasmic encephalitis (TE) has recently been mapped to the D region of the major histocompatibility complex (H-2). Since the gene for tumor necrosis factor alpha (TNF-alpha) is located 5' of the D region and TNF-alpha has been implicated as playing a role in neurological diseases, we were interested in determining the relationship of TNF-alpha production to TE resistance. We have demonstrated that resistance to TE in inbred mice can be correlated with specific restriction fragment length polymorphisms and microsatellite variants in the TNF-alpha gene. Mice that are susceptible to TE express elevated levels of TNF-alpha mRNA in brain tissue 6 wk after infection with the ME49 strain of Toxoplasma gondii. Resistant mice and all mice that are uninfected show no detectable TNF-alpha mRNA expression in brain tissue. Differences in the TNF-alpha gene between susceptible and resistant mice have been localized to the first intron, the promoter, and the 3' end of the TNF-alpha gene. These data implicate differences in regulation of TNF-alpha production in brain tissue as contributing to differences in susceptibility to development of TE.

Immunity and intracellular infection: resistance to bacteria in mice infected with a protozoan.

Mice infected with the intracellular parasite Toxoplasma gondii for periods of as long as 7 months were resistant to challenge with numbers of Listeria monocytogenes and Salmonella typhimurium that were uniformly lethal to normal mice. This resistance did not appear to depend on the strain of toxoplasma employed or the route of inoculation of either toxoplasma or bacteria. Onset of immunity to listeria was demonstrable as early as 1 to 2 days after infection with toxoplasma. Resistance to toxoplasma was not demonstrable in mice immune to listeria. Interferon did not appear to be a mediator of the immunity observed in toxoplasma-infected mice.

Interferon: protection of cells infected with an intracellular protozoan (Toxoplasma gondii).

Chick and mouse cell monolayers treated with interferon were significantly protected from destruction by toxoplasma. The results suggest that fewer numbers of organisms were released from interferon-treated cells. Interferons were uniformly more active in a viral assay than in a toxoplasma assay, and they had the same properties in both assays. The data suggest that interferon activity may be directed against organisms phylogenetically higher than viruses.

Control of carcinogenesis: a possible role for the activated macrophage.

Cytotoxic activity of activated mouse macrophages against mouse embryo fibroblasts was tested before and after spontaneous transformation of the fibroblasts in vitro. Activated macrophages caused little or no destruction of untransformed fibroblasts but were markedly cytotoxic to the same fibroblasts after spontaneous transformation. The efferent limb of this cytotoxic reaction appears to be nonimmunologic and to be related to abnormal growth properties rather than to the antigenic composition of target cells.

Interferon-gamma: the major mediator of resistance against Toxoplasma gondii.

Mice were injected with a monoclonal antibody to interferon-gamma to examine the importance of endogenous production of this lymphokine in resistance against infection with the sporozoan parasite Toxoplasma gondii. Mice with intraperitoneal infections of T. gondii that received no antibody survived and developed chronic T. gondii infection, whereas the infected mice that received the monoclonal antibody died of toxoplasmosis. The activation of macrophages, which kill T. gondii in vivo, was inhibited by administration of the monoclonal antibody, but the production of antibodies to T. gondii was not suppressed. The fact that an antibody to interferon-gamma can eliminate resistance to acute Toxoplasma infection in mice suggests that this lymphokine is an important mediator of host resistance to this parasite.

Phagocytic and bacterial properties of normal human monocytes.

The bactericidal and phagocytic capacities of monocytes for E. coli, Staphylococcus, Salmonella, and Listeria, and factors that influence these functions were evaluated and compared with those of the polymorphonuclear leukocytes of 30 normal human subjects. Monocytes killed a significantly smaller proportion of each of the bacterial species than did neutrophils from the same individuals. Whereas the neutrophils of all individuals demonstrated the ability to kill significant numbers of the four bacterial species, there was a marked variation in the effect of monocytes of different individuals on the growth curves of these same bacteria. When the bactericidal capacity of an individual's monocytes to more than one species of bacteria was examined in the same experiment, a significant difference in the effect of monocytes on the growth curve of one bacterial species as opposed to another was noted in 4 of 17 subjects. The bactericidal ability of monocytes of single individuals was consistent on different days in 9 of the 11 subjects whose monocytes were examined more than once against the same bacteria. Studies were performed to determine if the lesser bactericidal capability of monocytes was due to a difference in the ability of monocytes and neutrophils to phagocytize or to a difference in the ability of these cells to kill ingested bacteria or both. The results demonstrated that monocytes phagocytize bacteria significantly less well than neutrophils, but the intracellular killing capacity of both cell types is equal. Addition of phenylbutazone to cell suspensions completely inhibited intracellular killing by both monocytes and neutrophils, suggesting the possibility that the bactericidal mechanisms in both cell types might be similar. Monocyte killing of E. coli, Salmonella, and Listeria, but not of Staphylococcus, was significantly diminished in heat-inactivated autologous serum. Neither increasing the concentration of autologous serum from 10% to 25% nor replacement of autologous serum with pooled human serum had any effect on monocyte killing of any of the four bacteria. These studies demonstrate that peripheral blood monocytes are less bactericidal for the four bacterial species than neutrophils, solely because monocytes are less phagocytic. A baseline for further study of factors that influence monocyte function and for study of this cell in selected patient populations is provided.

Preferential activation and expansion of human peripheral blood gamma delta T cells in response to Toxoplasma gondii in vitro and their cytokine production and cytotoxic activity against T. gondii-infected cells.

Studies were conducted to determine if gamma delta T cells participate in the immune response to Toxoplasma gondii. Preferential expansion of human gamma delta T cells occurred when peripheral blood T cells from either T. gondii-seronegative or seropositive individuals were incubated with autologous PBMC infected with the parasite. That gamma delta T cells proliferated after incubation with infected cells was confirmed using purified of gamma delta T cells. These T. gondii-induced gamma delta T cell responses did not require prior exposure to the parasite since T cells obtained from umbilical cord blood from seronegative newborns also exhibited preferential expansion of gamma delta T cells. Cytofluorometric analysis of T cells obtained from either umbilical cord blood or peripheral blood from adults revealed that V gamma 9+ and V delta 2+ gamma delta T cells responded to stimulation with infected cells. Preferential expansion of gamma delta T cells was not restricted by polymorphic determinants of MHC molecules. PBMC that had internalized killed parasites but not PBMC incubated with T. gondii lysate antigens also stimulated preferential expansion and activation of gamma delta T cells as assessed by expression of CD25 and HLA-DR molecules. V gamma 9+V delta 2+ gamma delta T cells were cytotoxic for T. gondii-infected cells in an MHC-unrestricted manner, and produced IFN-gamma, IL-2, TNF-alpha, but not IL-4 when incubated with cells infected with the parasite. These results suggest that rapid induction of a remarkable primary gamma delta T cell response may be important in the early protective immune response to T. gondii.

Immunity to Toxoplasma gondii.

Recent advances in our understanding of the immunological mechanisms involved during infection with Toxoplasma gondii include evidence for the role of different subsets of lymphocytes and cytokines in acute infection as well as in reactivation of chronic infection. The mechanisms of presentation of T. gondii antigen have been clarified recently, and animal models of toxoplasmosis that mimic disease observed in AIDS patients developed.

Inhibition of mouse natural killer cell activity by zinc.

The effect of zinc on mouse natural killer (NK) cell activity was evaluated. The inhibition of NK cell activity with zinc was dependent on the concentration of zinc added (range tested: 0-40 micrograms zinc/ml) and occurred at both effector-to-target ratios tested. Zinc-induced inhibition of NK activity was observed with the use of peritoneal or splenic effector cells on Toxoplasma gondii-augmented NK activity. Maximal inhibition of activity was noted when zinc was present for the entire assay period. Inhibition was present but less marked with pretreatment of effector cells with zinc. Pretreatment of target cells with zinc had no measurable effect on NK cytotoxicity. Effector-to-target cell binding as measured by single-cell assays was not significantly altered by zinc. These results indicate that zinc is a potent inhibitor of NK activity.

Inhibition of target cell mitosis as a measure of the cytostatic effects of activated macrophages on tumor target cells.

Studies were carried out in vitro that showed that activated macrophages inhibited tumor target cells from undergoing mitosis. With the use of target cells synchronized in the mitosis phase of the cell cycle, this manifestation of macrophage-mediated cytostatic effects was preceded by the complete inhibition of target cell DNA synthesis as measured by incorporation of [3H]thymidine.

A role for the macrophage in in vivo and in vitro resistance to murine bladder tumor cell growth.

Studies were performed to determine the role of macrophages in inhibition of growth of the same tumor cell type both in vitro and in vivo. Bladder transitional epithelium tumor cells were injected s.c. into Toxoplasma-infected mice, previously shown to contain activated macrophages, and into uninfected controls. The subsequent growth of a solid tumor was significantly less in the Toxoplasma-infected animals. Bladder tumor cells from the same cell line were grown in vitro either alone or in the presence of peritoneal lymphocytes and/or macrophages from oxoplasma-infected and control, uninfected mice. [3H]Thymidine incorporation by the tumor cells was inhibited only in the presence of macrophages from the Toxoplasma-infected animals. Lymphocytes alone did not appear to be cytotoxic under the conditions used. Moreover, lymphocytes from Toxoplasma-infected mice did not convey cytotoxicity to macrophages from control animals under the experimental conditions used.

Tissue culture isolation of Toxoplasma from blood of a patient with AIDS.

A man with acquired immunodeficiency syndrome and multiple opportunistic infections developed central nervous system signs, and results of computed tomography suggested brain abscess. Histologic study and culture of material obtained from the brain biopsy specimen revealed Aspergillus. The clot from a peripheral venous blood sample obtained at the time of biopsy was processed and cultured on a monolayer of L cells. Five days later, Toxoplasma tachyzoites were identified in the cell culture. We bring this case to the attention of the medical community to point out that inoculation of tissue cell cultures as for viral isolation may aid in the diagnosis of Toxoplasma infection in these and other immunocompromised patients.

Tularemia presenting as community-acquired pneumonia. Implications in the era of managed care.

A case of pleuropulmonary tularemia was diagnosed by sputum culture and serologic studies in a patient who did not have classic epidemiological risks for tularemia. The patient had atypical pneumonia when initially seen and his condition slowly improved with antibiotic therapy that included erythromycin lactobionate. The diagnosis of tularemia was delayed because the gram-negative rod isolated from the patient's sputum was initially not speciated in an effort to reduce laboratory costs.

Intravenous sulfamethoxazole and trimethoprim for serious gram-negative bacillary infection.

Intravenous therapy with sulfamethoxazole and trimethoprim cured seven patients with serious gram-negative infection. Three patients had bacteremia, three had pneumonia, and one each had meningitis, peritonitis, pyogenic liver abscesses, and urinary tract infection. Sulfamethoxazole and trimethoprim was selected in three patients with renal failure to avoid aminoglycoside-induced nephrotoxicity, in three patients because of penicillin allergy, and in two cases because of bacterial resistance to other readily available antibiotics. Adverse drug reactions occurred in three cases and included oral monilia, transient leukopenia, and fluid overload. In contrast to the new broad-spectrum cephalosporin antibiotics, sulfamethoxazole and trimethoprim costs two to 2 1/2 times less and has not been associated with the emergence of bacterial resistance during therapy. This may favor the use of parenteral sulfamethoxazole and trimethoprim for some patients with serious gram-negative infection.