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).     

Effect of alveolar macrophages on Toxoplasma gondii.

As pulmonary involvement can occur in disseminated toxoplasmosis in immunosuppressed patients, studies were initiated to define local mechanisms of resistance of the lung to Toxoplasma gondii. Alveolar macrophages were obtained from normal mice and mice chronically infected with T. gondii by bronchopulmonary lavage and cultured in vitro. Although normal alveolar macrophages were difficult to infect with Toxoplasma, they supported intracellular multiplication of this organism. When exposed to Toxoplasma that had been pretreated with heat-inactivated serum containing specific antibody, the number of intracellular organisms increased remarkably, and the macrophages destroyed the coated parasites. After development of chronic infections with Toxoplasma, there was a transient period during which a striking increase in numbers of alveolar macrophages was observed in lavage specimens. These macrophages differed from those of normal alveolar macrophages. There was a greater percentage of large cells, a greater tendency to spread on glass, and an increased number of intracellular Toxoplasma, and the cells were activated to kill or inhibit multiplication of the parasite. During the period when activated macrophages were demonstrable in bronchopulmonary washings, histological changes in the lungs revealed a marked mononuclear cell infiltrate. These studies support a role for the activated alveolar macrophage as an effector in resistance of the lung to infection with Toxoplasma.     

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.     

Studies of macrophage function during Trichinella spiralis infection in mice.

Studies were made to investigate the quantitative and functional changes which occur in peritoneal macrophage populations obtained from mice infected orally with Trichinella spiralis larvae. C57BL/6 mice infected with T. spiralis larvae became parasitized with adult worms which were rejected from the intestine from 14 to 20 days after infection. Infected mice developed a striking increase in peritoneal exudate cells, composed largely of macrophages, which was maximal at from 16 to 18 days after infection. T. spiralis larvae and eosinophils were not seen in the peritoneal exudates. Macrophages from mice infected more than 11 days earlier inhibited DNA synthesis of syngeneic and allogeneic tumour cells, a property atributed to activated macrophages. In addition, macrophages from T. spiralis-infected mice had the functional ability to kill EL-4 tumour cells as measured by 51Cr release. Unlike activated macrophages, however, macrophages from infected mice did not develop the ability to inhibit multiplication of the intracellular pathogen Toxoplasma gondii. These studies demonstrate that T. spiralis infection in mice induces changes in macrophage function that differ from changes associated with infections by intracellular pathogens.     

Coumarin or warfarin treatment of mice does not increase the microbicidal or tumoricidal capacities of macrophages.

Benzopyrones have been shown to affect several functions of macrophages. We examined the effects of two benzopyrones, coumarin and warfarin, on the capacity of mouse macrophages to inhibit microorganisms and tumour target cells. Mice were treated with daily i.v. doses of either drug. Then the mice were challenged with lethal doses of Toxoplasma gondii or peritoneal macrophages from these mice were challenged in vitro with T. gondii or tumour target cells Survival of coumarin or warfarin-treated mice challenged with T. gondii was similar to that of control mice. Multiplication of T gondii and growth of tumour target cells were similar in preparations of macrophages from coumarin-treated, warfarin-treated, or control mice and were inhibited in preparations of activated macrophages from Corynebacterium parvum-treated mice that served as positive controls. Under our experimental conditions, benzopyrones did not activate mouse macrophages.     

Lymphokine activation of J774G8 cells and mouse peritoneal macrophages challenged with Toxoplasma gondii.

In vitro activation of macrophage cell line J774G8 and mouse peritoneal macrophages resulted in oxygen-dependent and oxygen-independent killing of intracellular Toxoplasma gondii. Activation was characterized by oxygen-dependent killing detectable by enhanced lysosome fusion and digestion of T. gondii. The toxoplasmacidal activity of activated J774G8 cells and peritoneal macrophages was prevented by adding the oxygen intermediate scavengers catalase or superoxide dismutase during culture. Activated J774G8 cells and peritoneal macrophages also inhibited replication of those Toxoplasma organisms which survived the initial microbicidal activity. The inhibition of Toxoplasma replication was not significantly affected by exogenous catalase or superoxide dismutase. Peritoneal macrophages from Toxoplasma-immune mice showed similar microbicidal and inhibitory responses, supporting the model that activation leads to destruction of intracellular parasites by two different mechanisms.     

Protection by a cyclic AMP-specific phosphodiesterase inhibitor, rolipram, and dibutyryl cyclic AMP against Propionibacterium acnes and lipopolysaccharide-induced mouse hepatitis

OBJECTIVE AND DESIGN: To study the effect of cellular cAMP-increasing agents on Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS)-induced mouse hepatitis. MATERIAL: Male BALB/c mice were used. Macrophages/Kupffer cells isolated from P. acnes-primed murine liver were used for the in vitro study. TREATMENT: Type IV phosphodiesterase (PDE)-specific inhibitor, rolipram, was administered (10, 30 mg/kg, p. o.). Dibutyryl cyclic AMP (dbcAMP) was injected (10, 100 mg/kg, i.p.) into the mice. METHOD: Plasma TNFalpha estimated by the use of an L-929 cell cytotoxic assay and plasma transaminase activities were measured for the in vivo study. The LPS-induced production of TNFalpha in vitro from the cultured macrophage/Kupffer cells was determined by ELISA. RESULTS: Rolipram suppressed the elevation of plasma transaminases induced by injection of LPS, and dbcAMP had a tendency to suppress them. Both agents attenuated the LPS-induced release of TNFalpha in vivo, and suppressed the TNFalpha production from the cultured macrophage/Kupffer cells. CONCLUSIONS: These results suggest that rolipram and dbcAMP have potential to inhibit TNFalpha production from activated macrophage/Kupffer cells, and it may be partially involved in the protecting effect in the P. acnes/LPS hepatitis model.     

Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated with Corynebacterium parvum.

In vivo and in vitro experiments were conducted to determine the effect of Corynebacterium parvum treatment of mice on resistance of Listeria monocytogenes and Toxoplasma gondii. Intravenous immunization with C. parvum conferred transient protection against intravenous challenge with Listeria or an avirulent strain of Toxoplasma but did not protect against a virulent strain of Toxoplasma. Compared with the level of protection conferred by C. parvum, a higher degree of resistance was noted when mice infected with Listeria or Toxoplasma were challenged with the homologous infecting organism. Peritoneal macrophages from mice immunized intravenously with C. parvum were activated to kill Toxoplasma in vitro. Whereas resistance to challenge in vivo was transient, this population of activated macrophages persisted. Peritoneal macrophages from C. parvum mice also markedly inhibited [3H]thymidine uptake by L cells.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

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.     

Toxoplasmacidal activity of macrophages activated by recombinant major surface antigen (P30) of Toxoplasma gondii.

Recombinant major surface antigen (P30), which was produced as a glutathione S-transferase (EC 2.5.1.18) fusion protein of Toxoplasma gondii, was found to be able to activate macrophages to kill T. gondii in vitro. The macrophage activation was due to P30 in the fusion protein, not to glutathione S-transferase.     

Role for Activated Macrophages in Resistance Against Trichinella spiralis

To determine whether activated macrophages are important in resistance against the intestinal phase of nematode parasites, we studied Trichinella spiralis infections in mice with normal macrophages and in mice with macrophages activated by either chronic Toxoplasma gondii or acute Listeria monocytogenes infections. The peak T. spiralis adult worm burden in the intestines of normal C57BL/6 or Swiss Webster mice occurred from 6 to 14 days after infection. Subsequent expulsion of worms from the intestines occurred from 8 to 20 days after infection. C57BL/6 mice chronically infected with T. gondii and then challenged with T. spiralis larvae had significantly lower peak intestinal worm burdens (P < 0.05) than normal C57BL/6 mice similarly challenged. Swiss Webster mice infected 7 or 13 days earlier with L. monocytogenes and then challenged with T. spiralis larvae had significantly lower peak worm burdens (P < 0.01) than uninfected mice. The time of expulsion of adult worms was not affected by either infection. Swiss Webster mice infected 42 days earlier with L. monocytogenes (i.e., possessing lymphocytes sensitized to L. monocytogenes but not possessing activated macrophages) did not have a lower worm burden than uninfected mice. Serum factors (e.g., antibody) did not appear to play a role because normal mice injected with serum from L. monocytogenes-infected mice had worm burdens similar to those of mice injected with normal serum. The histopathology of intestines of mice infected with T. gondii or L. monocytogenes was the same as that of normal mice. When T. spiralis larvae were incubated with normal macrophages or macrophages from T. spiralis-infected mice in vitro for 24 h, the number of larvae with adherent T. spiralis macrophages was significantly (P < 0.005) greater than the number of larvae with adherent normal macrophages. These studies suggest a role for activated macrophages in resistance to T. spiralis.     

Role of beta interferon in resistance to Toxoplasma gondii infection.

The role of recombinant murine beta interferon (rMuIFN-beta) and recombinant human IFN-beta (rHuIFN-beta) in resistance to Toxoplasma gondii was examined. rMuIFN-beta protected mice against a lethal infection with the parasite. The protective effect appeared to depend on the concomitant release of gamma interferon. rMuIFN-beta did not activate murine peritoneal macrophages to inhibit or kill T. gondii whether used alone or in combination with lipopolysaccharide (LPS). rHuIFN-beta did not activate human monocyte-derived macrophages to inhibit or kill T. gondii when 5-day-old monocyte-derived macrophages were used. In contrast, significant killing of T. gondii was noted when 10-day-old monocyte-derived macrophages were used. The addition of LPS enhanced this effect. These results revealed a role for IFN-beta in the mechanisms of defense against T. gondii and suggest its potential use in the treatment of toxoplasmosis in humans.     

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.     

Differential activation of resident macrophage subsets with two sources of lymphokine preparations

Resident murine macrophages were separated into subsets by Percoll density gradient centrifugation before treatment with lipopolysaccharide-stimulated lymphocytes or different lymphokine preparations. The lymphokines used were culture supernatants from lymphocytes obtained from lipopolysaccharide-injected mice or from purified protein derivative-treated lymphocytes from mice bearing an active BCG infection. The macrophage subsets were activated by the stimulated lymphocytes or lymphokine preparations to express C3b receptor-mediated ingestion or to inhibit the intracellular replication of Toxoplasma gondii or both. The results showed that the macrophage subsets were heterogeneous with respect to ingestion and T. gondii inhibition when activated with lipopolysaccharide-stimulated lymphocytes or lipopolysaccharide-derived lymphokines but were all homogeneous when activated with lymphokines from purified protein derivative-stimulated lymphocytes. When the macrophage subsets were allowed to remain in vitro for various times before lymphokine treatment, the relative pattern of subset activation changed when treated with lipopolysaccharide-derived lymphokines. In contrast, the macrophage subsets remained equally activated throughout the in vitro period when treated with the lymphokines from purified protein derivative-stimulated lymphocytes. The results suggested that functional macrophage heterogeneity depends not only on the nature of the activating signal but also on a state of receptivity of that signal by the macrophage population.     

gamma delta T cells play a crucial role in the expression of 65,000 MW heat-shock protein in mice immunized with Toxoplasma antigen.

Toxoplasma gondii is an obligate intracellular protozoan parasite and cellular immunity plays a crucial role in protection against infection with this pathogen. When mice are immunized with Toxoplasma homogenate, they readily acquire resistance against infection with a lethal dose of a low virulence Beverley strain of T. gondii. We have reported previously that expression of 65,000 MW heat-shock protein (hsp 65) in host macrophages closely correlates with protective potentials of hosts, while this protein is not expressed in Toxoplasma themselves. In this study, we examined the mechanism of expression of hsp 65 in mice immunized with Toxoplasma homogenate. Heat-shock protein was detected in peritoneal macrophages of BALB/c mice immunized 7 days previously by electroblot assay with a specific monoclonal antibody (mAb) for microbial hsp 65. Furthermore, an immunogold ultracytochemistry assay demonstrated that this protein was expressed on the cell surface of peritoneal macrophages in immune mice. This expression was not induced in those of immune athymic nude mice and SCID mice. Treatment of BALB/c mice with anti-Thy-1.2 mAb 1 day before immunization led to an almost complete loss of the expression of hsp 65. To determine the subsets of T cells responsible for induction of this protein, mice were depleted of gamma delta T cells, alpha beta T cells, CD4+ T cells or CD8+ T cells by treating with corresponding antibodies before immunization. From these experiments, gamma delta T cells were shown to be essential for the expression of hsp 65, although CD4+ alpha beta T cells also contributed to some extent. Thus, gamma delta T cells appear to play an important role in protective immunity against infection with T. gondii through mediating the expression of hsp 65 in host macrophages.     

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.     

Effects of Corynebacterium parvum treatment and Toxoplasma gondii infection on macrophage-mediated cytostasis of tumour target cells.

Injection of mice with Corynebacterium parvum or living or killed Toxoplasma gondii was studied to determine the efficacy of these treatments in activating peritoneal macrophages to inhibit the uptake of [3H]TdR (cytostasis) by tumour target cells in vitro. In the presence of activated macrophages from mice treated i.p. with a wide dose range of either C. parvum or living Toxoplasma, cytostasis was usually greater than 99%. This population of activated macrophages was transient in C. parvum-treated mice, but persists, probably for life, in Toxoplasma-infected mice. Whereas the i.p. route of administration of C. parvum was more efficient in activating macrophages than the i.v. route, the s.c. route appeared to be relatively ineffective. Treatment with killed Toxoplasma by any route was also relatively ineffective in activating macrophages. In contrast Toxoplasma infection resulted in highly activated peritoneal macrophages, regardless of the route of administration. Depending upon the route of initial treatment, the route of readministration of C. parvum had either no appreciable effect or resulted in a marked alteration in the cytostatic capacity of peritoneal macrophages.     

Interferon gamma fails to activate human monocyte-derived macrophages to kill or inhibit the replication of a non-pathogenic mycobacterial species.

The ability of interferon gamma (gamma) to activate human macrophages to kill mycobacteria was investigated using a mycobacterial species that does not cause disease in man. Although interferon activated human macrophage activity against other intracellular parasites, Toxoplasma gondii and Listeria monocytogenes, it failed to activate human monocyte-derived macrophages to kill not only Mycobacterium tuberculosis but also the non-pathogenic species, Mycobacterium phlei.     

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.