Peritoneal lavage cells of Indonesian thin-tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica

Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.     

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.     

Monocyte and macrophage functions in M-CSF-deficient op/op mice during experimental leishmaniasis

Mice with a naturally occurring Csfm(op)/Csfm(op) (op/op) gene mutation lack functional macrophage-colony stimulating factor (M-CSF) and are deficient of M-CSF-derived macrophages. They are severely monocytopenic, and their remaining M-CSF-independent macrophages were shown to differ in differentiation and distinct functions when compared with phenotypically normal mice of the same background. It is not known if osteopetrosis mice (op/op mice) are able to mount a specific immune response against intracellular pathogens, as this would require complex effector functions by macrophages. We therefore investigated the ability of op/op mice and their M-CSF-independent macrophages to combat infection with Leishmania major. op/op mice retained the ability to resist an infection with L. major by mounting a T helper cell type 1 cell response, eliminating parasites and resolving the lesions. Macrophages from op/op mice were able to sufficiently perform effector functions in vitro, such as phagocytosis, production of leishmanicidal nitric oxide (NO), killing of parasites, and release of interleukin (IL)-12. There were quantitative differences, as M-CSF-derived macrophages from hematopoietic organs of control mice showed significantly higher rates of phagocytosis and higher NO release after stimulation with lipopolysaccharides than corresponding macrophages from op/op mice. In contrast, when peritoneally elicited macrophages were used, those from op/op mice revealed a stronger response than those from control mice with regard to release of NO or IL-12. These differences suggest that M-CSF-independent maturation of op/op monocytes subsequent to their release from hematopoietic tissue exerts influence on their effector functions. However, M-CSF or M-CSF-derived macrophages are not necessary for an effective immune response against L. major.     

The role of antibody and complement in the cellular response to Trypanosoma congolense.

The in vitro cytotoxic response of bovine granulocytes and monocytes and of murine peritoneal macrophages against Trypanosoma congolense in the presence of antibody, antibody plus complement or complement alone was assessed using luminol aided chemiluminescence as a second parameter for effector cell activation. Neither cell type exhibited any trypanolysis exceeding that of antibodies and complement alone. The kinetics of the chemiluminescence response in the course of these reactions closely correlated with the trypanocidal activity of the antibody preparation used, suggesting effector cell activation as a response to antibody-mediated immobilization and damage of the trypanosomes. From these results and electron microscopic investigations we conclude that antibody- or complement-dependent cell-mediated cytotoxic reactions do not play a significant role in the defence of T. congolense infection, neither by extracellular lysis nor killing of ingested parasites.     

Killing of Giardia muris trophozoites in vitro by spleen, mesenteric lymph node and peritoneal cells from susceptible and resistant mice.

The ability of spleen, mesenteric lymph node (MLN) and peritoneal exudate (PEC) cells from susceptible (A/J) and resistant (B10.A) mice to kill trophozoites in vitro was determined. Both duration of incubation and cell density influenced giardicidal activity. Maximal killing was observed after 6 hr of incubation at the effector to target ratios of 30:1 and 50:1. Cells isolated from A/J and B10.A mice during the elimination phase of the infection killed more trophozoites than those isolated from mice during the acute phase of the infection. Cells isolated from mucosal sites (MLN) of donors infected for 15 days killed more trophozoites in vitro than those isolated from systemic sites (spleen, PEC). There were no differences in the giardicidal activity of cells from susceptible and resistant mice. Killing of trophozoites was mediated by plastic-adherent cells with macrophage properties. Non-specific stimulation with thioglycollate and the presence of immune mouse serum enhanced the capacity of macrophages to kill parasites. There was no apparent relationship between the capacity of A/J and B10.A mice to mount cell-mediated effector responses and their ability to control the infection with Giardia muris.     

Developmental differences determine larval susceptibility to nitric oxide-mediated killing in a murine model of vaccination against Schistosoma mansoni.

A persistent paradox in our understanding of protective immunity against Schistosoma mansoni infection in animals vaccinated with attenuated parasites has been that attrition of challenge parasites occurs during migration through the lungs in vivo, although parasites recovered from the lungs appear to be relatively resistant to cytotoxic effector mechanisms in vitro. We have compared the susceptibilities of different stages of larvae to killing by nitric oxide (NO), which was previously shown to be involved in the larvicidal function of cytokine-activated cytotoxic effector cells. Lung-stage larvae obtained 1 week after infection were not killed in vitro by NO generated either by a chemical NO donor or by activated cells. In contrast, parasites obtained from the portal system of control mice or from the lungs of vaccinated mice 2.5 weeks following challenge infection were killed by NO. As previously shown for mammalian cell targets, the effects of NO in susceptible larval stages may involve enzymes required for aerobic energy metabolism, since similar cytotoxicity was demonstrated by chemical inhibitors of the citric acid cycle or mitochondrial respiration. Taken together with previous observations of enhanced Th1 activity and expression of NO synthase in the lungs of vaccinated mice at 2.5 weeks after challenge infection, these observations elucidate the immune mechanism of vaccine-induced resistance to S. mansoni infection. Moreover, they suggest that conversion to a less metabolically active state may allow pathogens to escape the effects of the important effector molecule NO.     

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.     

Host-parasite interactions with peritoneal macrophages of mice and rats in vitro and in vivo.

This paper deals with the intracellular multiplication of mycobacteria in peritoneal macrophages from mice and rats immunized with tubercle bacilli or pretreated with Triton WR 1339. If unstimulated macrophages were used, almost unrestricted multiplication of mycobacteria was observed in macrophages from both vaccinated and pretreated hosts after infection of the cells in vitro. Only when the infection of the cells was perfored in the peritoneal cavity of vaccinated hosts did the macrophages display a high degree of inhibition. This striking difference in the behavior of macrophages infected in vitro and in vivo is explained by the local inflammation caused by the intraperitoneal infection, which leads to an influx of T-cell mediators. When macrophages from hosts pretreated with Triton WR 1339 were used, inhibition of the multiplication of mycobacteria within cells infected in vitro or in vivo was very slight, though this compound displayed a marked protective effect in the host. Addition of streptomycin to the culture medium caused a strong inhibition of intracellular mycobacteria even in small concentrations; there was no difference between normal and "immune" macrophages. When rats were infected with virulent tubercle bacilli, they were initially fully susceptible to the infection but showed rapid onset of a strong immune response.     

ANALYSIS OF ANTI-TRANSPLANTATION IMMUNITY OF TUMOR

In allogeneic immunity against tumors induced by methylcholanthrene in the W/KA strain rat, serum antibody agglutinated red blood cells of the W/KA strain rat. The allogeneic antiserum was cytotoxic to the lymphocyte, but not to the tumor cells induced by methylcholanthrene.
In vitro and in vivo studies of allogeneic immunization of tumor suggested that immune macrophages or histiocytes play a major role in the rejection of transplanted tumor. Direct adhesion of immune macrophages with target cells is necessary for destruction of allografted tumor.
In antitransplantation immunity of the syngeneic and autochthonous rat, there was no cytotoxic serum antibody demonstrable in so far as routine techniques were employed. However, this may not exclude the existence of specific serum antibody or non-cytotoxic reaction with target cells of specific serum antibody. On the other hand, immune macrophages were always cytotoxic to target tumor cells, thus showing tumor specifity.
Experiment in which diffusion chambers were inserted into syngeneic immune rats suggested the possible participation of a humoral factor which was released from the immune rats immediately after contact with target cells and transform normal macrophages to immunologically active macrophages. The ascites macrophages have the greatest effective cytotoxic action among the three types of cells. In allogeneic tumor immunity, one immune macrophage can damage one target tumor cell showing single hit interaction. ACTA PATH. JAP. 18: 207–225, 1968.     

Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species

LMPK, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is essential for the proliferation of the amastigote, the mammalian stage of the protozoan parasite. This has been demonstrated using deletion mutant promastigotes, the insect stage of the parasite: first, in vitro after differentiation to amastigotes, which subsequently lost their potential to proliferate; second, by infection of peritoneal macrophages, which were able to cope with the infection and cleared the parasites; third, by infection of BALB/c mice, which showed no lesion development. The lmpk deletion mutant promastigotes are a potential live vaccine because they infect macrophages, transform to amastigotes and deliver amastigote antigens to raise an immune response without causing the disease. In addition, inhibition of LMPK in a wild-type infection is likely to resolve the disease and as such, is an ideal target for drug development against leishmaniasis. Here we investigated the presence and copy number of lmpk homologues in Leishmania amazonensis, L. major, L. tropica, L. aethiopica, L. donovani, L. infantum, and L. braziliensis and discuss the results with regard to drug development and vaccination using kinase deletion mutants.     

The natural killer system in the rat: the relationship between natural and immune cell-mediated lysis of fibroblast targets.

Natural killer (NK) cell cytotoxic activity is thought to operate as an early defence system before the expression of specific cytotoxic immune effectors (Leibold & Peter, 1978). Apart from this temporal relationship it is likely that other mechanisms exist to regulate these two forms of host defence and their relationship to one another. Present studies firstly substantiate evidence that normal and immune effector cells represent different cell types even though they share many functional characteristics in vitro. Normal effector cells (NEC) cytotoxic for normal dermal-derived fibroblasts in vitro were present in rats 3-36 weeks old, were tissue-dependent (with low levels in lymph nodes and thymus, unrelated to cellular suppression), were nylon wool and glass non-adherent and sensitive to 400 rad of X-irradiation in vitro. Secondly, discrimination between normal and immune cell-mediated cytotoxicity by factor(s) present in normal rat serum (Lindsay & Allardyce, 1981) allowed various aspects of their in vitro and in vivo relationship to be examined. These studies suggested that the expression of NEC cytotoxicity was temporally related to the in vivo generation of immune cytotoxic effector cells.     

Role of antibody and complement in the control of Encephalitozoon cuniculi infections by rabbit macrophages.

The capacity of mononuclear peritoneal macrophages to phagocytose Encephalitozoon cuniculi was tested in vitro. Normal rabbit serum or cell culture medium had little effect on the rate of removal of organisms by rabbit peritoneal macrophages. Treatment with immune rabbit serum or immune rabbit immunoglobulin G significantly (P less than 0.001) increased phagocytosis of E. cuniculi. Guinea pig complement was found to significantly (P less than 0.001) enhance the phagocytosis of antibody-treated E. cuniculi. With few exceptions, induced (peritoneal exudate) macrophages were no more effective than unstimulated (resident) macrophages in the phagocytosis of E. cuniculi. Secondary lysosomes labeled with ferritin were seen fusing with phagosomes containing immune rabbit serum-treated parasites. Phagosome-lysosome fusion was not observed when parasites were treated with either normal rabbit serum or culture medium. The results of the present study suggest a role for antibody enhancement of phagocytosis and intracellular killing as a mechanism of resistance to encephalitozoonosis in rabbits.     

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 micrograms endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.     

Macrophage requirement for in vitro generation of specific, secondary cell-mediated cytotoxicity against SV 40-induced tumor-associated antigens in mice

The role of adherent phagocytic cells in an in vitro secondary cytotoxic response against Simian virus 40 (SV 40)-induced tumor-associated antigens was investigated. Spleen cells (responder cells), from mice primed with syngeneic SV 40-transformed cells, extensively depleted of macrophages by filtration through a Sephadex G-10 column followed by iron carbonyl treatment, had a markedly decreased capacity to generate in vitro secondary cytotoxic reactivity against syngeneic SV 40-transformed cells when cultured with the relevant stimulator cells. The secondary response was restored by the addition of adherent peritoneal cells from normal mice syngeneic to those immunized with the antigen. Within a certain dose range, small numbers of peritoneal cells completely reconstituted the response, whereas large numbers inhibited the reactivity. The restored cultures maintained specific cytotoxic reactivity against SV 40-induced tumor-associated antigens which was mediated by effector T cells as shown by sensitivity to anti-Thy-1.2 antiserum and complement. These results suggested a requirement for adherent phagocytic cells (accessory cells) in in vitro generation of a secondary, cytotoxic response to tumor-associated antigens.     

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.     

Macrophages direct tumour histology and clinical outcome in a colon cancer model

Macrophages generally constitute a major component of the tumour stroma. Although conventionally considered to be cytotoxic effector cells, macrophages have recently been described as promoters of tumour progression. The present study shows that selective depletion of peritoneal or liver macrophages prior to CC531 tumour cell inoculation resulted in highly differentiated tumours in rats. In contrast, tumours from control rats, in which macrophages are abundantly present, showed a desmoplastic stromal reaction with hallmark features of malignancy, such as neovascularization and matrix remodelling, indicating that the presence of macrophages is associated with malignant histopathological differentiation. Remarkably, macrophage-depleted rats, bearing highly differentiated tumours, had a worse prognosis, as they displayed a higher tumour load and poorer survival. Thus, while macrophages direct tumours towards malignant tumour histology, their role in anti-tumour defence is important. The selective inhibition of macrophage functions involved in malignant progression without interfering with cytotoxic ability may therefore identify important new targets for cancer therapy.     

Avian cellular immune effector mechanisms--a review

Considerable new knowledge has accumulated within the last few years on the immune functions of birds, and data show that the avian cellular immune effector mechanisms are quite similar to those of mammals. Cellular immunity in the chicken, the most widely studied avian species, is primarily mediated by T cells, macrophages and NK cells. The avian T lymphocytes express unique surface antigens that distinguish these cells from other cells. The T cells respond vigorously in vitro to T cell-specific mitogens and can perform cytotoxic and mixed lymphocyte reactions against allogeneic or tumour cell targets. The in vivo responses mediated by T cells include allograft and tumour cell rejection, graft-versus-host reaction and delayed type hypersensitivity reactions. T cells also serve as helper cells in enabling B Cells to produce antibody against thymus-dependent antigens. Avian T cells also perform immune regulatory functions and sub-populations of T suppressor cells can cause profound blockage of antibody production by B cells. Certain T cell functions require cooperation from other immune cells, particularly major histo-compatibility complex-compatible macrophages. Avian macrophages resemble their mammalian counterparts in being adherent and phagocytic, and they serve as accessory cells in a multitude of immune reactions as well as effector cells in their own right. Macrophages when co-cultivated in vitro with tumour target cells may non-specifically arrest proliferation of tumour cells or lyse tumour cells. They may also engage in specific anti-alloantigen cytotoxicity following hyperimmunisation. Under certain circumstances, macrophages can also be potent suppressors of immune reactivity. Birds appear to have a well developed NK cell system. NK cells of chickens share general characteristics with mammalian NK cells and lack the surface markers of T cells, B cells, or macrophages. Recent evidence indicates that the NK cell system plays a role in defence against Marek's disease. NK-like cells have also been shown to mediate ADCC in chickens and ducks. The immunoregulatory factors that may mediate avian immune effector cell functions are currently being actively studied.     

Interleukin-10 Inhibits Antimicrobial Activity Against Leishmania major in Murine Macrophages

The stimulation of macrophages is of importance to the defense against intracellularly replicating microorganisms such as Leishmania. In this study the direct effect of recombinant interleukin-10 (IL-10) on the leishmanicidal effector functions of murine peritoneal or bone marrow derived macrophuges was investigated. IL-10 almost completely inhibited the killing of intracellular leishmania at concentrations above 10ng/ml. This inhibitory effect was independent of the stimulus used as the activation of macrophages by IFN-γ and IL-7, recently shown to possess macrophage activating properties, were suppressed by IL-10. Kinetic experiments revealed that IL-10 must be present during the process of macrophage activation and that the leishmanicidal effector function of fully activated macrophages was not influenced. Furthermore, in the absence of exogenously added IL-10, the addition of neutralizing antibodies against IL-10 or IL-10-specific antisense phosphorothioate DNA-oligonucleotide led to an enhanced killing of parasites after stimulation with either IFN-7 or IL-7. In accordance with this, IL-10 mRNA was readily detectable in murine macrophages by PCR with reverse transcribed mRNA. These results indicate that IL-10, which is endogenously produced by macrophages, acts as an autocrine deactivating factor supporting the survival of the parasite.     

IgE antibody and resistance to infection. II. Effect of IgE suppression on the early and late skin reaction and resistance of rats to Schistosoma mansoni infection

Most helminth parasites induce a strong IgE antibody and elevated eosinophil response in their mammalian hosts and a number of in vitro studies have suggested that IgE, possibly in association with eosinophils, may be an essential element of the host protective immunity against helminth infections. To assess the role of IgE in protective immunity, we examined the effect of suppressing the IgE antibody response on rat immunity to Schistosoma mansoni. Suppression was achieved in neonates by injections of rabbit anti-epsilon chain gamma-globulins, control rats received injections of unspecific gamma-globulins. IgE suppression caused a marked reduction of the inflammatory reaction that developed in the skin of immune rats at the site of a cercarial challenge: the early (30 to 60 min) wheal and flare reaction was abolished, and the late cutaneous reaction (6 to 18 h) associated with intense pruritus, edema and local eosinophilia was greatly reduced. This shows that IgE was critical to the recruitment of effector cells and molecules in the skin during the first 24 h following parasite invasion. Worms were recovered 18 to 30 days after a primary infection and 18 days after a challenge infection from IgE-suppressed and control rats. IgE-suppressed rats cured a first infection as rapidly as the control rats; however, they were two to three times less efficient than the controls at eliminating a second or a third challenge. These observations demonstrate that IgE antibodies are essential for the full development of rat acquired protective immunity against Schistosoma mansoni.