Stephanie L. James

Role of T‐Cell Derived Cytokines in the Downregulation of Immune Responses in Parasitic and Retroviral Infection

Parasitic infection is frequently accompanied by a downregulation in host cell-mediated immunity. Recent studies suggest that this modulation of helper T cells and effector cell function can at least in part be attributed to the action of a set of inhibitory cytokines produced by T lymphocytes as well as by a number of other cell types. The best characterized of these inhibitory lymphokines are IL-4, IL-10 and TGF-beta. Interestingly, both IL-4 and IL-10 are produced by the Th2 but not the Th1 subset of CD4+ helper cells. The former subset dominates in many situations of chronic or exacerbated parasitic infection and is thought to suppress Th1 function as a consequence of the cross-regulatory activity of these two cytokines. The latter hypothesis is supported by recent experiments demonstrating that mAb-mediated neutralization of IL-10 reverses suppressed IFN-gamma responses and/or disease susceptibility in mice with parasitic infections. In vivo neutralization of TGF-beta has also been reported to increase host resistance to parasite challenge. In addition to suppressing T-cell differentiation, function or proliferation, IL-4, IL-10 and TGF-beta each inhibit the ability of IFN-gamma to activate macrophages for killing of both intracellular and extracellular parasites. Moreover, the three cytokines are able to synergize with each other in downregulating these parasiticidal effects. Interestingly, each of the cytokines inhibits the production of reactive nitrogen oxides, an effector mechanism previously demonstrated to play a major role in parasite killing by activated macrophages. In the case of IL-10, this suppression of nitrogen oxide production appears to result from an inhibition of TNF-alpha synthesis leading to defective macrophage stimulation. While distant from parasites in their biology and phylogeny, some retroviruses also appear to induce an over-production in downregulatory cytokines which is closely associated with the onset of immunodeficiency. Thus, in an animal model involving infection of mice with LP-BM5 MuLV and in human HIV infection, Th2 (IL-10 and/or IL-4) cytokine synthesis is increased while Th1 (IFN-gamma and/or IL-2) cytokine production is suppressed. These observations suggest that cytokine-mediated cross-regulation may play a role in the pathogenesis of acquired immune deficiency disease, contributing both to the progression of retroviral infection and the increase in susceptibility to opportunistic infections and malignancy. Observations of similar cytokine cross-regulatory activities in organisms as diverse as helminths, protozoa and retroviruses predict that comparable mechanisms may operate in a wide variety of infectious diseases.

NO as an affector molecule of parasite killing: modulation of its synthesis by cytokines

It has recently been appreciated that NO, a molecule previously known to play a physiologic role in blood pressure regulation, is a major effector molecule of macrophage cytotoxicity against a variety of microbial targets, including protozoan and helminth parasites. NO production by macrophages is arginine dependent and catalyzed by a cytokine-inducible form of the NO synthase. This activity is positively controlled by several up-regulatory stimuli (including IFN-gamma, TNF-alpha, IL-2) and negatively controlled by others (principally IL-10, IL-4, TGF-beta). Other cell types, such as endothelial cells and hepatocytes, display a similar capacity for NO production in response to cytokine stimulation. In murine models of leishmaniasis and schistosomiasis, in vivo NO synthesis correlates with protective immunity against infection. The effector molecule that plays a similar role in cell-mediated immunity in man has not yet been identified.

The role of nitrogen oxides as effector molecules of parasite killing

The many years of effort in the attempt to identify the effector molecules of lymphokineactivated macrophages have recently come to fruition with the discovery of immuneactive nitrogen oxide. Here, Stephanie James and John Hibbs discuss the novel mammalian biochemical pathways for the synthesis of inorganic nitrogen oxides that mediate antibody-independent killing of infectious agents as well as of tumor cells. Interaction with nitrogen oxides causes iron loss from critical target enzymes in invading organisms, resulting in metabolic failure of extracellular and even of intracellular parasites of the macrophages themselves.

Schistosome vaccines: current progress and future prospects

Vaccination against human schistosomes in laboratory hosts is now a reality. A number of different parasite molecules have been shown to confer partial protective immunity against challenge infection with Schistosoma mansoni or Schistosoma japonicum in rodent or primate hosts. These antigens are unusually diverse in their structure and stage specificity. Interestingly, although all of the vaccine molecules characterized are situated in the tegument, their exposure on the parasite surface, in most instances, is transient and/or non-essential. The properties of four of these immunogens, glutathione-S-transferase (P26,28), paramyosin (Sm97), GP38, and GP18 are discussed. Despite the identification and recombinant synthesis of several promising protective antigens, vaccination of humans against schistosomiasis remains in the realm of fantasy. At the technical level, a major problem is the failure of any of the current vaccine immunogens and immunization protocols to induce levels of resistance sufficient for significant reduction of human infection or disease. Once this important hurdle is passed, human immunization trials should be attempted as the potential beneficial impact of a vaccine against schistosomiasis remains enormous.

Effector functions of activated macrophages against parasites.

Evidence in experimental animals indicates a major role for cytokine-activated macrophages as effector cells in protective immunity against parasites. Research on cytokine function during this past year has contributed many insights into the immune mechanisms regulating murine macrophage function as well as the effector molecules employed by these cells to kill both intracellular and extracellular parasites.

Cell-mediated immune response to schistosomiasis.

Schistosomiasis is caused by infection with blood flukes of the genus Schistosoma, of which three species (S. mansoni, S. hematobium, and S.japonicum) are the principal causes of disease in man. Schistosomiasis is a major health problem in the developing world, with an estimated 200 million persons infected and 600 million living in endemic areas where they are at risk of infection of (BERGQUIST 1987). This helminth parasite is acquired by man or other mammalian hosts through contact with a larval form found in fresh water. Having penetrated the skin, the larvae migrate and mature into adult worms living in the veins of the intestinal or urinary tract. Paired male and female worms produce hundreds to thousands of eggs per day, which are either excreted or become trapped in the body tissues of the host. Schistosomiasis is primarily a chronic disease, the pathology of which is associated with reaction to these trapped eggs. However the estimated mortality rate of approximately 1% (BERGQUIST 1987) translates into hundreds of thousands of lives lost annually.

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae III. Identification of a mouse strain, P/N, that fails to respond to vaccination

Summary Eleven strains of inbred mice were examined for their ability to develop resistance to challenge Schistosoma mansoni infection as a result of previous exposure to homologous cercariae that had been attenuated by high‐dose irradiation. Two strains, C57B1/6J and BALB/c, demonstrated consistently high levels of vaccine‐induced immunity (means of 64% and 58% resistance, respectively, when compared to control groups of the same strain) and were designated as ‘high responder’ strains to vaccination. Six other strains fell into an intermediate category, demonstrating moderate, yet statistically significant, levels of immunity resulting from vaccination (means of 30–50% resistance). Only one of the strains examined consistently failed to respond to vaccination by the development of significant levels of immunity to challenge infection. Animals of the P/N strain demonstrated a mean of only 15% resistance to challenge in five experiments and have been classified as ‘low responders’ to vaccination. P/N mice have previously been characterized as deficient in their ability to mount delayed hypersensitivity reactions, produce lymphokine and display macrophage activation for cytolysis of extracellular and intracellular targets in other experimental systems, suggesting that these immune responses may be critical to the establishment of vaccine‐induced resistance to S. mansoni infection. The availability of high and low responder mouse strains should facilitate a genetic approach to characterization of the immune effector mechanism(s) of vaccine‐induced resistance to S. mansoni infection.

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells☆

Abstract Peritoneal macrophages from Schistosoma mansoni -infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro . In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro . However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ( 1 3 to 1 5 those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni -infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni -activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.

Post lung-stage schistosomula of Schistosoma mansoni exhibit transient susceptibility to macrophage-mediated cytotoxicity in vitro that may relate to late phase killing in vivo

Summary Studies of protective immunity against Schistosoma mansoni in immunized mice suggest that a proportion of challenge parasites may be eliminated after they have passed through the lungs of the host several days after infection; however, no potential immune effector mechanism of resistance against this stage of the parasite has yet been identified, since schistosomes have been shown to rapidly become resistant to antibody–dependent killing mechanisms. In this study, different development stages of S. mansoni were examined for their susceptibility to in vitro cytotoxicity by lymphokine–activated macrophages. As previously shown, newly transformed larvae were readily killed by lymphokine–treated peritoneal macrophages or the macrophage cell line IC‐21 (80 % mortality over 48 h in vitro), whereas 7 and 10 day old lung–stage parasites had become refractory to macrophage effects. However, after 2 to 2 1/2 weeks of development in vivo, juvenile parasites recovered from the liver were again susceptible to activated macrophage‐mediated cytotoxicity (25–65 % mortality). Ultrastructural studies of 2 1/2 week old parasites co–cultured with activated IC‐21 cells revealed that damage was largely restricted to the areas beneath the parasite surface and gut syncitia; surface membrane disruption was not evident. This late stage of suceptibility was transient and by 4 to 6 weeks liver–stage worms had again become refractory to macrophage killing. The interaction of post lung–stage parasites with activated macrophages was antibody independent. Furthermore, schistosomes isolated from the portal circulation 2 1/2 weeks after infection showed no evidence of surface‐bound immunoglobulin in a quantitative immunofluorescence assay, nor did antisera from chronically infected mice (CIS) or mice vaccinated with irradiated cercariae (VS) react with the surface of these parasites in vitro, making the possibility of direct antibody–dependent killing mechanisms unlikely. However, both CIS and VS did recognize excretory/secretory proteins synthesized by 21/2 week old liver‐stage schistosomes, including a major antigen of approximate Mr (x 10‐‐3) 220 (220K). It is therefore possible that such antigens might participate in protective immunity, for example via immune complex formation or activation of sensitized T cells. These observations support the role of macrophages as immune effector cells in mice immunized against Schistosoma mansoni, and provide the first physiologically relevant mechanism whereby the immune system might recognize and kill post‐lung stage schistosomes.

Ultrastructural studies of the killing of schistosomula of Schistosoma mansoni by activated macrophages in vitro

Summary Immunologically activated murine macrophages have been shown elsewhere to kill skin stage schistosomula of Schistosoma mansoni in vitro, in a manner analogous to the extracellular killing of tumour cell targets. In this study, the kinetics of the interaction between activated macrophages and larval targets and the resultant ultrastructural changes in parasite morphology that culminated in death have been analysed in detail. Unlike granulocyte‐mediated schistosomular killing, macrophage‐mediated cytotoxicity did not appear to be directed against the surface tissues of the parasite. Macrophages adhered only transiently following initiation of the cultures, yet changes in the subtegumental mitochondria and muscle cells of the larva were detected within the first hour of incubation. Progressive internal disorganisation followed rapidly, but the tegument and tegumental outer membrane remained intact, to form a ‘shell’ that maintained the general shape of the parasite. Such changes were recognised irrespective of whether the effector cell population comprised peritoneal macrophages activated by lymphokine treatment in vitro, or by infection with Mycobacterium bovis (strain BCG), or S. mansoni in vivo. That macrophages rather than contaminating granulocytes or lymphocytes, had mediated the observed damage was demonstrated by the use of a lymphokine treated macrophage cell line, IC‐21. The observation that macrophage cytotoxicity is directed against internal organelles rather than the tegumental outer membrane of this multicellular target, may help to elucidate the general mechanism of extracellular killing by these cells.