Morphological studies on the killing of schistosomula of Schistosoma mansoni by human eosinophil and neutrophil cationic proteins in vitro

Purified eosinophil and neutrophil cationic proteins isolated from the lysosomal secretion granules of human granulocytes, evoke characteristic, dose-dependent morphological changes in young schistosomula of S. mansoni. The first sign of damage is seen within 15–30 min of incubation and involves the formation of surface microvilli and blebs. Subsequently, tegumental evaginations of varying size are developed, but these appear to explode with rapidity, so that lengths of expanded tegumental outer membrane are deposited over the severely damaged surface of the parasite. Both types of granulocyte proteins are able to effect comparable damage at equimolar concentration. Other cationic proteins such as protamine and poly-L-arginine also damage the parasite surface but the pathological changes differ from those induced by the granulocyte proteins and they take longer to develop. In contrast, lysozyme-treated parasites are virtually similar to control schistosomula incubated in medium alone. These findings are discussed in relation to published data concerning the interaction of intact granulocytes with young schistosomula both in vitro and in viva.     

Cytotoxic effects in vitro of human monocytes and macrophages on schistosomula of Schistosoma mansoni

Human peripheral blood monocytes from normal donors were isolated by differential centrifugation and cultured in vitro in hydrophobic Teflon-coated tissue culture bags. Cells were harvested between 0 and 10 days and tested for their ability to kill schistosomula of Schistosoma mansoni in an in-vitro cytotoxicity assay. Freshly isolated, unstimulated monocytes demonstrated minimal cytotoxic capability. However, this was increased if the cells were pretreated with human recombinant gamma interferon (IFN-gamma), or with specific anti-S. mansoni antiserum. As the monocytes matured in vitro there were marked increases in the levels of antibody-independent killing of schistosomula. Monocytes grown in vitro with IFN-gamma (10(4) u/ml) took 2-3 days to develop almost maximal cytotoxicity (mean 94% kill of schistosomula). In contrast, unstimulated monocytes (no IFN-gamma) took between 5 and 7 days to achieve comparable cytotoxicity (mean 99% kill). Killing of the schistosomula was dependent upon a high effector to target ratio, and was a relatively slow phenomenon in vitro, parasite attrition occurring between 17 and 36 h. Supernatants from cytotoxic macrophages were ineffective in mediating cytotoxicity of the parasite.     

Neutrophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni in vitro: studies on the kinetics of complement and/or antibody-dependent adherence and killing

The capacity of rat peritoneal neutrophils to adhere to and kill schistosomula of Schistosoma mansoni in vitro has been investigated. Neutrophils adhere readily to schistosomula in the presence of antibody plus complement (C) (fresh immune rat serum), antibody alone (heat-inactivated immune rat serum) and C alone (fresh normal rat serum), but not with heat-inactivated normal rat serum. However, schistosomular killing is only achieved with neutrophils and fIRS or MRS. In the presence of hiIRS the cells detach after 6 h without producing a significant level of parasite death. The system involving neutrophils plus fIRS is the most efficient in terms of serum dilution and the rate of schistosomular killing. The complement-dependent antibody involved in this system belongs to the class IgG and occurs in rat serum at peak titres, 6–8 wk after a primary schistosome infection. Neutrophil adherence in the presence of MRS depends upon the generation of C3b molecules at the parasite surface via the alternative pathway of C activation. Studies on the antibody alone system indicate that the lack of significant schistosomular killing might result from the absence of factors which stimulate cell migration, since if a chemokinetic agent is introduced into the assay a 30% increase in mortality is recorded. The possible participation of neutrophils in the destruction of a primary and/or challenge infection in vivo is discussed.     

On the interaction between macrophages and developmental stages of Schistosoma mansoni: effect of muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) treatment on mice survival and the generation of schistosomulicidal macrophages

Schistosomiasis is a chronic disease afflicting hundreds of millions of people throughout the world against which there is as yet no effective vaccine. In the present study we tested the effect of the immunomodulator muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) on the survival of Schistosoma mansoni-infected mice and on the induction in them of schistosomulicidal macrophages. Mice exposed to 80 cercariae each and then treated with MTP-PE showed prolonged survival following either single or repeat infection. The treatment with MTP-PE, when initiated 70 days post the schistosome infection, diminished significantly the mortality of infected mice over an observed period of 110 days. In terms of treatment efficacy there was no evident difference between the intravenous and intraperitoneal mode of administration of the drug. MTP-PE treatment significantly reduced granuloma size and markedly diminished liver damaged as judged by the lower levels of alkaline phosphatase in the serum. Such treatment exerted no significant effect on the spleen or liver weight in infected mice nor on the worm burden resulting from either a single or double infection. In infected and non-treated mice, schistosomulicidal macrophages appeared after 8-10 weeks of infection. In infected mice treated with MTP-PE there was an accelerated appearance of such macrophages and these exhibited a greater cidal effect on the schistosomula. These immunostimulatory and life-prolonging effects of MTP-PE on S. mansoni-infected mice might indicate an effect of this reagent on cells involved in the granulomatous process.     

Factors affecting the levels of antibody- and complement-dependent eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro

In experiments designed to test why high levels of antibody-dependent, eosinophil-mediated killing of schistosomula are routinely observed in this laboratory, several factors that may contribute to variations in eosinophil activity were examined. The most important factors were: (1) the source of eosinophils, with marked variation being demonstrated not only, as previously shown, between individuals, but also between different cell preparations from a single individual; (2) the serum used as a source of anti-schistosomulum antibodies and (3) the age of the schistosomula at the time of assay. In contrast, addition of fresh normal serum as a source of complement had a relatively slight effect when the killing assay was carried out in round bottomed tubes. A more marked enhancement was observed in flat bottomed microtitre plates, and it is suggested that this enhancement may be attributable to the release of chemotactic complement components. No difference was observed between a laboratory maintained and a recently derived isolate of Schistosoma mansoni, either in initial susceptibility or in loss of susceptibility after 3.5 h of culture. In contrast to the marked effects of eosinophils under most conditions tested, there was no evidence for extensive neutrophil-mediated damage under the same conditions.     

Temporal variation in the carbohydrate and peptide surface epitopes in antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Changes in the surface antigenicity and susceptibility to eosinophil-dependent killing during in vitro development of schistosomula of Schistosoma mansoni, were examined using sera from rabbits and mice immunized with antigens that are shed from the schistosomulum in vitro (shed antigen), a carbohydrate extract of shed antigen (SAg/CHO) or a periodate-insensitive fraction of shed antigen (SAg/PEP). Anti-SAg/CHO antisera recognised mainly carbohydrate epitopes on the parasite surface, whilst anti-SAg/PEP antisera bound to periodate-insensitive, putative peptide, surface epitopes. Anti-SAg/PEP antibodies failed to recognise the surface of newly transformed schistosomula unless the parasite was first treated with sodium periodate, suggesting that these epitopes may be masked by periodate sensitive (i.e., carbohydrate) epitopes. There was an increase in anti-SAg/PEP antibody binding to the larval surface with age of the parasite in vitro; five-day-old lung schistosomula were also recognised by anti-SAg/PEP antisera. In contrast, anti-SAg/CHO antibody binding declined with parasite age, and failed totally to recognise lung schistosomula. This change in epitope expression was reflected in eosinophil-dependent cytotoxicity assays, with anti-SAg/CHO antisera killing young larvae and anti-SAg/PEP antisera only killing older larvae. Lung worms were not killed by either antisera. The difference in epitopes recognised by the antisera was also reflected in the antigens identified by immunoprecipitation and SDS-PAGE.     

Proteolytic cleavage of IgG bound to the Fc receptor of Schistosoma mansoni schistosomula

After the binding of IgG to the surface Fc receptor of Schistosoma mansoni schistosomula, the Fab portions of IgG are cleaved and small peptides are liberated in the culture medium. At least two types of proteinase activities have been demonstrated in the secretory products of schistosomula. One is an endoprotease with trypsin-like activity, with an optimum pH of 7 and an optimum temperature of 45°C. The other is a metalloaminopeptidase with an optimum pH of 7 and temperature of 37°C.     

Enhancement of eosinophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni by autologous mononuclear cells from patients

Adherent mononuclear cells (monolayer), when co-cultured with autologous peripheral blood eosinophils isolated from patients treated for Schistosoma mansoni infections, enhanced the eosinophil-mediated killing of antibody coated schistosomula. The monolayer increased the activity of the eosinophils by 225%, and was observed in 80% of the patients studied. Heat labile factors other than complement, present in immune serum, further enhanced the ability of eosinophils to kill schistosomula in the presence of the monolayer. On their own the adherent cells did not mediate obvious damage to the parasite. Eosinophils that had been pre-incubated with the monolayer (100 mins) and tested separately, killed equal numbers of schistosomula as in the co-culture assay; this excludes the possibility of concurrent schistosomula cytotoxicity by the two cell populations. The ability of the monolayer to activate eosinophils was not altered by inhibitors of protein synthesis. The monolayer was largely consistent of monocytes as demonstrated by an over 96% positive staining for non-specific esterases.     

日本血吸虫与曼氏血吸虫的致病差异

曼氏血吸虫和日本血吸虫是全球范围内两种主要的肠道血吸虫病病原体,所致基本病理均为虫卵引起的肝脏肉芽肿和纤维化,但两者在产卵方式以及肉芽肿的组成细胞等方面均存在明显差异。 目前,我国的血吸虫病研究主要以日本血吸虫病为对象,国外主要以曼氏血吸虫病为对象,而介绍两种血吸虫致病差异的综述较少。 为更好地理解两种血吸虫的致病差异,本文从血吸虫的基因组和进化路径、幼虫迁移、成虫寄生位置及产卵方式、局部组织病理、肉芽肿的形成机制以及肉芽肿的细胞组成等 6 个方面对日本血吸虫和曼氏血吸虫的致病差异进行了综述。     

Antibodies are involved in the protective immunity induced in mice by Schistosoma mansoni schistosomula tegument (Smteg) immunization

The Schistosoma mansoni schistosomula tegument (Smteg) plays an important role in triggering the host immune response and mice immunization with Smteg formulated with Freund's adjuvant or alum + CpG induce partial protection against S. mansoni infection associated with an increased antibody production. In this study, we investigated the role of these antibodies in parasite killing both in vitro and in vivo. We demonstrated that these antibodies were able to bind to the surface of S. mansoni recently transformed schistosomula and that these antibodies significantly increase the percentage of schistosomula killed in vitro by complement activation. Passive transference of immune sera decreased the parasite burden and the number of eggs trapped in the organs of mice that received sera containing anti‐Smteg antibodies. These results demonstrate that antibodies specific to surface tegumental antigens are involved in parasite elimination in mice immunized with Smteg.     

The use of mouse/human chimaeric antibodies to investigate the roles of different antibody isotypes, including lgA2, in the killing of Schistosoma mansoni schistosomula by eosinophils

We report the use of a matched set of mice/human chimaeric antibodies, directed against the 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) hapten, to investigate the roles of different human isotypes in antibody-mediated eosinophil-dependent killing of schistosomula. The chimaeric antibodies consist of mouse VH, VL and CL regions with human γ1, γ2, γ3 (2 allotypes), y4, α2, or e CH regions and were used in in vitro assays with human eosinophils and NIP-coated S. mansoni schistosomula. Some anti-NIP isotypes mediated high levels of killing, which was specific for NIP-coated larvae, and we suggest that these antibodies will be a valuable tool for studies on the role of antibody isotypes in anti-schistosome immune effector mechanisms. In particular, this method directly demonstrated, for the first time, that IgA is highly effective in mediating the killing of metazoan parasites by human eosinophils.     

Suppression of macrophage-mediated antibody-dependent killing of Schistosoma mansoni larvae by concanavalin A stimulated spleen cell-derived factor(s)

Culture supernatants (sup) from rat spleen cells stimulated with concanavalin A (Con A) sepharose 4B suppressed schistosomulum killing by macrophages in a system of antibody-dependent cell-mediated cytotoxicity (ADCC). (a) The suppressive effect of the sup was strongest when they were added to cultures at the start of the ADCC assay, and it was decreased when sup were added to cultures in which the ADCC assay had begun more than 3 h previously. (b) From the results of various experiments in ADCC reaction, it was suggested that the suppressive factor(s) inhibited at least in part the interaction between macrophages and antibodies which had been bound to schistosomula, and that the suppressive factor(s) was bound to the macrophage side. (c) The suppressive activity was partially inactivated by treatment at 56 degrees C for 30 min, and it was lost almost completely by the treatment at 100 degrees C for 10 min. (d) The suppressive factor(s) differed from known cytokines such as interleukin 1 (IL-1), interleukin 2 (IL-2), granulocyte-macrophage-colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF).     

Suppressor T cells in the specific control of the carrier response to schistosomula in mice infected with Schistosoma mansoni

The carrier effect, using TNP-labelled schistosomula was used to measure the helper T-cell activity against the schistosomula surface in CBA mice exposed to 30 cercariae of Schistosoma mansoni. After infection the helper T-cell activity reached a peak in 8--10 days, but by 6 weeks it had declined to background levels. Five x 10(7) spleen cells from chronically (12-week) infected mice when injected into 9-day infected mice caused a specific suppression of the helper T-cell response to schistosomula. Subsequent fractionation of the spleen cell population using a nylon wool column and specific depletion of T cells from the spleen cell population with anti-Thy 1.2 antisera and complement, showed that the suppressive activity was due to T cells. We conclude that during infection of mice with S. mansoni a population of suppressor T cells is generated which partially regulates antibody production against schistosome surface antigens.     

Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition

Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5-10 days post infection bound low or insignificant amounts of a variety of anti-schistosome antibodies including those from chronically infected and radiation attenuated cercariae-vaccinated mice, a vaccinated rabbit and rabbits hyper-immunized with non-living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6-day-cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti-parasite and anti-host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti-parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti-parasite antibodies coincided closely with the loss of 125I-labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6-day-cultured parasites recovered after 30 min in vivo failed to bind anti-host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently occurs independently of host antigen uptake, it is dependent upon an as yet unidentified host factor.     

Murine Schistosomiasis mansoni: anti-schistosomula antibodies and the IgG subclasses involved in the complement- and eosinophilmediated killing of schistosomula in vitro

Summary Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG_2a fraction and EAA in the IgG₁ fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG₁, IgG_2a and IgG_2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG₁ antibodies.     

Immobilization of Schistosoma mansoni miracidia by activation of the alternate complement pathway at unusually high serum dilution

Free-swimming Schistosoma mansoni miracidia were immobilized by adding normal mammalian serum to the water. Miracidial immobilizing activity (MIA) was shown to result from activating the alternate pathway of complement (APC). MIA in normal sera was heat-sensitive and anibody independent; it was greatly reduced in factor B-depleted or C6-depleted, but not in C1-depleted, human serum. Addition of purified factor B to B-depleted serum totally restored MIA. Half-maximal MIA in normal human, rabbit, and guinea pig sera was detectable at final dilutions exceeding 1/100, 1/200, and 1/500, respectively, and normal rat serum was particularly potent, with MIA at dilutions exceeding 1/2000. Detection of APC activity at such high dilutions is quite extraordinary and attributed to the hypotonic conditions. We confirmed and extended previous findings that heat-inactivated infection sera also display MIA. Immobilizing activity in irradiated-cercarial vaccine rat serum cofractionated with rat IgG and anti-SWAP antibody activity. Antibody-dependent MIA titres were much lower than for APC-dependent MIA. Based upon light microscope and transmission EM studies, immobilization of miracidia by APC activation was attributed to severe tegumental damage. Miracidia within egg shells were insensitive to MIA.     

Murine alloantigen acquisition by schistosomula of Schistosoma mansoni: Further evidence for the presence of K, D, and I region gene products on the tegumental surface

Previous studies have demonstrated that shistosomula, recovered from the lungs of Schistosoma mansoni -infected mice, acquire H-2K^k and both I^k and I^s gene products. We confirmed and extended those observations by identifying several individual antigenic specificities mapping not only to H-2K^k and I-A^k (Sher, Hall & Vadas 1978), but to H-2D^k, I-E^k, H-2K^b, H-2D^b and I-A^b as well. Private H-2 specificites 23 (H-2K^k) and 32 (H-2D^k) were identified on the tegumental surface of schistosomula isolated from C3H/Crgl mice (H-2^k). Larvae isolated from C57Bl/10Sn (B10) mice (H-2^b) were shown to acquire private specificities 33 and 2, which map to H-2K^b and H-2D^b respectively. I region associated (Ia) antigens coded for by genes mapping in the I-A subregion (Ia. 2 and 3) and I-E subregion (Ia. 7) were also acquired from C3H/Crgl mice. Schistosomula from B10 donors were shown to acquire Ia specificities 3 and 8, which are under I-A subregion control. Evidence that monoclonal antibodies recognize H-2 antigens on the larval surface is also demonstrated.     

Antigenicity and immunogenicity of the tegumental outer membrane of adult Schistosoma mansoni

The tegumental membranes of adult Schistosoma mansoni have been isolated and purified and shown to function as potent immunogens; they elicit an essentially identical immune response in rabbits, rats and mice. Anti-membrane antisera harvested from these animals consistently recognized common antigens, of relative molecular weight (mol. wt) 32 000 and 20 000, on the surface of young schistosomula, 5 day old lung worms and adult worm purified membranes. An additional molecule of 25 000 mol. wt was present on the surface of lung worms and adult worm membranes and was specifically recognised by serum from chronically infected mice and by serum from rabbits inoculated with adult worm purified membranes. The concept of antigenic identity between developmental stages that parasitize the mammalian host was further substantiated by the observation that anti-membrane antiserum bound to live schistosomula, lung worms and adult parasites as measured by indirect immunofluorescence. In complement-mediated in vitro cytotoxicity assays, the sera from rabbits inoculated with either adult worm purified membranes, or the 32 000 mol. wt antigen partially purified from adult worm membranes, mediated levels of schistosomula killing as high as those obtained with sera from chronically infected mice. These rabbit antisera also promoted eosinophil adherence and killing of newly transformed schistosomula, but lung stage parasites, despite binding the anti-membrane antiserum, were refractory to both humoral and cellular cytotoxicity. The significance of antigenic identity is discussed in relation to the concept of concomitant immunity.     

In-vitro antibody-dependent killing of schistosomula of Schistosoma haematobium by human eosinophils

Schistosomula of S. haematobium have been shown to be susceptible to in vitro killing by eosinophils in the presence of serum from an infected individual. The highest level of killing was found after 48 h in culture. Killing was related to the eosinophil to schistosomula ratio, being highest at 5000: 1. Killing was also related to serum concentration, being highest at a 1/10 final dilution, falling to background levels at a 1/120 final dilution. At a cell: target ratio of 2000: 1 and at a serum dilution of 1/10 eosinophils from subjects with high peripheral blood eosinophil counts were, cell for cell, more active in killing S. haematobium schistosomula than were eosinophils from subjects with lower counts. Sera taken from adults resident in an endemic area gave higher levels of killing in the presence of eosinophils than did sera taken from adults with no history of exposure.     

Antibody-independent binding and activation of complement by Schistosoma mansoni adult worms

The in vitro binding serum complement (C) components to Schistosoma mansoni worms was studied. By exposing frozen sections of adult male and female parasites to normal human serum, binding of both classical and alternative pathway C components was observed. By immunofluorescence (IFL) microscopy selective binding of Clq, C4 and C3 to the schistosomal tegument, internal structures, the intestinal tract and eggs was seen. The C4 and C3 binding was completely abolished in the presence of 10 mM EDTA. EGTA also completely inhibited binding of C4 and most of the C3 binding. These results suggest that C binding to S. mansoni adult worms occurs mostly by the classical activation pathway. However, the partial Ca2+ independence of C3 binding and the demonstrated binding of the regulatory protein beta 1H suggests that worms are capable also of C3 binding by the alternative pathway. No C binding occurred to intact worms. Although some in vivo bound immunoglobulin appeared to be present occasionally at the parasite surface and in the gut, this material did not account for the demonstrated extensive C deposition upon incubation of frozen sections with normal human serum in vitro. Antibody independence of the observed classical pathway C binding was further indicated by binding of isolated Clq to the same structures capable of binding C components from serum.