Release of leukotriene C4 (LTC4) from human eosinophils following adherence to IgE- and IgG-coated schistosomula of Schistosoma mansoni

The release of leukotriene C4 (LTC4) from human low-density eosinophils following adherence to live or formalin-fixed schistosomula of Schistosoma mansoni coated with parasite-specific IgE or IgG obtained from pooled human anti-S. mansoni serum has been studied. IgE-rich fractions were obtained after fractionation of pooled immune sera on fast-protein liquid chromatography (FPLC; polyanion SI-17 column) and were identified by parasite-specific RAST. Contaminating IgG was removed by adsorption on a Staphylococcus aureus-protein A affinity column. IgG-rich FPLC fractions were identified by a specific ELISA assay. IgG-dependent activities were confirmed by protein A adsorption. Low-density eosinophils adhered to live and formalin-fixed schistosomula coated with specific antisera and released 11.7 +/- 2.7 and 16.5 +/- 3.5 pmoles of LTC4/10(6) cells, respectively. LTC4 release induced by A23187 (5 x 10(-6) M) from the same cells was 80 +/- 24 pmoles/10(6) cells and 9.9 +/- 1 pmoles/10(6) cells in the presence of Sepharose particles (CNBr-activated 4B beads) covalently coated with normal human IgG. Fixed schistosomula coated with FPLC-purified IgE and IgG gave 7.6 +/- 0.4 and 6.0 +/- 0.1 pmoles of LTC4 per 10(6) low-density eosinophils, respectively. The same IgE- and IgG-rich fractions induced eosinophil-mediated cytotoxicity of live schistosomula in vitro. Removal of IgE by an anti-IgE affinity column abolished both the IgE-dependent release of LTC4 and the in vitro killing of larvae. Conversely, IgG-dependent activities were abolished by protein A, but not anti-IgE, adsorption. Normal density eosinophils generated undetectable amounts of LTC4 when incubated with IgE-coated schistosomula, whereas with IgG-coated larvae 4.6 pmoles/10(6) cells were obtained. Following preincubation with platelet-activating factor (PAF) (10(-7) M) and leukotriene B4 (LTB4) (10(-7) M), normal density eosinophils released LTC4 when in contact with larvae coated with antigen-specific IgE. Lyso-PAF had no effect in any of the systems tested. The synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) had no influence on IgE-dependent release of LTC4 from eosinophils. In contrast, FMLP (10(-7) M) enhanced the IgG-dependent LTC4 release, with PAF and LTB4 also showing a small enhancing effect. None of these agents substantially altered the release potential of low-density eosinophils in either IgE- or IgG-dependent events. Thus the results presented here indicate that in an IgE-dependent system, human low-density eosinophils can be induced to adhere to and kill IgE-coated helminthic targets and release biologically relevant amounts of LTC4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of the attack of eosinophils stimulated by blood mononuclear cell products on schistosomula of Schistosoma mansoni.

Purified human eosinophils were treated with peripheral blood mononuclear cell supernatants containing eosinophil cytotoxic enhancing activity (ECEA). Schistosomula of Schistosoma mansoni which had been coated either with antibody (Ab) from the sera of infected patients or with the lectin concanavalin A (Con A) were incubated with ECEA-treated and untreated cells for 2 minutes to 12 hours and examined ultrastructurally. Killing was assayed at 18 hours. ECEA caused an increase in the killing of Ab-coated worms, but Con-A-coated worms were not killed by either ECEA-treated or untreated cells. Eosinophils began to degranulate on Ab-coated worms within 2 minutes and continued to degranulate, so that by 12 hours about half of the parasites had greater than 50% of their surface covered by discharge material. The ECEA-treated cells degranulated more than the untreated cells. There was much less discharge material on Con-A-coated worms than on Ab-coated worms. Eosinophils adhered to discharge material on the surface of both Ab- and Con-A-coated parasites. At 3 and 12 hours, lysed cells and cell fragments were also seen adhering to discharge material. In the absence of discharge material the cells adhered to residual glycocalyx or to the tegumental outer membrane. These studies suggest that eosinophils kill schistosomula by progressively degranulating onto their surface over many hours and that the increased toxicity caused by ECEA is due to an increase in discharge.     

Schistosoma mansoni in baboons. Antibody-dependent cell-mediated damage to 51Cr-labelled schistosomula.

A technique is described for estimating cell-mediated damage to the larval stages of Schistosoma mansoni that occur in the mamalian host (schistosomula), by measuring release of 51Cr from labelled organisms. This technique, although widely used for assaying cytotoxicity to single cell suspensions or monolayers, has not previously been applied to a multicellular parasite. It is more objective than microscopical assays, and allows the processing of larger numbers of samples. The new method has been used for the detection and quantification of cell-dependent cytotoxic antibodies in infected baboons. The effector cell in normal baboon peripheral blood, as in man, is associated with a neutrophil- and eosinophil-enriched fraction. Cytotoxic antibodies appear in the serum about 4 weeks after a primary infection, rising to a peak at 7-10 weeks, and then declining. In some animals, a second rise towards 30 weeks was observed. There were no consistent differences between groups of baboons exposed to 1000 cercariae, 200 cercariae, and 200 cercariae administered at each of five monthly intervals. The possible relationship between cell-dependent cytotoxic antibodies and resistance to reinfection in infected animals is discussed.     

Selectin and Lewis(x) are required as co-receptors in antibody-dependent cell-mediated cytotoxicity of human eosinophils to Schistosoma mansoni schistosomula

Killing of Schistosoma mansoni larvae by human eosinophils via antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms requires adherence between effector cells and parasite targets. The role of adhesion molecules in this mechanism was investigated using blocking monoclonal antibodies (mAb) and soluble ligands. We show that, along with the Mac-1 alpha chain, interactions between selectins and LewisX-related structures, both expressed by eosinophils and parasite targets, play a critical part in the antibody-dependent cytotoxic function of eosinophils. To further elucidate the interactions between adhesion molecules and eosinophil Fc receptors, ADCC was performed with IgG1 or IgA mAb. We found that mAb directed against Mac-1 alpha chain or against LewisX could significantly inhibit the IgG1-, but not IgA cytotoxicity. This result might be explained, at least in part, by the inhibitory effect of these mAb on the release by eosinophils of eosinophil cationic protein, one of the major mediators involved in target killing. Taken together, these results suggest novel interactions between Fc receptors and selectins and LewisX-related structures which might act as co-receptors for eosinophil-mediated cytotoxicity.     

Eosinophil-enriched inflammatory response to schistosomula in the skin of mice immune to Schistosoma mansoni.

Exposure of the mouse skin to Schistosoma mansoni cercariae gives rise to acute, exudative inflammation in both normal and immune mice, but the immune response is anamnestically accelerated and is oesinophil-enriched, thereby enhancing opportunities for tegumental contact of schistosomula with host leukocytes, particularly with eosinophils. Many of the inflammatory changes occurring within the first 48 hours after exposure are due to cercarial products, e.g., "penetration tracts," but some remain demonstrable when schistosomula metamorphosed in vitro are injected intradermally and are therefore directed against the schistosomula themselves, such as the leukocyte "streaming patterns" seen in their pathways. In contrast to earlier observations in primates, cellular responses to schistosomula in the mouse lung 4 days after penetration are minimal in either normal or immune mice. Thus, immune cellular responses to schistosomula in mice are limited to an early time period after cercarial penetration and are morphologically suggestive of an antibody-mediated response rather than of delayed hypersensitivity. Our observations complement earlier evidence suggesting that antibody-mediated host leukocyte contact with schistosomula initiates the killing of challenge parasites in immune mice, with the eosinophil probably playing a crucial role.     

Rat IgE directed against schistosomula-released products is cytotoxic for Schistosoma mansoni schistosomula in vitro

The immunogenicity of molecules shed by schistosomula into culture medium (antigens present in schistosomula-released products, SRP-A) has been studied. The results obtained show that SRP-A preferentially induce an IgE response when injected into rats, without the need for adjuvants. Moreover, anti-SRP-A IgE is cytotoxic in vitro for the larvae in the presence of macrophages, eosinophils or platelets, which have previously been demonstrated as being the three efficient killer cells for schistosomula in the presence of specific IgE. Immunofluorescence analysis locates the target antigens at the schistosomulum surface. Among the antigens recognized by anti-SRP-A IgE, two molecules of 26 and 22 kDa have been identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by western blotting.     

Schistosoma mansoni: phospholipid methylation and the escape of schistosomula from in vitro cytotoxic reaction

The ability of Schistosoma mansoni schistosomula to evade in vitro cytotoxic activity of antibodies plus complement is shown to be increased by incubation with Concanavalin A (Con A) or with non-immune inactivated human serum. This effect was not observed if S-adenosyl-homocysteine (SAH) a methyltransferase inhibitor was added to the incubation medium. Methyl group incorporation occurs in schistosomulum phospholipids if parasites are incubated in Earle's balanced salt solution. This incorporation is increased by Con A addition and this increase is inhibited by SAH. Supernatants of schistosomula incubated in culture media containing Con A were able to promote phospholipid methylation, showing that methyltransferases were liberated into the culture media. The possible roles played by these phenomena in host-parasite interactions are discussed.     

Enhancement of neutrophil- and eosinophil-mediated complement-dependent killing of schistosomula of Schistosoma mansoni in vitro by leukotriene B4

We have studied the ability of leukotrienes and other lipoxygenase products of arachidonic acid (AA) to influence complement-dependent killing of schistosomula of Schistosoma mansoni in vitro by human neutrophils or eosinophils. These lipid mediators, which included LTB4, LTC4, LTD4, 5-HETE and 5-HPETE, had no apparent effect, by themselves, on schistosomular motility or viability. However, in the presence of granulocytes and fresh serum (as a source of complement) LTB4 (but not LTC4, LTD4, 5-HETE or 5-HPETE) enhanced neutrophil- and (to a much lesser extent) eosinophil mediated, complement-dependent killing. These effects varied with the concentration of LTB4, the dilution of complement and time of incubation. The percentage of LTB4-induced enhancement obtained with neutrophils was greater than that observed with eosinophils (although the latter were obtained from patients with helminthic parasitic disease). The synthetic bacterial analogue f-Met-Leu-Phe, also known to amplify complement associated granulocyte events, was comparable to LTB4 in its ability to enhance neutrophil- and eosinophil-mediated, complement-dependent killing of schistosomula. These results indicate that LTB4, which is released in mast cell associated reactions and promotes cell locomotion and enhancement of complement receptors in vitro, increases neutrophil- and eosinophil-mediated, complement-dependent damage of schistosomula, possibly through enhancement of C3b receptors and that this may be an important amplification mechanism in IgE related immunity to migrating helminthic larvae.     

Immunity to schistosomiasis mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae: I. Humoral responses against skin-stage schistosomula.

The anti-schistosomular humoral responses of guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosoma mansoni have been investigated in vitro. The sera of vaccinated animals contain schistosomulicidal complement-fixing antibodies which peak in titre at week 5 after vaccination and predominantly consist of IgG2 and IgM antibodies. The ability of the serum to arm macrophages from normal animals to bind to schistosomula, also peaks in titre at week 5 and is associated with IgG2 antibodies. Basophils from normal animals can be sensitized in vitro by vaccine serum to degranulate in the presence of schistosomular antigens. This anaphylactic antibody activity is associated with IgG1 but not IgE antibodies, and peaks in titre at week 10. Three antigens (14 kD, 20 kD and 43 kD) are specifically and transiently detected by vaccine serum on Western blots of schistosomular proteins; these antigens are first discernible at week 4, but were virtually undetectable at week 12.     

Evidence for pili-mediated adherence of Klebsiella pneumoniae to rat bladder epithelial cells in vitro.

The possible role of pili in the pathogenesis of urinary tract infections caused by Klebsiella pneumoniae was studied in an in vitro mixture of a phosphate-buffered saline suspension of rat bladder epithelial cells and phosphate-buffered saline-washed K. pneumoniae. Nonpiliated and piliated populations derived from a single K. pneumoniae strain were obtained by controlling the total time of growth in broth medium. The piliated phase demonstrated a significant increase in adherence when compared to the nonpiliated phase. Incubation of the bacteria and epithelial cell mixture at 4 and 37 degrees C resulted in no differences in adherence; optimal adherence occurred at pH 5. Pretreatment of the bacteria with enzymes to destroy the pili resulted in a decrease in adherence, as did killing the bacteria by various means before adherence testing. Pretreatment of the epithelial cells with certain saccharides inhibited bacterial adherence. Finally, a 96% decrease in adherence was observed after coincubation of bacteria and epithelial cells with papain-treated antipili antibodies. Thus, it appears that pili on the surface of K. pneumoniae mediate attachment of the bacteria to rat bladder epithelial cells.     

Cytotoxicity of human and baboon mononuclear phagocytes against schistosomula in vitro: induction by immune complexes containing IgE and Schistosoma mansoni antigens.

Normal human blood monocytes, pre-incubated at 37 degrees C with sera from patients infected with Schistosoma mansoni, strongly adhered to S. mansoni schistosomula in vitro, whereas no significant adherence was induced by sera from uninfected individuals. Comparable adherence occurred with normal baboon blood monocytes or peritoneal macrophages when these cells were incubated with sera from S. mansoni-infected baboons. Adherence of macrophages to schistosomula was associated with damage to the larvae, as estimated by a 51Cr release technique. Neither adherence nor cytotoxicity was induced by pre-incubation of the schistosomula, instead of the monocytes, with immune serum. The relevant factor in immune serum was heat-labile, but was not a complement component. Absorption and ultracentrifugation experiments showed that immune complexes, containing S. mansoni-specific IgE antibody and soluble parasite antigens, produced monocyte or macrophage adherence and cytotoxicity. Similar observations have been reported previously in the rat model. Since the production of large amounts of IgE is a predominant feature of schistosome infections in man and experimental animals, it is possible that this new mode of mononuclear phagocyte activation could act as an immune effector mechanism against S. mansoni.     

Surface antigens on Schistosoma mansoni. II. Adsorption of a Forssman-like host antigen by schistosomula

A study was made of the nature of mouse (host) antigens adsorbed by schistosomula of Schistosoma mansoni. Using the mixed antiglobulin test, extracts of a number of individual mouse tissues were tested for their ability to coat schistosomula. All were effective to some extent, with the greatest activity being found in extracts of the lung and spleen. Antibodies against the schistosomulum-coating antigen as well as surface host antigens of adult Schistosoma mansoni were removed by absorbing with erythrocytes from a number of Forssman-positive but not Forssman-negative animal species. These antibodies were also absorbed by Forssman-positive guineapig kidney extract and methanol soluble (Forssman-positive) but not insoluble fractions of sheep erythrocyte stromata and mouse lungs. Schistosomula could be coated in vitro with methanol soluble fractions of mouse lung and erythrocytes and sheep erythrocytes. Though both mouse and sheep coating antigens reacted with anti-mouse and anti-sheep antibodies, reactions were stronger with the homologous antiserum. It was concluded that schistosomula of Schistosoma mansoni adsorb from mice an antigen similar but not identical to the Forssman antigen of sheep erythrocytes, and that this antigen is also found on the surface of adult worms.     

Bacterial adherence to human endothelial cells in vitro.

Differences in the ability of bacteria to adhere to normal valvular endothelium may account for the predominance of particular species as pathogens in acute endocarditis. An in vitro adherence assay was developed to simulate the host surface encountered in acute bacterial endocarditis by using confluent monolayers of human endothelial cells. Adherence of 32 gram-positive and -negative blood culture isolates to this surface was compared. All five Staphylococcus aureus strains tested were highly adherent to endothelial cells, as was one gram-negative strain (Serratia marcescens). The remaining gram-positive and -negative isolates, including four viridans streptococci, were relatively nonadherent. Transmission electron microscopy demonstrated attachment of Staphylococcus aureus and invagination of the underlying endothelial cell membrane at 1 h followed by engulfment of large numbers of bacteria after 3 h. The intracellular bacteria appeared to be contained within vacuoles. Preferential attachment of some strains of bacteria, in particular Staphylococcus aureus, to human endothelial cells occurred in vitro, suggesting that adherence is an important determinant of bacterial pathogenicity in acute endocarditis. Active uptake of bacteria by endothelial cells may help account for the virulence of Staphylococcus aureus in endovascular infections and for the ability of this organism to establish multiple metastatic foci of infection.     

Freshly isolated and cultured human monocytes obtained by plasmapheresis kill schistosomula of Schistosoma mansoni.

The efficacy of human peripheral blood monocytes (PBM) in killing of schistosomula is controversial. The purpose of this study was to determine the schistosomulacidal activity of human monocytes isolated by two different techniques. Peripheral blood monocytes were obtained either by venipuncture (PBMv) or plasmapheresis (PBMp), purified on Ficoll-Paque, and cultured briefly. The cells then were incubated with schistosomula (cell parasite ratio of 10(4):1) for 16 to 18 hours with or without interferon-gamma IFN-gamma (600 U/ml) or sera from patients with schistosomiasis as a source of antischistosomal antibodies (HASA). Freshly isolated PBMv treated with IFN-gamma or HASA did not kill schistosomula. Freshly isolated PBMp alone killed 22 +/- 13% (mean +/- standard deviation [SD]; n = 9) of worms over background and after incubation with IFN-gamma and HASA, 30 +/- 17%. PBMp cultured in vitro for 7 days killed 50 +/- 15% (mean +/- SD; n = 12) of the schistosomula. Pretreatment of the cells with IFN-gamma and incubation with HASA did not significantly enhance the parasite killing beyond this level. Electron microscopy showed that freshly isolated PBMp attached to the worms and fused occasionally with the outer tegumental membrane. Granules constituted 1.4% of the cytoplasmic volume. Degranulation onto the parasite surface was not observed. Peripheral blood monocytes obtained by plasmapheresis accumulated glycogen during in vitro culture with the parasite and released threefold more H2O2 than PBMv after exposure to phorbol myristate acetate. Thus plasmapheresis increases the schistosomulacidal activity of PBM, enhances the generation of H2O2 and promotes the accumulation of glycogen.     

The acquisition of antigens in the intercellular substance of mouse skin by schistosomula of Schistosoma mansoni.

Serum from patients suffering from the autoimmune skin disease pemphigus vulgaris has been used to demonstrate the presence of intercellular substance (ICS) on the surface of these chistosomula of Schistosoma mansoni which has penetrated mouse skin in vitro or during a percutaneous infection. ICS was absent from mechanically transformed schistosomula or those formed in the peritoneal cavity of mice. Schistosomula which penetrated mouse skin rapidly in vitro acquired very little of the ICS. It was found during a percutaneous infection that schistosomula recovered from the skin after 10 min had no detectable ICS, while those recovered after 2 hr and 24 hr gained increasing quantities of the material. It is concluded that schistosomula which are delayed in their exit from the skin acquire more ICS. However, this material must be shed during subsequent migration since schistosomula from lung and liver, and 7-week-old worms do not posses it. The implications of the findings are discussed.     

Receptors for antibody-opsonic adherence on the eosinophils of guinea pigs.

Eosinophils have recently been implicated in antibody-dependent cell-mediated damage to schistosomula. Because of this, eosinophils of the guinea pig have been examined for surface receptors capable of giving antibody opsonic adherence; a rosetting reaction has been used. The eosinophils were shown to possess Fc receptors for homologous immunoglobulin. No selective difference between IgG1 and IgG2 was observed. In marked contrast to macrophages, guinea pig eosinophils failed to show opsonic adherence to red cells sensitized to a comparable degree with rabbit antibody. With red cell antibodies made in the pig, however, the reciprocal situation held, namely opsonic adherence was stronger with eosinophils than with macrophages.     

Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation

Despite their significance in human and veterinary medicine, and the ability to maintain the parasites in the mouse, relatively little functional detail is available regarding the biology of schistosomes. This deficit is due largely to the lack of well-developed molecular tools for manipulating gene expression in these parasites. Here, we describe an electroporation protocol that provides a routine approach for efficiently introducing nucleic acids into schistosomes. Using luciferase-encoding RNA for electroporation, and luciferase activity as a read-out, we established 400 microg/ml of RNA, and a 20 ms pulse at 125 V using a square wave electroporation generator to be optimal for electroporating schistosomes. Under these conditions schistosomula from 1 hr to 18 hr old could be successfully electroporated, the majority of parasites within a population expressed the introduced RNA, and acute mortality was negligible. Electroporation, as described here, makes possible experimental studies using transiently expressed constitutively active and/or dominant negative mutant proteins, etc. In addition, the finding that electroporation can be used to introduce RNA into schistosomula raises the possibility of using this approach to introduce either DNA constructs or dsRNA sequences, both of which might be expected to have longer-term, ideally inheritable, effects.     

Purification and characterization of proteases secreted by transforming schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni which are mechanically transformed at 4 degrees C and are then incubated at 37 degrees C in defined medium spontaneously secrete two proteases, a major one of 28 kDa and a minor one of 60 kDa. These were purified by ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel AcA 54 with yields of 33% and 29%, respectively. Both appeared as single bands by silver staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The 28 kDa protease is a glycoprotein that has a pI of 11 or higher and an optimal activity around pH 9.0. It cleaves casein, gelatin and human C3 and C3b. It is metal-ion independent and is inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, soy-bean trypsin inhibitor, alpha 1 antitrypsin, Zn2+ ions, sodium dodecyl sulphate and normal human serum. The 60 kDa protease is a glycoprotein with a pI of 9.2. It can also cleave casein and gelatin and its activity is inhibited by phenylmethanesulfonyl fluoride but not by diisopropyl fluorophosphate or sodium dodecyl sulphate. We suggest that these proteases may play a role during cercarial penetration of the skin and in shedding of the cercarial glycocalyx.     

Ultrastructural observations on the in vitro interaction between rat eosinophils and some parasitic helminths (Schistosoma mansoni, Trichinella spiralis and Nippostrongylus brasiliensis).

Rat eosinophils form an intimate association with the surfaces of parasitic helminths, in vitro, in the presence of immune serum. The parasite presents a non-phagocytosable surface to the cell. The initial response of the eosinophil is degranulation which leads to the formation of large cytoplasmic vacuoles. Peroxidase, an enzyme localized in the matrix of the crystalloid secretion granules, is discharged into these vacuoles as a consequence of degranulation. The vacuoles eventually become connected to the adherent basal plasma membrane of the eosinophil, and peroxidase is secreted directly onto the surface of the parasite. There is no morphological evidence to suggest that this particular secretion affects the integrity of the parasite surface.     

The reversible effect of glucose on the energy metabolism of Schistosoma mansoni cercariae and schistosomula.

This study on isolated cercarial bodies demonstrates that the biological transformation from cercaria to schistosomulum and the biochemical transition from an aerobic to an anaerobic energy metabolism are separate processes, which are not necessarily linked. The metabolic transition depends on the external glucose concentration and is fully reversible. In the presence of only a tracer amount of [6-14C]glucose, carbon dioxide was the major end product, but at higher glucose concentrations mainly lactate was formed. This effect could be demonstrated in cercarial bodies in water as well as in fully transformed schistosomula. In non-transformed cercariae a change towards a more anaerobic energy metabolism could be induced by an increase in the external glucose concentration, which demonstrated that the biochemical transition can occur in the absence of the biological transformation. Furthermore, the biological transformation can occur without a concomitant biochemical transition: in the presence of 5 mM glucose, lactate production by cercarial bodies during transformation was increased 50-fold, whereas in the presence of only a tracer amount of glucose the metabolic profile remained that of cercariae. Also, in fully transformed schistosomula, this transition to a more anaerobic energy metabolism was induced by increased glucose concentrations, but at low glucose concentrations carbon dioxide was the major end product, as in cercariae. The effect of external glucose on the metabolism was fully reversible. After a high glucose concentration had induced a more anaerobic metabolism in cercariae in water, the metabolism returned to an aerobic one upon removal of the glucose. Likewise, the metabolism in schistosomula switched back and forth between anaerobic and aerobic patterns, following successive changes in the glucose concentration.(ABSTRACT TRUNCATED AT 250 WORDS)