Human neutrophil-mediated killing of schistosomula of Schistosoma mansoni: augmentation by schistosomal binding of eosinophil peroxidase

Eosinophil peroxidase (EPO) is a major component of the large cytoplasmic granules of eosinophils, and is released onto the surface of schistosomula when eosinophils adhere to antibody and complement coated organisms. EPO is a strongly cationic protein, which can bind to the surface of schistosomula with retention of peroxidatic activity. The binding per se was not toxic to the organisms under our conditions, but EPO-coated schistosomula were rapidly killed when H2O2 and halide were added, under conditions in which uncoated schistosomula were unaffected. The toxicity of the surface-bound EPO system was not significantly inhibited by albumin (20 mg/ml), in contrast to the complete inhibition by this concentration of protein when the EPO was free in solution. Purified polymorphonuclear leukocytes (PMNs) from normal donors were toxic to uncoated schistosomula in medium containing antischistosomal antibody and complement, and this toxicity was significantly increased when EPO was bound to the surface of the organisms. The toxicity of PMNs to EPO-coated schistosomula was inhibited but not abolished by the hemeprotein inhibitor azide. This is compatible with the involvement of surface-bound EPO in an enzymatic attack on the organism, utilizing H2O2 generated by PMNs stimulated by adherence to antibody and complement-coated schistosomula. PMN adherence to schistosomula is increased by surface-bound EPO, and this also may contribute to the enhancement of neutrophil-mediated toxicity by EPO. These findings indicate a mechanism by which two inflammatory cells, the eosinophil and neutrophil, may interact to enhance the destruction of a target organism.     

小鼠感染后不同时间血清对日本血吸虫童虫细胞介导？..

The tests of antibody-dependent cell-mediated cytotoxicity (ADCC) were performed to observe the kinetics of in vitro killing of Schistosoma japonicum schistosomula mediated by eosinophils, macrophages or neutrophils with infected mouse sera of different duration. The results showed that when complement was not involved, the killing rates of schistosomula mediated by eosinophils or macrophages began to increase significantly at 4 wk post-infection, reached a plateau in 5-7 wk and 6-8 wk respectively, and then declined to the levels of that at 4 wk till 11 wk. In case of neutrophils, there was no significant cytotoxicity detected. When complement was involved, all the three effector cells could mediate cytotoxicity to schistosomula, with the killing rates higher than those without complement at the correspondent time intervals. These indicate that ADCC appears to be an important ingredient in the acquired resistance to S. japonicum, and demonstrate that eosinophil- and macrophage-mediated cytotoxicity to schistosomula is complement-independent, whereas neutrophil-mediated cytotoxicity to schistosomula is complement-dependent. This method may be of some value for choosing optimal time for vaccination and estimating the effectiveness of a candidate vaccine.     

小鼠感染后不同时间血清对日本血吸虫童虫细胞介导的细胞毒作用

用抗体依赖、细胞介导的细胞毒试验(ADCC)观察了小鼠感染后不同时间血清在体外杀伤系统中对日本血吸虫童虫的作用规律。未加补体时,嗜酸粒细胞或巨噬细胞介导的童虫杀伤作用于感染后4wk出现明显作用,分别于5—7wk和6—8wk达高峰,然后下降,至11wk时降至4wk时的水平;而未加补体时无明显的中性粒细胞介导的杀伤作用。在补体参与下,这三种细胞均能介导对童虫的杀伤作用,且作用增强。证实ADCC是日本血吸虫获得性抵抗力的一个重要组分,嗜酸粒细胞、巨噬细胞介导的对童虫的细胞毒作用是不依赖补体的,而中性粒细胞介导的作用是依赖补体的。结果对选择免疫预防适宜时间和评价候选疫苗效果有参考意义。     

Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils.

Eosinophils, which are prominent cells in asthmatic inflammation, undergo apoptosis and are recognized and engulfed by phagocytic macrophages in vitro. We have examined the ability of human small airway epithelial cells (SAEC) to recognize and ingest apoptotic human eosinophils. Cultured SAEC ingested apoptotic eosinophils but not freshly isolated eosinophils or opsonized erythrocytes. The ability of SAEC to ingest apoptotic eosinophils was enhanced by interleukin-1alpha (IL-1alpha) or tumor necrosis factor alpha (TNFalpha) in a time- and concentration-dependent fashion. IL-1alpha was found to be more potent than TNFalpha and each was optimal at 10(-10) mol/L, with a significant (P <.05) effect observed at 1 hour postcytokine incubation that was maximal at 5 hours. IL-1alpha stimulation not only increased the number of SAEC engulfing apoptotic eosinophils, but also enhanced their capacity for ingestion. The amino sugars glucosamine, n-acetyl glucosamine, and galactosamine significantly inhibited uptake of apoptotic eosinophils by both resting and IL-1alpha-stimulated SAEC, in contrast to the parent sugars glucose, galactose, mannose, and fucose. Incubation of apoptotic eosinophils with the tetrapeptide RGDS, but not RGES, significantly inhibited their uptake by both resting and IL-1alpha-stimulated SAEC, as did monoclonal antibody against alphavbeta3 and CD36. Thus, SAEC recognize apoptotic eosinophils via lectin- and integrin-dependent mechanisms. These data demonstrate a novel function for human bronchial epithelial cells that might represent an important mechanism in the resolution of eosinophil-induced asthmatic inflammation.     

东方田鼠感染日本血吸虫后肺和肝的组织细胞反应及虫体的扫描电镜观察

目的：通过东方田鼠（Microtus fortis,Mf）感染日本血吸虫（Schistosoma japonicum,Sj）后肺和肝的组织细胞反应以及虫体受损情况，探寻Mf体内杀伤Sj的效应细胞和Sj的受损原因。方法：以Sj尾蚴经腹部皮肤感染Mf5d和11d后剖杀，分别取肺和肝，固定，切片，光镜下观察虫全周围的组织细胞反应，并与同时期的感染小鼠组织切片进行比较，同时，对Mf体内的Sj童虫进行扫描电镜观察。结果：Mf肺脏虫体周围有一定量的嗜酸性粒细胞浸润，而肝脏虫体周围有大量的嗜酸性粒细胞和少量的中性粒细胞，巨噬细胞等浸润，同时期的感染小鼠组织内虫体周围炎症反应不明显，电镜观察显示Mf体内的Sj存在明显的畸形和发育延迟，结论：嗜酸性粒细胞可能是Mf体内杀伤Sj的重要效应细胞，Sj在Mf体内不能正常发育。     

Studies on antibody-dependent cell-mediated cytotoxicity to Schistosoma japonicum schistosomula in mice

In the presence of antischistosomular serum (Ab), the adherence of macrophages (M?) eosinophils (Eos) and neutrophils(Neu) to and the killing effect on schistosomula of Schistosoma japonicum were studied with a mouse model. In vitro experiment showed that all the three kinds of effector cells could adhere to the surface of the schistosomula when opsonized with specific Ab resulting in the significant increase in the percentage of dead schistosomula. When SPA was added to the system, the percentage of schistosomula with adherent cells decreased markedly. It was revealed that the adherence of cells was at least partially through the binding of Fc fragment of IgG to Fc receptor on the cell surfaces. After having been incubated with Ab and/or cells for 18 h in vitro, the schistosomula were inoculated into the peritoneal cavity of mice. The adult recovery rate 6 weeks after inoculation in groups Eos + Ab and M? + Ab were significantly lower than that of the control group (P less than 0.01).     

Comparative toxicity of purified human eosinophil granule cationic proteins for schistosomula of Schistosoma mansoni

The human eosinophil granule contains several distinctive cationic proteins that have been purified to homogeneity, including major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). Two earlier studies have shown that MBP and ECP both damage schistosomula of Schistosoma mansoni in vitro in a dose-dependent fashion. The present study expands upon these observations by comparing the toxicity of MBP, ECP, as well as EDN when tested at equimolar concentrations (0.03-2 X 10(-5) M). On a molar basis, ECP was 8 to 10 times more potent than MBP, and the ECP-mediated killing of schistosomula was qualitatively different than that of MBP. Purified ECP produced complete fragmentation and disruption of schistosomula, whereas MBP produced a distinctive ballooning and detachment of the tegumental membrane. In contrast, EDN was only marginally toxic at high concentrations and caused crinkling of the tegumental membrane. Heating MBP and ECP for four hr at 56 degrees C caused precipitation and loss of toxicity for MBP, but not for ECP. Native MBP (with reactive sulfhydryl groups intact) and stabilized, reduced and alkylated MBP had comparable toxicity. To determine the relative contribution of MBP, ECP and other potentially helminthotoxic eosinophil granule constituents to schistosomulum damage, fractions of acid soluble granule extracts prepared by chromatography on Sephadex G-50 columns were analyzed for toxicity to schistosomula and for MBP and ECP levels by radioimmunoassay. Schistosomula were killed by fractions containing MBP, and to a much lesser and more variable extent by fractions containing EDN and a 21,000 dalton protein, but not by fractions coincident with the elution of ECP, which contained concentrations of ECP below that required to produce significant killing of schistosomula by the purified protein. Therefore, although ECP is a more potent helminthotoxin for schistosomula than MBP on a molar basis, MBP, by virtue of its abundance in the granule, accounts for the bulk of the toxicity in fractions of acid solubilized granules obtained from eosinophils of patients with marked eosinophilia.     

Effects of recombinant tumour necrosis factor on antibody-dependent eosinophil-mediated damage to Schistosoma japonicum larvae

Human recombinant tumour necrosis factor (rTNF) enhanced monoclonal IgE-dependent eosinophil-mediated cytotoxicity to schistosomula of Schistosoma japonicum in a dose-dependent manner. The enhancing effect of rTNF was also observed for the antibody-dependent cytotoxicity of a human eosinophilic leukemia cell line, EoL-3, but not of another cell line, EoL-1. Observation by a slow-motion movie camera demonstrated that activated EoL-3 cells adhered to the surface of schistosomula by 6 h after incubation, which triggered intracellular movement of eosinophil granules. The granules were concentrated toward the surface of the larvae and then degranulation started. The cell membrane was left as a balloon-like remnant. Cell sorting analysis by FACStar indicated that the expression of receptors for C3bi (CR3) and low affinity FcR for IgE (Fc epsilon RII) increased on the surface of EoL-3 cells after stimulation with rTNF, while this was not observed for EoL-1 cells.     

Studies on the development of anti-schistosomular surface antibodies by mice exposed to irradiated cercariae, adults and/or eggs of 5. mansoni

Levels of antibody a binding to the schistosomulum surface and b mediating in vitro eosinophil-dependent cytotoxicity of schistosomula were studied and compared to the in vivo levels of resistance to cercarial challenge in mice infected with irradiated cercariae, unirradiated cercariae of single or mixed sex, or injected with eggs. Antibody-binding was assessed by counting the number of IgG-Fc-receptor bearing cells (P388D1 cells) adhering to mechanically-transformed schistosomula. Significant levels of adherence occurred with sera taken from 1--2 weeks following exposure to irradiated cercariae, the level increasing gradually thereafter and being enhanced by repeated exposure. Sera from the bisexual infection showed a dramatic increase in binding activity between weeks 5--8, and with the single sex infections there was a steady rise up to week 10 followed by a sharp rise between weeks 10--12. Weekly injections of eggs produced a steady rise in serum binding activity. Sera taken just before challenge were also tested for their ability to mediate killing of schistosomula by eosinophil-enriched preparations of heterologous rat peritoneal exudate cells in vitro. Significant levels of killing occurred with all sera, but the greatest lethal activity was found in sera from the egg-injected, chronically-infected and 200 male cercariae-infected groups. This ranking did not correlate with in vivo resistance against challenge as assessed by worm recovery, the egg-injected and single sex-infected groups failing to manifest significant resistance.     

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.     

Stimulation of human granulocyte function by monoclonal antibody WEM-G1.

From a panel of monoclonal antibodies (MAb) produced against purified human neutrophils, MAb WEM-G1 was selected for its ability to stimulate granulocyte function as assessed by the capacity to kill antibody-coated tumor target cells. MAb WEM-G1 bound to neutrophils and eosinophils and not to monocytes, lymphocytes, or erythrocytes, and thus identified a granulocyte differentiation antigen. It enhanced killing by both neutrophils and eosinophils in a dose-dependent fashion, with a range of effector-to-target ratios and dilutions of anti-target cell antibody. The effect of MAb WEM-G1 was additive with that of colony-stimulating factor (CSF-alpha). Similarly, the ability of neutrophils to exhibit enhanced cytotoxicity in the presence of WEM-G1 was increased by preincubation of neutrophils with CSF-alpha. Immunoprecipitation analysis showed that WEM-G1 identified a cell-surface protein on human neutrophils of approximate molecular weight 110,000. It is suggested that this structure is involved in regulation of the function of human granulocytes.     

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.     

Human eosinophil hematopoiesis studied in vitro by means of murine eosinophil differentiation factor (IL5): production of functionally active eosinophils from normal human bone marrow

The production of human eosinophils in vitro from normal bone marrow by using murine eosinophil differentiation factor (mEDF/interleukin 5) is described. Eosinophil production was selective and first detectable after 14 days and reached a peak between 21 and 35 days when they were the predominant cell type (41% to 89%). Until day 14, all the eosinophils were typical myelocytes, developing thereafter into metamyelocytes and mature cells. All cell types had characteristic light- and electron-microscopic features, apart from the absence of granules with crystalline cores. The eosinophils produced were readily recovered, and both immature myelocytes and mature cells were functionally active in an antibody-dependent, cell-mediated cytotoxicity assay. mEDF added into the assay enhanced the cytotoxicity but to a lower degree than previously reported for peripheral blood eosinophils, which suggests that they may be partially activated. The possibility that eosinophils could be deactivated was tested by removing mEDF from the culture medium. The eosinophils retained viability and functional activity, however, and showed no increased ability to be activated by mEDF for up to six days after removing the mEDF. The liquid culture of human bone marrow was shown to be an alternative assay for eosinophil differentiation factors to colony formation.     

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.     

The role of pulmonary cellular reactions in the resistance of vaccinated mice to Schistosoma mansoni

A histopathological and ultrastructural study was made of schistosomula and associated inflammatory reactions in the lungs of normal mice, and mice previously vaccinated with irradiated cercariae. In normal mice at day 7 post-infection all schistosomula were located in blood vessels. From day 11 onwards an increasing proportion of schistosomula were intra-alveolar (80% from day 20). No cellular reactions were evident around intravascular parasites in normal mice but at later sampling times large compact foci were associated with alveolar parasites. Initial reactions, probably in response to non-specific tissue damage, were approximately 50% polymorphonuclear, and 50% mononuclear. Mononuclear cells predominated at later times. In spite of inflammation, no damage to the schistosomula was observed. There was no evidence for re-entry of schistosomula into blood vessels, and it was assumed entry into alveoli occurred accidentally as parasites attempted to traverse pulmonary blood vessels. The pattern of localization of schistosomula in vaccinated mice was similar to that in normal mice, the proportion in alveoli increasing with time (64% from day 20). The most significant difference was that intravascular schistosomula attracted foci of host leucocytes which were always 85% or more mononuclear, containing both lymphocytes and macrophages. The infiltrating cells enlarged the intersititium, separating the vascular endothelium from the alveolar epithelium. Fibrous protein was also deposited in the interstitial region. In some instances the complete blood-air barrier was destroyed by the infiltrates. Unusual paracrystalline inclusions were observed in alveolar macrophages and giant cells. The differences in cellular responses in vaccinated and normal mice suggest that challenge schistosomula stimulated an anamnestic immune response. The resulting inflammation, by impeding movement through the vasculature, terminated migration in the lungs, and accounted for the observed resistance to reinfection. The reactions in vaccinated mice have many of the features of a delayed hypersensitivity response implying that lung phase resistance in vaccinated mice may be T-cell rather than antibody-mediated.     

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.     

Eosinophils do respond to fMLP

Eosinophils were isolated from normal human blood by separation over Percoll gradients, which resulted in eosinophil suspensions of a purity higher than 95% and recoveries of about 65%. Normal human eosinophils were found to respond to formyl-methionyl-leucyl-phenylalanine (fMLP) at concentrations greater than 10(-7) mol/L with an increase in the concentration of intracellular free calcium, oxygen consumption, nitroblue tetrazolium reduction, and chemiluminescence. The maximal response of eosinophils to fMLP was lower than that of neutrophils isolated from the same blood samples and required at least ten times as much fMLP as was needed for neutrophils. Low fMLP concentrations (approximately 10(-8) mol/L), which in themselves did not stimulate O2 consumption by either eosinophils or neutrophils, primed these cells to respond to a suboptimal concentration of another stimulus. Purification of eosinophils after treatment of whole blood with fMLP showed that these eosinophils had lost their ability to respond to fMLP. We conclude that normal eosinophils do respond to fMLP and that therefore fMLP should not be used to isolate eosinophils.     

Eosinophilia and resistance to Schistosoma haematobium in man

We have measured the levels of infection with Schistosoma haematobium in children resident in an endemic area of The Gambia before and 3 months after successful chemotherapy and following reinfection. An exposure index was calculated from data collected on water contact, cercarial densities and infected snail densities at water contact sites. Peripheral blood eosinophil levels were recorded and the ability of serum (heat inactivated) from the children to allow killing of schistosomula of S. haematobium was examined. Of 50 children with a post-treatment egg count of less than 1 ovum/10 ml urine, 26 were classified as reinfected, acquiring greater than 1 ovum/10 ml urine over the transmission season. Twenty-four were classified as not reinfected, acquiring less than 1 ovum/10 ml of urine over the same period. These two groups did not differ with respect to their estimated age, weight or pretreatment egg counts. Children who were reinfected had significantly higher levels of exposure and significantly lower peripheral blood eosinophil counts than children who were not reinfected. At all levels of exposure children with high eosinophil counts were less likely to be reinfected than those with lower counts. But antibody-dependent, complement-independent killing of schistosomula of S. haematobium by eosinophils was barely detectable and did not differ between reinfected and non reinfected groups. These observations suggest that subjects with elevated counts are less susceptible to reinfection but the mechanisms involved are not apparent.     

Schistosoma mansoni: loss of the ability of schistosomula to bind mouse complement following intravenous injection into mice

The ability of freshly prepared schistosomula to become opsonized by the alternative pathway of mouse complement in vivo was investigated. Skin schistosomula were intravenously injected into mice and recovered shortly afterwards from their lungs. Following an in vivo residency of a few minutes, most schistosomula had considerably less C3b detectable by immunofluorescence on their surface than worms which had been incubated for the same time with mouse serum in vitro. Deposition of C3b was undetectable on all schistosomula following an in vivo residency of a few hours. Irradiation or treatment with puromycin of the schistosomula prior to injection did not alter the difference between in vitro and in vivo complement deposition. Moreover, schistosomula which had been passaged briefly through a mouse, lost most of their ability to deposit mouse complement on their surface during a subsequent in vitro incubation with mouse serum. It is suggested that opsonization of freshly transformed schistosomula with C3b of murine complement is less efficient in vivo than in vitro.     

Clinical correlates of eosinophiluria

We assessed the clinical correlates of eosinophils in the urine in 65 patients. In 16% of 470 patients whose urine was specifically examined, eosinophils were noted in the urine sediment. Review of the 65 patients with eosinophiluria demonstrated that when eosinophils were expressed as a percentage of total urine white blood cells, 85% (55/65 patients) had less than 5% urine eosinophils and 45% (29/65 patients) had less than 1%. Infection of the upper and lower urinary tract accounted for 45% of the clinical conditions associated with eosinophiluria. In nine (14%) of the 65 patients a diagnosis of acute interstitial nephritis could be made by clinical criteria or from renal biopsy specimens. We conclude that the finding of urine eosinophils is associated with a variety of clinical conditions and may be most useful when expressed as a percentage of total white blood cells in the urine. At a low-percentage positive (less than 5%), it may not be a good predictor of acute interstitial nephritis, but at a higher level (greater than 5%) it may be a more valuable predictor.