抗体和(或)补体依赖细胞介导的对周期型马来丝虫微丝蚴细胞毒作用的体外研究

用碘六醇(Iohexol)分离液从大鼠腹腔渗出液中分离出效应细胞用于体外试验。结果表明,在特异性抗周期型马来丝虫微丝蚴(mf)血清存在时,中性粒细胞(Neu)和巨噬细胞(Mφ)对mf都有强烈的粘附和杀伤作用;嗜酸粒细胞(Eos)只表现粘附效应。正常鼠血清存在时,Neu、Mφ对脱鞘mf的粘附作用要高于Eos。血清加入EDTA可明显地抑制效应细胞的粘附和细胞毒作用,而加入EGTA则无类似现象,提示补体通过激活替代途径参与了该反应。用葡萄球菌A蛋白(SPA)或兔抗大鼠IgG免疫球蛋白处理抗血清后,粘附细胞的虫体数显著下降,表明细胞的粘附效应主要依赖于IgG抗体。通过相差显微镜和电镜观察,显示效应细胞粘附于虫体表面,并对其产生损伤作用。     

Involvement of complement and fibronectin in eosinophil-mediated damage to Nippostrongylus brasiliensis larvae

By using IL-5 transgenic mice, it has been shown that eosinophils might play a key role in elimination of larval stages of nematode infections. The present study was carried out to clarify molecular mechanisms involved in the eosinophil-mediated killing of Nippostrongylus brasiliensis larvae. The larvicidal activity was observed in the presence of normal serum in vitro. Electron microscopic observations revealed firm attachment of eosinophils to the cuticular surface of larvae, which was damaged by electron-dense materials released from eosinophils. The larvicidal activity was abrogated by heat- or zymosan-treatment of the serum, whereas depletion of IgG or IgM from the serum did not interfere with eosinophil adhesion and killing. Moreover, pretreatment of eosinophils with monoclonal antibodies against CD11b or VLA-4 inhibited the eosinophil-mediated killing of larvae. Immunofluorescent staining demonstrated the deposition of C3c and plasma fibronectin on the cuticle of the larvae. These results indicate that interactions between CD11b and VLA-4 and their respective counter-ligands deposited on the cuticle are essential in eosinophil-mediated adhesion and damage to larvae of N. brasiliensis.     

Adherence of human eosinophils to infective filariform larvae of Necator americanus in vitro

Eosinophilia is common in hookworm infection but the interaction between eosinophils and the larval stage of the parasite is poorly understood. The present study was conducted to test the ability of the eosinophils to adhere to infective filariform larvae of Necator americanus in vitro. Adherence of eosinophils to the larvae was found to be serum dependent. Antibody facilitated eosinophil adherence but this was maximal in the presence of complement. The adherence was greatly diminished by EGTA treated normal human serum (NHS) and was completely abolished when NHS was treated with either EDTA or heat-inactivation, suggesting that the process can be facilitated through complement activation via the alternative pathway. As with other nematodes, the surface of hookworm larvae appeared to be both antigenic and complement-activating. Although it is not known whether eosinophil adherence has any larvicidal effect, the present study demonstrated for the first time a definite interaction between human eosinophils and hookworm filariform larvae.     

Antibody-mediated in-vivo cytotoxicity to Trichinella spiralis newborn larvae in immune rats

The antibody-dependent cell-mediated larvicidal response of AO rats against Trichinella spiralis newborn larvae was studied in vivo. Rats were immunized with 2000-3000 muscle larvae orally and then challenged 6-20 days later with 10,000-20,000 newborn larvae intraperitoneally. Newborn larvae recovery from the peritoneal cavity decreased significantly and was accompanied by cuticular cell adherence and killing of newborn larvae by day 9 of infection. Similar effects were observed when newborn larvae were incubated with blood obtained from immunized rats. The cell adherence and larvicidal responses reached their peak by day 16 of the primary infection. Passive transfer experiments demonstrated that newborn larvae infectivity was substantially impaired once cell adherence occurred. Cuticular adherence took place in vitro only when immune serum was added to the incubation medium. Complete destruction of newborn larvae in vivo after passive transfer, as measured by muscle larvae burden was only evident after exposure to both immune serum and immune cells, not to either alone. Non-specific stimulation of the peritoneal cavity with a sterile intestinal infection failed to induce cuticular adherence or larval killing in these rats. We conclude that a stage-specific antibody-dependent cell-mediated larvicidal response is rapidly generated in vivo after the host is exposed to newborn larvae. It is a systemic response which impairs the infectivity of newborn larvae and can destroy them before they reach muscle tissue.     

Cytotoxic activity of rat granulocytes against Mesocestoides corti

Rat eosinophils or neutrophils were purified from peritoneal washings which had been enriched either for eosinophils by infection with the parasite Mesocestoides corti or by intravenous injection with Sephadex G200 particles, or for neutrophils by the intraperitoneal injection of glycogen. Neither eosinophils nor neutrophils attached to or damaged live M. corti parasites in vitro although they did lyse chick erythrocytes in the presence of rat anti-chick red blood cell antibody, with the neutrophils showing the highest level of cytotoxicity and the eosinophils from the infected rats the lowest. Neutrophils gave a specific antibody-dependent cytotoxic response to chick erythrocytes coated with solubilized M. corti extract, a response not seen with eosinophils. The cytotoxicity shown by neutrophils could not be blocked by adding eosinophils or sera obtained from chronically infected rats although it was reduced by incubating the neutrophils with cell-free supernatants obtained from the spleen cells of infected rats following stimulation with solubilized parasite extract in vitro. Eosinophils from infected rats expressed fewer membrane Fc receptors for antibody than did neutrophils or eosinophils from uninfected animals. Incubation of neutrophils and eosinophils from uninfected rats with the immune spleen cell supernatants reduced Fc receptor expression to levels similar to those seen with eosinophils from infected animals. These same supernatants had no effect on the expression of granulocyte complement receptors. It is suggested that infection of rats with M. corti can lead to the production of an antigen-specific suppression capable of impairing the antibody-dependent activity of granulocytes in vitro.     

Immune cytotoxic activity of human eosinophils against Trichinella spiralis newborn larvae

The ability of human eosinophils to kill the newborn larvae (NBL) of Trichinella spiralis of different maturation status, in the presence of antibody, was studied. A cytotoxic in vitro test was performed using NBL less than 2h of age (NBL₂) or NBL maintained in culture at 37°C for 20 h (NBL₂₀), peripheral blood eosinophils, anti-Trichinella serum and human fresh serum as source of complement. Under these experimental conditions eosinophils from normal individuals attached to NBL₂ as well as to NBL₂o but only the latter were killed. On the other hand, eosinophils from volunteers with eosinophilia killed NBL regardless of larval age. Neither adherence nor significant mortality was observed in the absence of immune serum. These results indicate that NBL maturation and eosinophil activation status are crucial for antibody-dependent cellular cytotoxic reaction (ADCC).     

Candidacidal activity of Crohn''s disease neutrophils.

The ability of normal and Crohn's disease neutrophils to kill Candida albicans has been studied using neutrophils isolated from peripheral blood and suspended in phosphate buffered saline at 5 x 10(6) cells per ml. C albicans was grown to a stationary phase in broth culture and suspended in phosphate buffered saline at 10(7) organisms/ml. Neutrophils and Candida were then incubated together at 37 degrees C in a shaking water bath in the presence of fresh serum. At 30 and 60 minutes samples were withdrawn, neutrophils lysed, and Candida survival assessed by colony counting. Results were compared with control suspensions of Candida incubated with serum alone. After 30 and 60 minutes in the presence of autologous serum normal neutrophils had killed significantly more Candida than Crohn's disease neutrophils (mean (SD) 61.0 (16.7)% v 40.5 (16.2)% at 30 minutes, p less than 0.0001; 83.2 (7)% v 70.8) 16)% at 60 minutes, p less than 0.005). The results did not alter significantly when normal neutrophils were incubated with Candida in the presence of Crohn's disease serum instead of normal serum. When Crohn's disease neutrophils were incubated with Candida in the presence of normal serum instead of autologous serum there was some improvement in candidacidal ability at 30 minutes (48.9 (20.6)% v 40.5 (16.2)%, p less than 0.03) but not at 60 minutes. Phagocytosis, measured using a radiometric assay, was normal. Neutrophils from patients with Crohn's disease have an impaired ability to kill this granuloma provoking organism. It is not due to serum inhibitors or defective phagocytosis.     

Serum dependent cell-mediated immune reactions to Brugia pahangi infective larvae

Fresh normal rat serum (fNRS) promoted adherence and cytotoxicity of albino rat neutrophils and macrophages to Brugia pahangi infective larvae (L3) in vitro. EDTA and not EGTA abolished the adherence activity suggesting the involvement of complement components via the alternate pathway. C3 molecules were detected on the surface of the parasite by immunofluorescence. fNRS depleted of complement by treatment with Zymosan A or of factor B by heating at 50 degrees C for 20 min, failed to promote cell adherence to the parasite. fNRS and cells from albino rat were more potent in inducing cytotoxicity to L3 than those from jird or Mastomys which may reflect the greater resistance offered by the albino rat to B. pahangi infection. In the presence of IgG and a heat labile factor, possibly complement, of immune serum, neutrophils and macrophages and to a lesser extent eosinophils adhered to and killed the larvae. Immune sera raised against microfilariae of different filarial parasites promoted cell-mediated cytotoxicity to B. pahangi L3 suggesting sharing of antigens between the two stages.     

Human immunoglobulin G mediates protective immunity and identifies protective antigens against larval Strongyloides stercoralis in mice

Protective immunity to larval Strongyloides stercoralis in mice has been shown to be dependent on antibody, complement, and granulocytes. The goals of the present study was to determine the following: (1) whether human serum could passively transfer immunity to mice, (2) the mechanism by which the serum mediated killing, and (3) whether the antigens (Ags) recognized by the protective human antibody could induce protective immunity in mice. Immunoglobulin G (IgG) from a S. stercoralis-seropositive individual passively transferred immunity to mice. The antibody required granulocytes, but not eosinophils, and complement activation to kill the larvae. Antibody-dependent cellular cytotoxicity was not required for larval killing. Immunization of mice with soluble larval Ags isolated by use of the protective immune IgG resulted in protective immunity. In conclusion, immunity could be transferred to mice by IgG from immune humans, and Ags identified by the immune human IgG induced protective immunity in mice, which thereby suggests their possible use in a vaccine against this infection.     

Immunoglobulin G-dependent classical complement pathway activation in neutrophil-mediated cytotoxicity to infective larvae of Angiostrongylus cantonensis

The participation of antibody and complement in cell-mediated adherence and cytotoxicity to infected larvae (L3) of Angiostrongylus cantonensis was investigated in vitro. Of the different cell types involved in the reaction, neutrophils were seen to have a predominant role in immune serum--dependent adherence and cytotoxicity to L3. In the presence of immune serum, cytotoxicity to L3 by neutrophils from infected rats was twice that of neutrophils from normal rats. Although mononuclear cells and eosinophils from infected rats significantly increased the adherence to L3, they had little lethal effect on L3. A further study using gel filtration (Sephacryl S-200) and affinity chromatography (protein A) revealed that immunoglobulin G (IgG) alone was responsible for the complement activation in neutrophil-mediated killing of L3. Neither adherence nor cytotoxicity to L3 by neutrophils were affected when immune serum was heated to 50 degrees C or treated with zymosan, but they were markedly decreased when immune serum was treated with Mg2(+)-ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results of this study indicate that the neutrophil-mediated adherence and cytotoxicity to L3 of A. cantonensis are mediated through IgG-dependent classical complement pathway activation.     

Increased activity of macrophages from mice infected with Brugia pahangi''. in vitro adherence to microfilariae

After the inoculation of infective larvae of Brugia pahangi into the peritoneal cavities of CBA/Ca mice adult worms developed, but by 12 weeks post-infection the parasites were usually dead and surrounded by granulomatous tissue. Macrophages were the most common cell type in these granulomas and were also found in increasing numbers free in the peritoneal cavity. We have investigated the ability of macrophages to damage microfilariae in vitro, in a system where microfilariae were cultured together with cells and serum taken from either uninfected female CBA/Ca mice or at various intervals after infection. Macrophages adhered to and killed microfilariae in this system and there was an increase in vitro adherence of these cells during the course of infection. The peak level of in vitro activity of these cells coincided with the phase of parasite killing in vivo. The presence of serum also affected the degree of macrophage adherence and subsequent death of the parasite. Analysis of serum components using EGTA, zymosan or heat inactivation suggested that complement and possibly heat labile antibody were involved. Immunoglobulins were shown by immunofluorescence to be present on the surface of microfilariae cultured in serum from infected mice. It is concluded from this study that macrophages are actively involved in the termination of murine filarisis.     

Inhibition of caspase-dependent spontaneous apoptosis via a cAMP-protein kinase A dependent pathway in neutrophils from sickle cell disease patients

Sickle cell disease (SCD) is a chronic inflammatory condition characterized by high leucocyte counts, altered cytokine levels and endothelial cell injury. As the removal of inflammatory cells by apoptosis is fundamental for the resolution of inflammation, we aimed to determine whether the leucocyte apoptotic process is altered in SCD. Neutrophils from SCD individuals showed an inhibition of spontaneous apoptosis when cultured in vitro, in the presence of autologous serum for 20 h. Intracellular cyclic adenosine monophosphate (cAMP) levels were approximately twofold increased in SCD neutrophils; possible cAMP-upregulating factors present in SCD serum include interleukin-8, granulocyte-macrophage colony-stimulating factor and prostaglandin. Accordingly, co-incubation of SCD neutrophils with KT5720, a cAMP-dependent protein kinase (PKA) inhibitor, abrogated increased SCD neutrophil survival. Caspase-3 activity was also significantly diminished in SCD neutrophils cultured for 16 h and this activity was restored when cells were co-incubated with KT5720. BIRC2 (encoding cellular inhibitor of apoptosis protein 1, cIAP(1)), MCL1 and BAX expression were unaltered in SCD neutrophils; however, BIRC3 (encoding the caspase inhibitor, cIAP(2)), was expressed at significantly higher levels. Thus, we report an inhibition of spontaneous SCD neutrophil apoptosis that appears to be mediated by upregulated cAMP-PKA signalling and decreased caspase activity. Increased neutrophil survival may have significant consequences in SCD; contributing to leucocytosis, tissue damage and exacerbation of the chronic inflammatory state.     

Reactive oxygen intermediates from eosinophils in mice infected with Hymenolepis nana

A large number of eosinophils were recruited to the intestinal villi after infection with Hymenolepis nana . Eosinophil numbers were increased more rapidly in challenged mice than in primary infected mice. Local intestinal eosinophils from challenged mice showed more extracellular oxygen radical release, as assessed by histochemical mothods using nitro blue tetrazolium, accompanied with tissue injury and larval degradation. Intestinal eosinophils isolated from the lamina propria induced specific oxygen radical generation in response to H. nana oncosphere extract as measured by luminol-dependent chemiluminescence. This response was stronger in challenged mice than in primary infected mice. Radical generation from uninfected mice was negligible. Lipid peroxidation in the small intestine, as measured by formation of malondialdehyde, was increased during H. nana challenge infection, the peak activity coinciding with the elimination of challenge larvae. Continuous administration of a NADPH oxidase inhibitor to sensitized mice interfered with the degeneration of challenge larvae. These results suggest that intestinal eosinophils may be the major contributor to oxygen radical production in response to H. nana and that reactive oxygen species may play a part of effector molecule in the resistance to reinfection with H. nana     

支气管哮喘患者外周血炎性细胞凋亡的研究

目的　观察支气管哮喘患者外周血嗜酸粒细胞 (EOS)、淋巴细胞和中性粒细胞对地塞米松及孟鲁司特凋亡诱导作用的反应性 ,并探讨差异的分子机制。方法　 18例支气管哮喘患者外周静脉血分离淋巴细胞、EOS和中性粒细胞 ,体外培养时分别加入地塞米松和孟鲁司特 ,流式细胞仪检测细胞凋亡率和Fas受体表达率 ,用酶联免疫吸附测定 (ELISA)法检测细胞中半胱氨酸天冬氨酸蛋白酶 3 (caspase 3 )水平。结果　 ( 1)凋亡率 :体外培养时淋巴细胞、EOS和中性粒细胞的自发凋亡率分别为 ( 6 9± 0 7) %、( 3 1± 11) %、( 3 2± 3 0 ) % ,地塞米松刺激后的凋亡率分别为 ( 17 1± 10 8) %、( 4 4±2 2 ) %、( 3 5± 2 4) % ,孟鲁司特刺激后的凋亡率分别为 ( 2 2 5± 17 6) %、( 50± 2 7) %和 ( 55± 2 2 ) % ,地塞米松和孟普司特组淋巴空白细胞和EOS凋亡率与空白对照组比较 ,差异有显著性 (P <0 0 1、0 0 5) ;而中性粒细胞与EOS比较 ,差异无显著性 (P >0 0 5)。 ( 2 )Fas表达率 :淋巴细胞、EOS和中性粒细胞的Fas基础表达率分别为 ( 1 50± 0 0 7) %、( 2 2 0± 0 10 ) %和 ( 1 2 1± 0 0 9) % ,地塞米松刺激后分别为( 6 58± 2 10 ) %、( 7 52± 3 2 0 ) %和 ( 3 2 4± 2 3 4 ) % ,孟鲁司特刺激后分别为 ( 5     

Expulsion of Nematospiroides dubius from the intestine of mice treated with immune serum

This paper describes experiments which demonstrated that the survival of Nematospiroides dubius was severely impaired in mice treated with immune serum. CFLP donor mice were given a series of infections ranging from 25 to 200 infective larvae, at weekly intervals for 6 weeks. The mice were treated with anthelmintic on day 21 and/or day 28 to prevent the accumulation of lethal numbers of parasites in the intestine, and were bled between day 42 and day 49. Female NIH recipient mice were given a total of 2.0-2.5 ml of immune serum i/p., in several separate smaller doses at various times in relation to the day of infection. Between the administration of immune serum begun during the first 4 days of infection and the animals being killed within the next 3 weeks, the mice harboured fewer worms than control animals, the worms were stunted and their fecundity was greatly reduced. Furthermore, these worms were subsequently lost from the intestines of treated mice, during and after the fourth week of infecton. These effects on N. dubius were not observed when mice were given normal serum nor when immune serum was administered after day 6 of the infection. The delayed rejection of adult worms from mice treated with immune serum is of particular significance and suggests that immune serum contained factors which facilitated the expression of a second component in worm expulsion not nornally effective in a primary infection. The possible immunologcal mechanisms underlying these findings are discussed and related to the immunosuppression which N. dubius is known to induce in the host.     

Temporal recruitment of neutrophils and eosinophils to the skin in a murine model for onchocercal dermatitis.

The parasitic helminth Onchocerca volvulus causes ocular onchocerciasis (river blindness) and onchocercal skin disease. To understand the immunologic basis for early stage skin disease, we developed a model in which C57B1/6 mice were immunized subcutaneously and injected intradermally (in the ear) with soluble O. volvulus antigens (OvAg). We found that ear thickness increased significantly after intradermal injection of OvAg and remained elevated for at least 7 days. Dermatitis was dependent on prior immunization, and was associated with an intense cellular infiltrate in the dermis. Neutrophils were the predominant inflammatory cells in the dermis 12 hr after intradermal injection, with only occasional eosinophils present. Conversely, increased ear thickness at later time points was associated with eosinophils, and neutrophils were only rarely detected. Both cell types were present at intermediate time points. These data indicate that recruitment of neutrophils and eosinophils to the skin is temporally regulated.     

Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation

For a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1-ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.     

Products of neutrophils and eosinophils increase the responsiveness of human isolated bronchial tissue

This study examines the possibility that products of neutrophils and eosinophils could increase the responsiveness of human isolated bronchial tissue. Neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers. The cells were incubated with 1 microM calcium ionophore A23187 for 10-15 min then centrifuged, the supernatant collected and stored at -70 degrees C. Human bronchial rings (2-3 mm diameter, 3-4 mm long) were prepared from specimens resected at thoracotomy. The tissues were suspended in organ baths under a 1 g load and changes in tension measured isometrically. Stable contractions to bolus doses of histamine (0.1-10 microM) or to electrical field stimulation (40-100 V, 4-16 Hz, 1 ms for 20 s) were established. Supernatant from 106 neutrophils or 105 eosinophils was then added and tissue responsiveness reassessed. Neutrophil supernatant increased tissue responsiveness to histamine and electrical field stimulation by 54 +/- 17% (n = 5, p less than 0.05) and 18 +/- 7% (n = 6, p less than 0.05), respectively. Eosinophil supernatant increased the histamine response by 60 +/- 23% (n = 8, p less than 0.05) while tissue responsiveness to electrical field stimulation was unchanged (n = 3). Thus, as neutrophils and eosinophils can change the responsiveness of human bronchus in vitro it is possible that they do this in vivo and may not simply be temporally related to the development of bronchial hyperresponsiveness.     

Activation of the alternative complement pathway in normal human serum by Loa loa and Brugia malayi infective stage larvae

Infective stage larvae (L3) of Loa loa and Brugia malayi upon in vitro incubation with normal human serum activated the alternative complement pathway. C3 conversion products were detected on larval cuticles by eosinophil adherence and by immunofluorescence with C3c antiserum. No evidence for cuticle binding of IgG, IgA, IgM, Clq, or C4 was found by immunofluorescence. L3-induced C3 activation was inhibited by 10 mM EDTA but unaffected by 10 mM Mg++-EGTA. Human sera deficient in C2, C4, or C6 incubated with L3 resulted in C3 activation. However, sera treated with zymosan or heated for 1 h, 56 degrees C were unreactive with L3. Immunoelectrophoresis of fresh serum exposed to L3 for 1 h at 37 degrees C showed C3 cleavage products. The results indicate that these nematode L3 activate the alternative complement cascade via cuticular surface components. Larval viability was unaffected by complement activation or by adherence of eosinophils.