Monocytes activate eosinophils for enhanced helminthotoxicity and increased generation of leukotriene C4

© ELSEVIER Paris 1987

Ann. Inst. PasteurlImmunol.

1987, 138, 97-116

MONOCYTES ACTIVATE EOSINOPHILS FOR ENHANCED HELMINTHOTOXICITY AND INCREASED GENERATION OF LEUKOTRIENE C4

by P. Eisas (1), T.H. Lee (2), H.L. Lenzi (3), and A.J. Dessein (1) (1)

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9 (France) (2) Guy's Hospital, London SEI 9RT (UK), and (3) Depto de Patologia, Fiocruz, Rio de Janeiro (Brazil)

SUMMARY

Recent observations have shown that eosinophils are activated in certain clinical conditions and that activation may enhance the role of eosinophils in immune protection against helminth parasites and in the pathogenesis of certain diseases associated with high eosinophilia. Our laboratory has attempted to identify the immunological mechanisms causing such an activation. The data summarized here show that eosinophils can be activated in vitro with supernatants of resting or stimulated monocytes. The supernatants enhance eosinophil helminthotoxicity by increasing cell degranulation; they also enhance the generation of leukotrienes in eosinophils by exerting a permissive effect on an early step of arachidonic acid metabolism. Biochemical analysis of the enhancing activities suggests that they are carried by a unique molecule or a unique set of molecules whose biochemical and functional properties are different from those of previously described monokines such as IL-l, IFN-o:,~, CSF and TNF. Studies on individuals with chronic schistosomiasis suggest that such regulatory interactions between eosinophils and macrophages may take place in the hepatic granulomatous reactions in patients with hepatosplenic schistosomiasis. KEY-WORDS: Monocyte, Eosinophilia, Leukotriene, Allergy, Helminthotoxicity; Immunoregulation, Schistosomiasis.

Received November 24, 1986.

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Introduction. The eosinophil leukocyte is located in the connective tissues under the epithelial layer of the skin, bronchi, gastrointestinal tract and the wall of the uterus [1]. In healthy individuals, only a small number of eosinophils, estimated at 11400 of the total eosinophil pool of the organism, circulate in the blood, where they remain for a short (3 to 8 h) period before entering the tissues. Most infections cause an increase in neutrophil number without increasing blood and tissue eosinophilia. Some bacterial infections cause eosinopenia (reviewed in [2]). Blood and tissue eosinophilia are markedly augmented in individuals with allergic diseases or with helminth infections [3]. In certain of these subjects, eosinophiIs represent up to 90 010 of the blood white cells and may be observed in large numbers in certain organs, i.e. in the bronchi of certain individuals undergoing allergic crisis or in parasitized tissue [2, 4]. The association between atopic diseases, helminthiasis and high eosinophilia have suggested that eosinophils take part in the regulation of allergic reactions, in particular in hypersensitivity type I reactions and in immune protection against helminth parasites [2]. Eosinophils secrete a variety of mediators which might affect the development of the inflammation. The most important of these mediators are probably the 5-lipoxygenase metabolites of arachidonic acid: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4 (LTC 4) [5-9]. 5-HETE is a potent chemotactic factor for neutrophils, LTC4 is a smooth muscle constrictor and a vasospastic and venopermeability factor [10-12]; LTC4 , with its cysteinylglycyl and cysteinyl analogues LTD 4 and LTE 4 , constitutes the activity previously termed slow-reacting substance of anaphylaxis [13]. A critical role of eosinophils in protective immunity against parasites is suggested by numerous studies: eosinophils are strongly helminthotoxic in vitro [14] and can be found attached and degranulating on disintegrated helminth larvae in tissues of immune-infected hosts [15]. Several eosinophil degranulation products such as the major basic protein (MBP), eosinophil cationic protein (ECP) and eosinophil peroxidase are highly toxic for the parasite, either because they dismember parasite tegument [16] or because they generate oxygen radicals [17].

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arachidonic acid. antibody. antibody-dependent cellular cytotoxicity. complement. colony-stimulating factor. eosinophil-activating factor. eosinophil cytotoxicity-enhancing factor. eosinophil cationic protein. eosinophil peroxidase. hypereosinophilic syndrome. 5-hydroxyeicosatetraenoic acid.

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high-performance liquid chromatography. interferon. interleukin-l. leukotriene. major basic protein. minimal essential medium. monokine. nordihydroguaiaretic acid. peripheral blood mononuclear cell. reverse phase HPLC. tumour necrosis factor.

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Since eosinophils appear to be a major component of the host's response to helminth infections, factors modulating their helminthotoxic capacity have received much attention. Lymphokines secreted by cells from Schistosoma mansoni egg granulomas [18] and mast-cell-derived mediators, such as the eosinophil-chemotactic factor of anaphylaxis [19, 20, 21], were shown to potentiate the helminthotoxicity of eosinophils in vitro. Early reports have shown that eosinophils, in certain hypereosinophilic states such as in the hypereosinophilic syndrome (HES) or in tropical eosinophilia, appear sometimes degranulated and vacuolated [22, 23]. The proteins, normally stored in eosinophil granules, were detected in abnormal quantities in the blood of certain of these patients [24]. Since these substances are toxic for mammalian cells, it has been suggested that abnormal degranulation of the eosinophils in hypereosinophilic states may cause or aggravate organ damage associated with certain hypereosinophilia. More recently, eosinophils have also been implicated in the pathogenesis of certain asthmatic conditions. The mechanism(s) causing degranulation of eosinophils in hypereosinophilia is unknown. However, recent observations suggest that it may result from the activation of the cells: eosinophils from individuals with eosinophilia from various aetiological origins exhibit enhanced helminthotoxicity [25], low surface charge, augmented metabolism and high lysosomal enzymatic activities [26]. Finally, eosinophils from hypereosinophilic subjects could be fractionated on density gradients into two fractions, eosinophils in the less dense fraction demonstrated enhanced helminthotoxicity and altered oxidative metabolism, and they may represent an activated cell population [27]. The association between eosinophilia and eosinophil activation has suggested that eosinopoietic factors may have the dual function of stimulating stem cells and activating mature blood eosinophils. This hypothesis has received some support from the demonstration that semi-purified colony-stimulating factor (CFS-ex) of placental origin markedly enhanced the helminthotoxicity of blood eosinophils, probably by enhancing cell degranulation [28]. These observations have been recently confirmed and extended in experiments performed with cloned recombinant CSF-ex. During the course of the CSF studies, we observed that supernatants of resting PBMC also markedly enhanced eosinophil cytotoxicity. Further analysis of this phenomenon showed that it was caused by a monokine which exerts profound regulatory effects on several eosinophil functions. These studies and their possible biological implications are discussed here. Resting human blood mononuclear cells produce, in culture, factors which enhance ADCC by human blood eosinophils. Human peripheral blood mononuclear cells (PBMC) purified by centrifugation of heparinized blood on «Ficoll Hypaque» were cultured (2 x 10 6 cells/ml) in serum-free defined medium; the supernatants were collected 24 h

100

P. ELSAS AND COLL.

later and added to human eosinophils (5 III of conditioned medium for 10 6 eosinophils) which had been purified (> 85 0,10 pure; contaminating cells were neutrophils) from the blood of individuals with 2 to 15 % eosinophilia. After a short incubation period (5 to 60 min) in the conditioned medium, eosinophils were tested in an ADCC reaction on S. mansoni larvae in the presence of heat-inactivated human antischistosomular antibodies. At low Ab concentrations, control medium-treated eosinophils only adhered in small numbers to the larvae (fig. 1) and killed a small fraction (2 to 10 %) of schistosomula (fig. 2). In contrast, eosinophils treated with the conditioned media adhered in large numbers to schistosomula and demonstrated high helminthotoxicity (40 to 80 %). PBMC supernatants were not toxic for the larvae, as assessed by microscopic examination and maturation of worms when injected in

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mice [29, 30]. The factor(s) causing enhancement of eosinophil cytotoxicity was termed ECEF (eosinophil cytotoxicity-enhancing factor). Control eosinophils neither adhered to nor killed schistosomula in the absence of antibodies. However, eosinophils treated with ECEF adhered to the larvae without Ab [31]. The number of adhering cells was low, however, as compared to cells reacted with Ab-coated larvae; nevertheless, this observation suggested that ECEF changed eosinophil surface properties, thus modifying their adherence capabilities. The effect of ECEF on eosinophils from normal individuals and from eosinophilic individuals with atopy, helminth infections, tropical or idiopathic eosinophilia is shown in [31]. Eosinophils were collected on the 23-24 % metrizamide gradient layer. Less dense eosinophils which sedimented on the 22 and 21 070 metrizamide gradient layer were contaminated with neutrophils and/or mononuclear cells and were not included in the study. Mononuclear cell supernatants enhanced the cytotoxicity of eosinophils from almost all normal and eosinophilic individuals. Though few preparations failed to demonstrate enhanced cytotoxicity after incubation in the conditioned medium, this property could not be ascribed to any particular clinical group of individuals. ECEF production in chronic schistosomiasis.

Chronic infections by Schistosoma mansoni present a spectrum of clinical manifestations which are related to the intensity of infection. On one extreme, light infections have no severe clinical manifestations despite chronic egg

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elimination in the faeces. On the other hand, heavily infected individuals develop progressive disease with involvement of the liver and spleen (hepatosplenomegaly), characterized by severe inflammatory reactions of the periportal areas leading to scarring, fibrosis and portal hypertension. These hepatic inflammatory reactions are infiltrated by large numbers of macrophages and eosinophils and usually undergo modulation as the infection becomes more chronic. Modulation is mediated by suppressor T lymphocytes and is accompanied by a diminution of cell-mediated immunity to schistosome antigen (reviewed in [32]). It has been suggested that the progressive tissue destruction characteristic of hepatosplenomegaly may result from a failure to modulate the hepatic inflammatory reactions caused by S. mansoni eggs and to limit the harmful side-effects of products released by activated macrophages and eosinophils [32]. Initial studies in a hyperendemic area for S. mansoni showed decreased production of ECEF in PBMC cultures from most S. manson i-infected individuals relative to healthy controls and to subjects harbouring other helminth parasites. Only 27 and 23 070 of PBMC cultures from S. mansoni individuals in two independent studies produced detectable ECEF activity, whereas PBMC cultures of 84 % of healthy controls produced ECEF. Low ECEF production in the infected subjects could not be attributed to the culture conditions or to the presence of an ECEF inhibitor or a substance interfering with eosinophil activation. ECEF production was studied in patients with different clinical status and egg excretion: PBMC from individuals with light or medium infections produced less ECEF than those of healthy controls; in contrast, PBMC from patients with heavy infections (> 500 eggs/gm of faeces), some of them with developing hepatosplenomegaly, produced ECEF at high levels. This suggested that regulatory mechanisms limited the production of ECEF in light infections and that these controls were no longer operative in individuals with severe hepatic inflammation. These studies also revealed a possible association between ECEF and the inflammatory process, which is consistent with the effect of ECEF on the generation of 5-HETE and LTC 4 by eosinophils, as analysed below. Origin and biochemical properties of ECEF.

Conditioned media prepared with plastic adherent and plastic non-adherent blood mononuclear cells contained enhancing activity [33, 34]. The activity was also produced by non-adherent mononuclear cells depleted of T lymphocytes by rosetting with sheep erythrocytes. Finally, PBMC and plastic adherent cells depleted of T and B lymphocytes by treatment with anti-Leu-l and anti-Ig Ab + C conserved their ability to produce the enhancing activity [33]. These results suggested that ECEF was produced by monocytes. This conclusion was supported by the observation that the human macrophage-

MONOCYTES, EOSINOPHILS, HELMINTHS AND LTC4

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like histyocytic lymphoma U937 and the promyelocytic leukaemia bipotential line HL-60 produced ECEF-like factor(s) [33]. The enhancing activity in PBMC and adherent mononuclear cell supernatants was shown to be destroyed by trypsin treatment and to be stable at 70°C for 1 h. Sephadex-G 100 gel chromatography and isoelectrofocussing of the mononuclear cell supernatants showed that the enhancing activity was associated with molecules having an apparent MW of 14,000 to 60,000 daltons and PI of 3.8, 4.2, 4.5 and 4.8.

Mechanism of the enhancing effect. The bridging of eosinophil Fc receptors by antibodies bound to schistosomula causes eosinophils to secrete the contents of their granules, including the basic proteins MBP and ECP and EPa on the parasite tegument. Tegumental damage is caused by oxygen radicals, by MBP and probably ECP. ECEF has no effect on eosinophil helminthotoxicity in anaerobic conditions [34]. This shows that oxidative metabolism is required for the enhancement of eosinophil cytotoxicity and led us to test the effect of ECEF on the production of oxygen radicals by eosinophils [34]; ECEF neither enhanced superoxide anion production nor increased iodination by resting or stimulated eosinophils. In contrast, CSF-cx, which also enhanced eosinophil helminthotoxicity [28], caused a marked increase in the generation of superoxide anions and iodination by eosinophils [34]. These observations suggested that the enhancement of eosinophil cytotoxicity by mononuclear cells was not primarily due to a stimulation of eosinophil oxidative burst and to the generation of reactive oxygen radicals. The observation that ECEF-treated eosinophils, unlike control cells, were capable of adhering to schistosomula in the absence of antibodies [31,35] suggested that ECEF may enhance cell degranulation and that ECEF-treated eosinophils adhered to degranulation products on the larvae. These products have been shown to act as a « glue» between the eosinophil plasma membrane and the larval tegument [36]. To assess the effect of ECEF on cell degranulation, the extent of eosinophil discharge on Ab-coated schistosomula was measured by light microscopy after labelling EPa products, or by electron microscopy, by estimating the larval surface area covered by electron-dense material of eosinophil origin. Both measurements indicated an increased release of MBP and EPa on the larval tegument by ECF-treated eosinophils, suggesting that the enhanced cytotoxicity is the consequence of an enhanced ability to degranulate [35]. These results, however, did not fully explain the strict oxygen dependency of the enhancement of cytotoxicity caused by ECEF [34]. Since cell degranulation involves important remodeling of the cell membrane and rapid turnover of membrane lipids, and since previous studies have also shown that oxygenation products of arachidonic acid stimulated degranulation of neutrophils [37, 38], we decided it would be useful to evaluate the possible role of arachidonic acid oxygenation metabolites in the activation of eosinophils by ECEF. We tested the effect of inhibitors

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of 5-lipoxygenase (NDGA) or of cyclooxygenase (Indomethacin) on the enhancement of cytotoxicity by monocyte supernatants (fig. 3). Indomethacin had no detectable effect on the enhancement of cytotoxicity. NDGA prevented it when added to eosinophils at the beginning of incubation with ECEF. NDGA, however, did not reduce the helminthotoxicity of control eosinophils, showing that it was not toxic to the cells and that it did not inhibit the killing mechanism itself. These observations, although they should be interpreted with caution since NDGA could affect other cellular oxygenation reactions, suggest that the enhancement of eosinophil cytotoxicity caused by monokines requires normal functioning of the 5-lipoxygenase pathway. This suggested that the monokine may regulate the 5-lipoxygenase pathway in eosinophils.

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Effect of eosinophil-activating monokines on the generation of arachidonic acid metabolites. Arachidonic acid, released from membrane phospholipids after cell activation, is metabolized by the cyclooxygenase pathway to prostaglandins and thromboxane and/or by the 5-lipoxygenase pathway to leukotrienes, according to the cell type. 5-lipoxygenation of arachidonic acid, which occurs

MONOCYTES, EOSINOPHILS, HELMINTHS AND LTC4

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in each type of human granulocyte, produces 5(S)-hydroperoxy-6-trans8, 11, 14-cis-eicosatetraenoic acid; a portion of this compound is reduced to 5-HETE and the remainder is enzymatically converted to an unstable intermediate, 5,6-oxido-7 ,9-trans-ll ,14-cis-eicosatetraenoic acid (leukotriene A 4 , LTA 4 ). LTA 4 is processed in eosinophils into one major product, 5(S)hydroxy-6(R)-S-glutathionyl-7 ,9-trans-ll, 14-cis- eicosatetraenoic acid (LTC 4) by a glutathionyl-S-transferase termed LTC 4 synthetase. In the neutrophil, LTA 4 is converted predominantly to 5(S),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB4) by an epoxide hydrolase [5-9]. The effect of PBMC culture supernatants on the release of LTC 4 by eosinophils activated with the calcium ionophore is shown in figure 4. When the supernatants and the calcium ionophore were added simultaneously to eosinophils, no effect of the supernatants on LTC 4 production was observed. In contrast, preincubation of eosinophils with the supernatants for a few minutes was sufficient to cause a marked increase in LTC 4 generation by CA2 + ionophore-activated eosinophils. Maximal enhancement was observed with eosinophils pre-incubated in the PBMC supernatants for 3 to 5 min. This suggested that no protein synthesis was required for enhancement. The kinetics of LTC 4 generation by control and PBMC supernatant-treated eosinophils which were activated with the Ca2 + ionophore is shown in figure 5: LTC 4 production by supernatant-treated cells was accelerated as compared to con-

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Symbols represent different eosinophil donors. The data represent (1,10 enhancement of LTC 4 generation relative to controls incubated in culture medium containing LPS for the indicated periods of time.

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troIs and reached higher maximal levels. The enhancing effect was observed at all Ca2 + ionophore concentrations [39] ; it was of larger magnitude when the cells were activated with concentrations (1 to 2.5 flM) of calcium ionophore which were suboptimal for the generation of LTC 4 by untreated eosinophils. Enhancement of LTC4 levels in eosinophil supernatants resulted from increased synthesis and not from enhanced release of LTC4 from the cells, since cell-associated LTC4 (measured in methanol extracts of experimental and control eosinophil pellets) represented a minor fraction of total LTC4 (in pellets + supernatants) and did not significantly differ between experimental and control groups.

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The data are from one representative experiment. Each point represents the mean of duplicate determinations on a single experimental sample.

To identify the source of the enhancing activity, PBMC were fractionated according to their adherence properties and surface membrane markers (Ig and Leu-l antigen). The fractionated cells were tested for the production of the enhancing activity in vitro (fig. 6). The enhancing moieties were produced by resting or LPS-stimulated adherent mononuclear cells which were negative for surface immunoglobulins and for the Leu-l antigen. These

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Total or fractionated PBMC from 2 normal individuals were treated with (+) or without (-) anti-Ig and anti-Leu-l antibodies (Ab) and complement (C) and were subsequently cultured at a density of 2 x 10 6 cells/ml in MEM (Eagle's) with 5 IJ.g/ml LPS for 24 h. Supernatants from these cultures were tested on eosinophils from 2 separate donors for enhancement of LTC 4 production after ionophore stimulation. Dashed lines indicate LTC 4 generation by control eosinophils treated with medium + LPS. Supernatants from PBMC cultured in the absence of LPS did not significantly increase LTC 4 production above this level.

findings strongly suggested that the enhancing activity was produced by monocytes. The enhancing effect of various monokine (MK) preparations on LTC 4 generation by eosinophils purified from the blood of 11 different donors is shown in figure 7. MK enhanced LTC 4 production in all but one eosinophil preparation, and the magnitude of the enhancement was inversely correlated

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Panel A, control (-MK) and enhanced (+ MK) LTC 4 production for each one of the 11 individual experiments. Panel B, correlation of the percent enhancement of LTC 4 production by MK preincubation with LTCi production by eosinophils pre-incubated in control medium before stimulation by ionophore. The data in panel B are replotted from the experiments depicted in panel A. Where obtained, the percent enhancement of the responses by eosinophils from the same donor to more than one monocyte supernatant were averaged and depicted as a single point.

(r = - 0.79, P < 0.01) with the amounts of LTC 4 released by eosinophils in the absence of monokine. It was suggested that the MK might have less effect on eosinophils which had been exposed to activating stimuli in vivo, i.e. during an inflammatory process in certain eosinophilic donors. To further analyse the effects of the monokine on AA metabolism, eosinophils were labelled with 3H-arachidonic acid prior to treatment with MK or control medium and stimulation by Ca2 + ionophore. RP-HPLC analysis of the released products showed that MK treatment increased counts at the retention times of LTC 4 (119 and 137 070), 5-HETE (313 and 35 %) and free arachidonic acid (10 and 45 %) in 2 independent experiments, one of which is depicted in figure 8. These results suggested that MK exerts a positive effect on fatty acid hydrolase activity. The enhancing activity was not reduced by heating at 56°C for 30 min and was only partially decreased by heating at 90°C for 30 min. TSK-250 gel filtra-

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tion of the monocyte supernatants yielded four peaks of enhancing activity (150,50,20 and 5 Kd). Isoelectrofocussing of the monocyte supernatant revealed that the enhancing activity was associated with molecules of PI 4.2, 4.5 and 4.9 (fig. 9). This isoelectrofocussing profile showed close similarities with that of ECEF. All peaks of LT-generation-enhancing activity correspond to peaks of eosinophil-cytotoxicity-enhancing activity (fig. 9). Charge heterogeneity has been reported for other cytokines and may be accounted for by postranslational modifications, by limited differences in amino-acid sequences or by several unrelated molecules [40·43]. In summary, monocytes release factors(s) which enhance two biologically important eosinophil functions: helminthotoxicity and generation of AA-5-lipoxygenation metabolites. Although the data do not demonstrate that these effects are caused by a unique molecule or a family of related molecules, several observations point to this suggestion: the two enhancing activities are

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produced by monocytes in the same culture conditions; they are associated with molecules having comparable heat stability, size and charge heterogeneity. In addition, the data suggest that the enhancement of eosinophil helminthot6xicity depends on the normal functioning of the 5-lipoxygenase pathway. Since others have reported that 5-HETE enhanced neutrophil degranulation [37, 38] and that LTC4 and LTB 4 increased eosinophil helminthotoxicity [44], it is possible that the enhancement of cytotoxicity is a consequence of the increased 5-lipoxygenase metabolism of AA. We were unable, however, to

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A. Enhancement of LTC 4 production: eosinophils from 2 different donors were preincubated (2 separate experiments) with the fractions of isoelectric focussing of supernatants prepared with 2 different mononuclear cell preparations; then eosinophils were stimulated and assayed for LTC4 production. LTC 4 production in samples incubated in control medium with LPS was 12 ng/ml (.A.) and 0.6 ng/ml (.)/2 x 10 eosinophils in the respective experiments. B. Enhancement of eosinophil cytotoxicity: eosinophils from 2 different donors were incubated with schistosomula, antischistosomular antibody and fractions from isoelectric focussing. Cytotoxicity was scored after 18 h. No enhancing activity was found outside the pI range 3.5-5 (data not shown).

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confirm the effects of LTC 4 and LTB 4 on eosinophil cytotoxicity and/or to activate the 5-lipoxygenase pathway with Ab-coated larvae. Several findings show that the MK which enhances eosinophil cytotoxicity and LT generation is distinct from previously described monokines: CSF-CI. and interleukin-1 (lL-1), interferons CI. and ~ (IFN-Cl.-and~) and tumour necrosis factor-CI. (TNF), which are also secreted by human monocytes in short-term cultures. IL-1 has been reported to increase the metabolism of exogenously added arachidonic acid in murine thymoma and to stimulate thromboxane synthesis in endothelial cells [45]. However, neither IL-1 nor the interferons increase eosinophil cytotoxicity [46]. Furthermore, IL-1 activity is heat-labile and its activity is destroyed after heating at 70°C for 30 min., unlike the activites described here [47]. IL-1 was also separated from the eosinophilcytotoxicity-enhancing activity in RP-HPLC [48]. The two species of interferons secreted by monocytes, IFN-CI. and IFN-~, are destroyed by heating at 95°C for 30 min. This treatment only slightly affected the enhancing activities described here; furthermore, LPS does not induce large IFN secretion by monocytes (M. Criscuollo, personal communication). Recent reports indicate that TNF-CI. increased eosinophil cytotoxicity [49] and leukotriene production by neutrophils and eosinophils (R. Roubin, P. Elsas, W. Fiers and A. Dessein, manuscript submitted). However, TNF-CI. was not as potent as ECEF for the enhancement of eosinophil cytotoxicity and, unlike ECEF, TNF-CI. enhanced more arachidonic acid metabolism in neutrophils than in eosinophils. Moreover, TNF-CI. enhanced eosinophil respiratory burst, whereas ECEF did not. Finally, high eosinophil cytotoxicity and LT-generation-enhancing activity is released by U937 cells, which are poor producers of TNF-Cl.. A monokine with properties very similar to those of ECEF, termed eosinophil-activating factor (EAF) has been reported independently by Butterworth and collaborators [46,50-52]. EAF is a 40-Kd protein of pI 4.4 produced by monocytes from individuals with moderate eosinophilia. It was shown to increase eosinophil cytotoxicity against schistosomula and against antibodycoated virus-infected mammalian cells. It also increased eosinophil degranulation, superoxide production and hydrogen peroxide production in response to immunological stimulants. It is heterogeneous in molecular weight, with a major component of about 40 Kd and a minor fraction of less than 10 Kd. It is also heterogeneous in charge, as assessed by TSK-DEAE chromatography. Recently, EAF was shown to be different from both CSF-CI. and TNF-CI. in TSK-DEAE-5PW chromatography [46]. Since EAF was shown to account for most of the eosinophil cytotoxicity-enhancing activity in monocyte supernatants, it is likely that EAF and ECEF are the same molecules. It is possible that other activities in these supernatants, i.e. CSF, account for the stimulation of eosinophil respiratory burst by EAF. The understanding of the biological role of these factors in vivo and their structural and functional relationships to other cytokines requires purificaton to homogeneity of the substances. This work is made difficult by the size and

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charge heterogeneity of the active moieties and by the small quantity of these factors in monocyte culture supernatants. The demonstration that U937 cells produce EAF which exhibit functional and biochemical properties identical to those of the blood monocyte product enables us to undertake these studies [48]. RESUME AUGMENTATION DE L'HELMINTHOTOXICITE ET DE LA PRODUCTION DE LEUKOTRIENE C 4 PAR DES EOSINOPHILES ACTIVES PAR DES MONOCYTES

Les eosinophiles sont normalement presents en faible nombre dans Ie sang et les tissus des individus sains; leur nombre est par contre tres augmente dans certaines allergies et dans les helminthiases. Quoique les fonctions biologiques des eosinophiles ne soient pas totalement comprises, Ie role de ceux-ci dans la destruction des helminthes et dans la regulation des reactions d'hypersensibilite immediate est suggere par de nombreuses observations in vivo et in vitro. De recents travaux montrent que les eosinophiles isoles de certains patients sont dans un etat active, et plusieurs groupes de chercheurs, dont Ie notre, se sont interesses aux mecanismes immunologiques responsabIes de cette activation. Nous resumons ici, les resultats de nos etudes concernant Ie role possible des monocytes dans l'activation des eosinophiles humains par des facteurs proteiques solubles. II est rapporte que les surnageants de culture de monocytes humains augmentent tres fortement les capacites helminthotoxiques des eosinophiles ; ceux-ci apres incubation, durant quelques minutes, avec les surnageants sont capables de detruire les larves de Schistosoma mansoni en presence de quantites d'anticorps mille fois inferieures a celles requises par les eosinophiles temoins. Cette augmentation de la cytotoxicite est la consequence d'un effet «potentiateur» exerce par les produits des monocytes sur la degranulation de l'eosinophile. Les surnageants de monocytes augmentent egalement la production de leukotrienes par les eosinophiles; ceux-ci apres traitement durant quelques minutes avec les surnageants sont capables de produire jusqu'a 100 fois plus de leukotrienes que les temoins. Cette augmentation est la consequence d'un effet permissif exerce par les surnageants sur une des premieres etapes du metabolisme de l'acide arachidonique, probablement celles impliquant phospholipases ou 5-lipoxygenase. La ou les molecules produites par les monocytes et capables d'activer les eosinophiles ont ete caracterisees biochimiquement. Cette analyse montre que les differents effets activateurs des surnageants sont tres probablement causes par une seule molecule ou par une famille de molecules et que cette ou ces molecules sont distinctes de IL-l, CSF, INF-
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vations suggerent egalement que l'augmentation de la cytotoxicite des eosinophiles pourrait etre la consequence de l'effet exerce par Ie (1es) facteur(s) sur Ie metabolisme de l'acide arachidonique. Finalement, l'etude de la production de ces activites chez des sujets atteints de schistosomiase chronique suggere que les monocytes pourraient activer les eosinophiles et potentialiser leur fonction inflammatoire chez les sujets atteints de formes hepatospleniques severes. MOTS-CLES: Monocyte, Eosinophilie, Helminthotoxicite, Allergie, Leukotriene; Immunoregulation. ACKNOWLEDGEMENTS We would like to thank Dr Irmeli Penttila for reading the manuscript and Drs 10hn David and K. Frank Austen for helpful advice and discussion during the course of these studies.

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