Stimulus-secretion coupling and leukotriene formation in the triggering of immediate hypersensitivity reactions

186

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and during IgE anaphylaxis in the rabbit. J. Immunol., 1979, 123, 28352841. ~281 MOIILEY, J., SANJAR, S. • PAGE, C. P., The platelet in asthma. Lancet, 1984, 1I, 1142-1144. [29] RANK, J. A., MITCHCOCK, M., MERmLL, W., BACH, M. K., BRASHLER, J. R. & ASKENASE, P. W., Ig-E-dependent release of leukotriene C4 from alveolar macrophages. Nature (Lond.), 1982, 297, 329-331. I30] SI~DIVY, P., CAILLARD,C. G., CARHUETTE,A., DI~RI~GNAUCOURT,J. & MONDOT, S., 48740 RP: a selective anti-PAF agent, in ,, Inflammation: Proceedings of the I I I r d International Congress ,,. (M. Chignard, J. M. Mencia-Huerta & F. Russo-Marie). Raven Press, New York, 1985. ]31] SHEN, T. Y., HWANG, S. B., CHANG, M. N., DOEBBE~, T. W., LAM, M. H. T., Wu, M. S., WANG, X., HAN, G. W. & LI, R. Z., Characterization of a platelet-activating factor receptor antagonist isolated from haifenteng (Piper futokadsura): specific inhibition of in vitro and in vivo plateletactivating factor-induced effects. Proc. nat. Acad. Sci. (Wash.), 1985, 82, 672-676. '[32] Slaoxs, P., RoY, J. P., TETnAULT, P., BOnGEAT, S., PICAnD, S. & COaEV, E. J , ProM. Med., 1981, 7, 327. I33] SONGSImDEJ, V., BussE, W. \V. & BUCKNER, C. K., Pharmacological alteration of antigen-induced contraction of lung parenchymal strips isolated from actively sensitized guinea-pig. Earop. J. Pharmacol., 1983, 92, 215-222. .[34] STI.~tLER, N. P., BACH, M. K., BLOOr~, C. L. & HUGLI, T. E., Release of leukotrienes from guinea-pig lung stimulated by CSa des arg anaphylotoxin. .1. Immunol., 1982, 128, 2247-2252. I351 STIMLER,N. P., BLOOT, C. M., MUGEI, T. E., WYKLE, R. T., McCALL, C. E. & O'FLAHERTY, J. T., Anaphylactic actions of platelet-activating factor. Amer. d. Path., 1981, 105, 64-69. I36] VARGAFTIG, ]~. B., LEFORT, J., CHIGNARD, M. & BENVENISTE, d., Plateletactivating factor induces a platelet-dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives. Europ. J. Pharmacol., 1980, 65, 185-192. I37] VARGAFTIO, ]3. n., LEFORT, J. & MURPHY, R. C., Inhibition by aspirin of bronchoconstriction due to leukotrienes Ca and D~ in the guinea-pig. Europ. J. Pharmacol., 1981, 72, 417-418. 11381 VZSS~N, M. F., BOUnGOIN, S., LmDZR, D. & HARBON, S., Lipoxygenase and cyclooxygenase products of arachidonic acid in uterus. Selective interaction with the cAMP and gAMP systems. ProM. Leuko. Med., 1984, 13, 75-78.

:STIMULUS-SECRETION COUPLING AND L E U K O T R I E N E FORMATION IN T H E T R I G G E R I N G OF IMMEDIATE H Y P E R S E N S I T I V I T Y REACTIONS by P. Braquet Q), P. Borgeat (2), A. Etienne (1) and M. Braquet (a)

41) lnstilnl Henri-Beau/our, 17, avenue Descartes, 92350 Le Plessis-Robinson (France), (2) Groupe de Recherches sur les Leucotri~nes, Centre Hospitalier de l'Universild Laval, 2705, boulevard Laurier, Quebec G I V 402 (Canada), and (a) C R S S A , Centre de Recherches du Service de Santd des Armdes, Division de Radiobiochimie, 1 bis, rue Raoul-Balany, 92140 Clamart (France) INTI~ODUCTION Immediate type 1 allergic reactions ~)ccur following the interaction be-

tween allergen, immunoglobulin E (IgE) and mast cells or basophils [1]. Other immunoglobulins such as IgG and IgA do not combine with these

L E U K O T R I E N E S IN A L L E R G Y cells, or else they bind with much lower affinity and are not able to mediate reaginic hypersensitivity reactions. The specific binding of IgE molecules to the target cells is due to unique structures in the Fc portion of IgE molecules [2l. These receptors (Fc.,R) are found on most cells involved in the immune response, such as mast cells [3, 4], basophils [5, 6], macrophages [7[ and eosinophils [8]. The immediate reaction of cell-bsund IgE antibody with allergen triggers the release from mast cells or basophils of either preformed mediators stored in secretory granules (i. e. histamine, serotonin, glycosamino-glycans, neutral proteases...) or newly found mediators (PGD2, platelet-activating factor (PAF, AGEPC) and slow-reacting substances (SRS, SRS-A)) (for review, see [9]). Irate IgE-dependent cutaneous and pulmonary reactions elicit an influx of eosinophils, neutrgphils and m~nonuclear cells. Among the various mediators released in immediate-type hypersensitivity reactions, SRS constitutes one of the most potent families. SRS activity was first recognized by Feldberg el al. [10] in 1938 in perfusates of c~bravenom-challenged guinea-pig and later on in diffusates of human lung fragments [11] and antigen-challenged guinea-pig lung [12]. However, it was not until 1979 that SRS was identified for the tirst time as 5-hydroxy-6-(S)glutatbionyl-7,9,11,14-eieosatetrae~loie acid, termed leukotriene C [13]. This discovery followed the identification of novel 5-hydroxylated compounds synthesized by rabbit polymorphonuclear leukocy~es in 1979 by Borgeat and Samuelsson [14]. Figure I illustrates the structure and the formation of leukotrienes. Under the action of a 5-1ipoxygenase, the biosynthesis by transformation of arachidonic acid (AA) into 5-(S)hydroperoxyeicosatetraenoie acid (5HPETE) and leukotriene A4 (LTA4) (5,6-oxido- 7,9,11,14 - eicosatetraenoic acid) [14, 16, 17] by the action of a specific hydrolase, and, since it is the precursor of the SRS LTC4, by the action of a specific glutathione-S-transferase [18].

187

LTC4 is then cleaved by 7-glutamine transpeptidase to yield I,TD4 which, in turn, is cleaved by an LTD dipeptidase to yield LTE4 [18]. These latter transformations are accompanied by the loss of the residues ~,-glutamyl and glycyl. Besides LTA4 formation, 5-HPETE also yields 5-hydroxyeicosatetraenoic (5-HETE). Variations in fatty acid composition influence leukotriene structure and function: eicosatrienoic acid (n-9), eicosapentaenoic acid (n-3) and eieosatrienoic acid (n-6) yield, respectively, LTCs, LTC~ and 8,9-LTC3. These leukotrienes are liable to analogous degradations of the tripeptide part. On the other hand, depletion of glutathione results in diminished levels of peptidoleukotrienes and enhanced synthesis of H E T E in mouse macrophages. Since the subject is extremely vast, we have limited ourselves in this paper to recalling the putative mechanisms of the membrane signal leading to the formation of leukotrienes in IgE-stimulated mast cells. We will subsequently see which cells involved in this immediate-type hypersensitivity are the most powerful producers of leukotrienes in relation to tile different potential stimuli. In a final stage, the role of leukotrienes in the modulation of specific cellular immunity will be reviewed. ~IECHANISM OF IGE-TRIGGERED MEMBRANE SIGNAL LEADING TO LEUKOTBIENE FORMATION.

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189

appreciated t h a t I g E - m e d i a t e d histam i n e release and l e u k o t r i e n e generat i o n are difficult to separate (fig. 2). I n several systems, evidence has b e e n gathered which s u p p o r t s the hypothesis t h a t the r e e e p t o r / l i g a n d i n t e r a c t i o n in the m e m b r a n e stimulates the t u r n o v e r of p h o s p h a t i d y l i n o s i -

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tol, and it is this turnover which leads to the formation of calcium channels in the membrane [19]. It appears that stimulation of rat mast cells with antigen, anti-IgE, eoncanavalin A, compound 48/80 and the iuophore A23187 produces increased turnover of phosphatidylinositol [20, 21] (fig. 2). The stimulus-secretion coupling which intervenes in mast cells and leads to phosphatidylinositol turnover is independent of calcium and has a time-course similar to that for secretion [20]. As might be expected, concentrations of ligand-inducing phospholipid turnover are somewhat lower than those inducing histamine secretion, indicating that a certain level of turnover has to be achieved before a secretory response is manifest. It was pointed out above that a certain level of calcium entry needs to be achieved before secretion occurs. Apart from phosphatidylinositol, phosphatidic acid and phosphatidycholine have also been reported to be turned over at increased rates in the stimulated mast cells, but phosphatidylserine, phosphatidylethanolamine and sphingomyelin do not show such increased turnover [21, 22]. Another hypothesis which has been put forward to explain the relationship between phospholipid metabolism and calcium in the activation of histamine secretion concerns the methylation of phosphatidylethanolamine in the membrane [23, 24]: inhibitors of S-adenosyl-L-methionine-mediated methylation, such as S-isobutyryl-3-deazaadenosine or 3-deaza-adenosine, inhibit 45Ca influx and release [25]. As demonstrated by Hirata and Axelrod [26], the methylation process leading to Ca ~+ influx involves two transferases: methyltransferase-I (MT-I) faces the intracellular compartment and transfers one methyl group from Sadenosyl methionine to phosphatidylethanolamine (PE) to form phosphatidyl-N-monomethylethanolamine (PME). The second enzyme, methyltransferase II, faces the external surface and adds two more methyl groups successively, resulting in the formation of phosphatidylcholine (PC)

aud its translocation from the cytoplasmic to the extracellular side of the membrane. This re-orientation of phospholipids within the membrane decreases bilayer microviscosity and the increase in membrane fluidity may allow for coupling of IgE-Fc receptors to adenylate cyclase and an increase in Ca 2+ flux through calcium channels into the cells (fig. 2). The activation of phospholipid inetaboli~m is accompanied by a. rapid and transient monophasic rise in cyclic 3',5'-adenosine monophosphate (cAMP). This increase appears in the first ten seconds and is followed by a fall either below or back-to-basal level in the following 60 [27, 29]. The transitory increase may be in part related to a change in phospholipid metabolism: the binding of the IgE-antigen complex to F e a r would activate the regulatory unit of adenylate cyclase leading to cyclase triggering (fig. 3 ; see [30] for details), cAMP could influence the membrane signaling of IgE-activated mast cells in two ways: (1) activation of protein kinase leading to phosphorylation and release, and (2) a reduction in the calcium permeability of the membrane [31]. 2. - - Non-antigen receplors in masl cell signaling processes.

It is classically accepted that basic proteins are able to trigger secretions from PMNL or mast cells ]32, 33]. The reasons for this stimulation were unclear for a long time, but recent results are helping to understand this phencinenon. Elsewhere, a series of works by Stanwcrth's team [34, 35] showed that histamine release upon IgE challenge could be explained by the existence in the IgE chain of a basic, peptide fragment ]35]: e. g. Lys-Thr-Lys-Gly-Ser-Gly-Ser-GlyPhe-Phe-Val-PHe-Nh2, which would generate a signal on the membrane at the level of a second receptor other than Fc~R (fig. 4). Contact between this basic peptidic fragment and the membrane could possibly occur by a confcrmational change within the cellbound anaphylactic antibody molecule. This assumption was confirmed by the use of basic, peptidic fregments

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comparable to the natural ones and which were able to trigger hi'~tainine release from mast cells with the same efficiency [35]. We recently demonstrated t h a t Inelittin, a 25-ainino-acid peptide, was a powerful stimulator of the formation of leukotrienes and hydroxyeieosatetraeuoie acids in human leukocytes ]36]. The main Inetabolites produced were LTB4, LTC4, 5-HETE, 12H E T E , 15-HETE, 5S,12S-DiHETE and 5S,15S-DiHETE. The mechanism b y which melittin causes lipoxygenase product formation probably involves the ability of basic peptide to stimulate phospholipase A~ and AA release, since the synthesis of Inetabolites was observed in both the absence and pre~,ence of exogenous AA. Therefore, given our previous data, it could be postulated that leukotriene synthesis induced by an immunologic challenge of mast cells partially derives from such an interaction. ~SYNTHESIS OF LEUKOTRIENES BY THE DIFFERENT WHITE BLOOD CELL SUBSETS INVOLVED IN IMMEDIATE-TYPE ItYPERSENSITIVITY.

The synthesis of leukotrienes by mast cells, basophils, PMNL, eosinophils, monccytes and Inacrophages which are all types of ceils involved in hypersensitivity reactions has been extensively studied during the past five years. Very close immunological and biochemical relationships between tile different subsets of leukocytes are involved in such phenomena.

1 . - Cells involved in iininediale IgEdependenl reactions (masl cells and basophils ). Mast cells and basophils share several notable properties. Both contain pronfinent cytoplasmic granules with affinity for certain basic dyes and with electron-dense content shown by electron microscopy. Both mast cells and basophils display plasma membrane receptors (Fc~R) which specifically bind the Fc portion of IgE antibody with l:igh affinity (for review, see [38]). After active or passive sensitization

with IgE, exposure to specific multivalent antigen causes both cell types to undergo an integrated, non-cytolytic series of biochemical and ultrastructural alterations often referred to as anaphylactic degranulation or exocytosis. Both types of cells represent a major source of potent chemical mediators involved in a wide spectruin of inflammatery and immunologic processes. Exocytotie degranulation of basophils and mast ceils may be induced in response to a long and growing list of iinmunologically non-specific agents including certain complement fragments, naturally occurring or synthetic basic peptides, lyinphokines, lectins and some neurotransinitters. Taken together, these observations have demonstrated that mast cells and basophils ~re central participants in diseases of immediate hypersensitivity. Araehidonate Inetabolites are triggered by immunologic (IgE) activation o~ both rat and human mast cells: PGD2 is the predominant Inetabolite [39, 40], whereas only human inast cells generate appreci-ble amounts of 5-1ipoxygenase products which include 5 - H E T E and SBS, the latter being composed of a spectrum of leukotrienes [41, 42]. The main metabolites synthesized after A-23187 challenge appear to be LTC4 and LTD4, whereas LTB, seems to be produced in a lower amount. As with other cell types, Inurine bone-marrow-derived mast ceils, differentiated in vitro and presuIned to be Inucosal-type mast ceils, elaborate 20-50 ng LTC4 and 4-8 ng LTB4/10 ~ cells after IgE-dependent activation, or less than one-half of the leukotrienes obtained from the same cells activated with c~lciuIn ionophore [43]. These Inurine mast ceils generate almost no PGD2, whereas that is the predominant product of oxidative metabolism of araehidonic acid by rat and human connective tissue mast cells activated by an immunologic mechanisin. Furthermore, the main difficulty with mast cells or basophils is to obtain pure cell preparations. Further investigations should thus be performed in order to assess previous findings

LEUKOTRIENES IN ALLERGY (Mencia Huerta el al., personal communication). 2 . - Cells involved in late I qE-dependent reactions. a) Neutrophils. The metabolism of arachidonic acid through lipoxygenase pathways in the neutrophil PMNL has been investigated quite extensively in the past years. In fact, the 5-1ipoxygeuase metabolites were detected for the first time in rabbit peritoneal PMNL stimulated by glycogen [44]. Shortly after, 5-1ipoxygenase and 15-1ipoxygenase products were also isolated from human blood PMNL: a suspension of the latter constituted by 95% neutrophils mainly released LTB4 and 5-HETE during incubation in the presence of the ionophore A23187. Conversely, these metabolites were not produced (or in traces only) upon incubation with exogenous arachidonic acid. This data indicated that the 5-1ipoxygenase of human PMNL does not have spontaneous activity and must be activated in order to catalyze the lipoxygenation of arachidonic acid [45]. Figure 5 shows the classical pattern of lipoxygenase products in human neutrophils stimulated with the ionophore A-23187: LTB~ is the major leukotriene formed. A time-dependent study showed a rapid disappearance of LTB~ from the medium [46]: indeed, an appreciable amount of co-OH-LTB~, a metabolite of LTB4, is formed rapidly in these conditions; co-oxidation continues and also yields the complete oxidized metabolite, o~-COOH-LTB~. Indeed, exogenous LTB4 added directly to human leukocytes is rapidly metabolized into to-OH-LTB4 and to-COOH-LTB4. This type of experiment indicated that non-activated human leukocytes catalyze the u-oxidation of LTB4, similar to ionophore-stimulated cells. Careful quantitation of LTB~ and its two metabolites in these experiments indicated a nearly quantitative oxidation of LTB4 into o~-0H-LTB4 and ~COOH-LTB~; this suggested that, under the experimental conditions used, LTB4 did not undergo any other detectable transformation. Ann. Inst. Pasteur/Immunol., 136 D, n ~ 2 , 1 9 8 5 .

193

It is interesting that other oxidative mechanisms leading to the inactivation of LTB4 and LTC4 have been recently described in phorbol-esteractivated PMNL [47, 48]; these processes, however, did not take place in unstimulated PMNL. The relative importance of the various catalytic pathways of LTB4 under physiological conditions remains unknown. Plasma may dramatically modify the picture of LTB4 metabolism obtained from in vitro experiments performed in serum-free media; it is indeed possible that the binding of LTB4 to serum albumin would protect the compound from catabolic processes. PMNL are weak producers of peptidoleukotrienes: the quantity of LTC4 synthesized upon challenge by A23187 represents only 10% or less than that of LTB~ [49] (fig. 5). The other 5-1ipoxygenase metabolites released by A-23187-stimulated PMNL are the trans-isomers of LTB4 (A6trans-LTBa and A6-trans-12-epi-LTB4) and the 5,6-di-HETE derived from the non-enzymatic hydrolysis of LTA~, the 5-hydroxy-12-methoxyeicosatetraenoic acids derived from methanolysis of LTA, I50] (fig. S). The neutrophils also produce the 15lipoxygenase products 15-HPETE and 15-HETE when incubated with exogenous arachidonic acid [51] and the 5(S),IS(S)-di-HETE when incubated with both exogenous substrate and the ionophore A-23187. The 5(S),I2(S)-diHETE is another lipoxygeuase metabolite frequently detected in incubation media of neutrophils. Its synthesis requires either the addition of exogenous 12-HETE or 12-HPETE to A23187-stimulated PMNL or the presence of an appreciable amount of platelets in the PMNL suspension. Human PMNL alone do not contain 12-tipoxygenase activity. As LTB, is the main metabolite obtained after stimulation, PMNL may participate in the following events involved in immediate hypersensitivity reactions: adhesion of leukocytes to endothelial cells, stimulation of chemotaxis and chemokinesis of other PMNL including eosinophils, stimulation of the degranulation and release lit

194

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of lysosomal enzymes and an increase in the CA ~~ mobilization, all of which are reactions which contribute to the (zdema of the bronchial epithelium and which potentiate and prolong asthmatic attack.

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L E U K O T R I E N E S IN ALLERGY granulocytes, the cells originate in the bone marrow from a common stem cell. Their development is regulated by genetic factors and by products of T lymphocytes. Chemotactic factors of the complement cascade (C5a), lymphokines and eosinophil chemotactic leukotrienes (ECL) stimulate their migration to tissue sites. Generation and biological effectiveness of eosinophil chemotactic factors can be modulated in numerous ways, thus changing the outcome of the inflammatory event. In general, the allergic diseases associated with eosinophilia are those in which an immediate-type hypersensitivity reaction is predominant, i. e., those reactions involving mast cells sensitized by IgE and triggered by specific allergen. Common examples are hay fever and other forms of allergic rhinitis, extrinsic bronchial asthma and general anaphylactic reactions, for example, in subjects sensitive to certain drugs and stinging insects. Present evidence suggests that the interaction of sensitized mast cells with specific antigen leads to the release of factors which are chemotactic for eosinophils, and t h a t eosinophils migrate into the site of degranulated mast cells as a result of chemotaxis and ]or chemokinesis. The use of purified eosinophils from normal subjects as well as an eosinophil-rich PMNL preparation obtained from patients with hypereosinophilia revealed some analogies but also some maj or differences in the transformation of arachidonic acid through lipoxygenase pathways in eosinophils and neutrophils [52-55] (fig. 6). Both white cell subsets display 5- and 15-1ipoxygenase pathways. In addition, similarly to the neutrophil, the eosinophil shows little if any spontaneous 5-1ipoxygenase activity and must be activated by the ionophore A-23187 to generate metabolites. Figure 6 shows the classical pattern of an A-23187-stimulatede osinophilrich PMNL preparation: the major leukotriene formed is LTC,, whereas LTB4 is produced in smaller quantity; this in contrast with the pattern recorded with neutrophils (fig. 5) where

195

LTB4 was the main metabolite formed. In fact, several studies with purified preparations of eosinophils suggested that, among PMNL, eosinophils are the source of LTC4, whereas neutrophils are the source of LTB4 [54]. Furthermore, Shaw el al. [55] showed that the amounts of LTC4 produced by unseparated leukocytes were directly proportional to the percentage of eosinophils in the total cell suspension. Another argument in favor of the dichotomy of the production of LTB4 and LTC~ seemed to be demonstrated by the fact that eosinophils do not catalyze the co-oxydation of LTB4 [54]. High 15-1ipoxygenase activity in the eosinophil also appears to be a feature of arachidonic acid transformation in these cells [53]. In a recent study on 30 eosinophil-rich PMNL preparations, a correlation was found between the present content in eosinophils and the synthesis of 15-HETE (Borgeat, unpublished data). Therefore, eosmophils which accumulated in tissues partly as a result of the response to neutrophil-derived LTB4 contribute to the production of peptidoleukotrienes with subsequent amplification of the acute allergic response. Through this phenomenon among others, markedly elevated numbers of eosinophils can cause auto-aggression against the body's own cells, such as the Purkinje cells of the brain, cardiac muscle cells or epithelial cells of the skin and the bronchial tree. e) Monocyles. It is only recently that leukotriene production by stimulated monocytes has been identified [56-58]. Similarly to neutrophils and eosinophils, the blood monocytes do not show spontaneous 5-1ipoxygenase activity. Monocytes contain both-5 and 15-1ipoxygenase activities: LTB4 is the major leukotriene formed, but LTC4 is also an important metabolite (fig. 7). In contrast to neutrophils, monocytes do not metabolize LTB~ into co-OH and co-COOH metabolites (fig. 7). d) Macrophages. Macrophages are produced in tile marrow, transported to tissues by way

196

9e FORUM D'IMMUNOLOGIE Further, they are capable of ingesting and modifying antigens so as to increase their immunogenicity, thereby amplifying the immune response. Several studies have already shown that the alveolar macrophage contains some 5-1ipoxygenase activity [59, 61]. Figure 8 shows the products formed by ionophore A-23187 and arachidonic-acid-stimulated cells. The profile shows the presence of LTB4, the lransisomers of LTB4 and 5-HETE. Alveo-

of the blood and subsequently lodge and reside in tissue for several weeks. They take up their stations and they serve as non-specific destroyer cells once a signal from the other parLs of the immune system, the trigger mechanism, is received. Because they can produce massive non-selective digestion of the material around them, macrophages can be a major source of inflammation and mediators. Macrophages also possess IgE receptors.

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LEUKOTRIENES

IN A L L E R G Y

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197

of significant spontaneous 5-1ipoxygenase activity; indeed, alveolar macrophages produce detectable quantities of 5 - H E T E and LTB4 when incubated with exogenous arachidonic acid alone. The stimulation of the cells with exogenous substrate and the ionophore A-23187 results in the synthesis of larger amounts of the two metabolites, indicating that the cells contain the active and the inactive forms of the

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ELUTION TIME (rain) FIG. 7. - - Reversed-phase I t P L C analgsis of arachidonic acid metaboliles formed by human blood mononuclear cells (and plalelets) stimulated with 2 FM ionophore A-23187. The cell suspension consisted of 5.3 • l0 s m o n o n u c l e a r cells in 1 ml of PB$ of which 30 % were monocytes (esterase-positive cells), 69 % l y m p h o c y t e s and 1 % PMNL. The platelet contamination was one free platelet/leukocyte, plus 2-3 rosetted platelets/monocytes. See figure 5 for e x p e r i m e n t a l details.

198

9e F O R U M D ' I M M U N O L O G I E gocytes is not clear at the present time, but one m a y speculate t h a t it is related to the state of cell activation. Finally, it is interesting t h a t D a m o n el al. [62] reported the formation of LTDa by cultured alveolar macrophages (adherent cells). The discrepancy between this study and those mentioned above [61] (fig. 8) concern-

5-1ipoxygenase. This is in contrast with blood phagocytes (PMNL and monocytes) which almost exclusively contain the inactive form of the 5-1ipoxygenase and must be activated with the ionophore to produce 5 - H E T E and L T (see above). The meaning of this difference in the levels of spontaneous 5-1ipoxygenase activity between the tissue phagocytes and the blood pha-

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-

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The cell suspension consisted of 1 • 106 mononuelear cells in 1 rnl of PBS, of which 69% were macrophages (esterase-positive cells), 29 % lymphocytes and 2 % PMNL. See figure 5 for experimental details9

LEUKOTRIENES ing LTD4 synthesis in alveolar macrophages may be explained by the different experimental conditions used, i. e. adherent cells and cell suspensions. L E U K O T R I E N E S , M O D U L A T O R S OF S P E CIFIC C E L L U L A R IMMUNITY.

Taking into account the important activity of LTB~ on several functions of neutrophils and the probable role of LTB4 in Ihe immediate hypersensitivity phenomenon, the effects of LT on human lymphocytes have also been recently investigated. Now, it seems proven that LT may act as immunoregulating mediators. 1 . - lnlroducliou o/ suppressor lgmphocyles.

When LTB4 or, to a lesser extent, LTD4 is added to cultures of human mononuclear leukocytes stimulated by mitogenic agents (concanavalin A (or ConA, phytohaemagglutinin A or PHA), a significant inhibition in the lymphoproliferative response is obtained at concentrations as low as 10 -14 M [63]. Similar results were reported by Webb el al. [64], who demonstrated that LTD4 and LTE4in concentrations as low as 10-1~M caused > 50% inhibition of PHA-induced 3H-thymidine incorporation in mouse splenic T cells, while much higher concentrations (10 -7 M) caused inhibition of the formation of antibody-forming cells against sheep erythrocytes in Mishell-Dutton cultures. At the same time, Bailey el al. [65] have found that another lipoxygenase product, 15-HETE, induced a similar effect. From these first observations, it appeared that the activation of suppressor ceils could be involved in these responses. Recently, Atluru and Goodwin [66] demonstrated that LTB4 (10-12-~10-~M), but not LTC4 or LTD,, inhibited polyclonal IgG and IgM production in PWM-stimulated cultures of human peripheral blood lymphocytes. The inhibition was apparently due to the induction OKT8 + radiosensitive suppressor cell by LTB,. The precursor of this suppressor cell may be an OKT4 + cell since exposure of OKT4 + T cells to

IN A L L E R G Y

199

LTB4 resulted in an increase in the percentage of cells bearing OKT8 § markers. Several low molecular we~ight mediators are able to induce T suppressor cells in vitro: Webb el al. [67] reported that glass-adherent mouse splenic T cells which were incubated with 10 -s M P G E became suppressive of subsequent in vitro assays of humoral and cellular immunity, and this PGEinduced suppressor cell acted b y secreting a suppressor factor. Fischer el al. [68] has reported t h a t 10 -e M P G E induces suppressor T cells from normal peripheral blood T cells. Other investigators have reported an analogous suppressor system activated b y 10 -4 M histamine [69, 70]. The level of activity reached by the suppressor cells in the presence of LTB4 was comparable to t h a t reached in the presence of ConA or histamine. However, LTB4 is active at concentrations much lower than those required for histamine (10 -~ -+10-3M). Therefore, LTB4 is three to six orders of magnitude more potent than either P G E or histamine [63]. 2. - - Increase in nalural cyloloxicity.

Suppressor cells can be produced at the same time as T cytotoxic cells [71]; both types possess common membranous markers [72]. Several authors report that PGE inhibits T cytotoxic cells [73, 74] and that, at low concentrations, indomethacin increases the natural cytotoxic activity [73, 75]; in contrast, Rola-Pleszczynski et al. [75] found that nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase pathways, inhibited more than 80% of the natural cytotoxic activity and that this inhibition could be reversed by the addition of LTB4. In a co-culture of mononuclear leukocytes and target cells, LTB4 increases the natural cytotoxic activity in a significant manner, i. e. from 60 to 280% [75, 76]. This increase is not significantly modified by addition of indomethacin, indicating that a plateau is apparently reached in the stimulation of this activity. The amplitude of this increase is similar to that produced b y interferon or interleukin-2 [77].

200

9e F O R U M

D'IMMUNOLOGIE

Re[erences.

[1] ISHIZAKA, T. & ISHIZAKA, K., A c t i v a t i o n of m a s t cells for m e d i a t o r release t h r o u g h l g E receptors, in ~( Progress in a l l e r g y >>, vol. 34 (p. 188-235), K a r g e r , Basel, 1984. [2] FROESE, A., Receptors for IgE on m a s t cells a n d basophils, in (( Progress in allergy ,, vol. 34 (p. 142-187), Karger, Basel, 1984. [3] LAWSON, D., FEWTRELL, C., GOMPERTS, B. & RAFF, M. C., A n t i - i m m u n o g l o bulin-induced h i s t a m i n e secretion of rat p e r i t o n e a l m a s t cells s t u d i e d b y i m m u n o - f e r r i t i n electron microscopy. J. exp. Med., 1975, 142, 391-402. [4] TIGELAAR, R. E., VAZ, N. M. • OVARY, Z., I m m u n o g l o b u l i n receptors on mouse m a s t cells. J. Immunol., 1971, 106, 661-672. [5] ISHIZAKA, K., TOMIOKA, H. & ISHIZAKA, T., Mechanisms of passive sensitization. - - I. Presence of I g E and IgG molecules on h u m a n leucocytes. J . Immunol., 1970, 105, 1459-1467. [6] KULCZYCKI, A., ISERSKY, C. & METZGER, H., The i n t e r a c t i o n of IgE with r a t basophilic leukemia cells. - - I. Evidence for specific binding of IgE. J . exp. Med., 1976, 139, 600-616. [7] CAPRON, A., DESSAINT, J.-O., CAPRON, M. & BAZlN, H., Specific IgE a n t i b o d i e s in i m m u n e adherence of n o r m a l m a c r o p h a g e s to Schistosoma mansoni schistosonules. Nature (Lond.), 1975, 253, 474-475. [8] HUBSCHER, T. & ErSEN, A. H., Allergen binding to hnnmn peripheral leucocytes. Int. Arch. Allergy, 1971, 71, 689-693. [9] SCHWARTZ, L. B. & AUSTEN, K. F., S t r u c t u r e a n d function of the ~hemical m e d i a t o r s of m a s t cells, in (( Progress in allergy ,, vol. 34 (p. 274-321), K a r g e r , Basel, 1984. [10] FELDBERG, W. & KELLAWAY, C. H., L i b e r a t i o n of histamine and f o r m a t i o n of a lysolecithin-like s u b s t a n c e b y cobra venom. J. Physiol. (Lond.), 1938, 94, 187-226. [11] HIGHSMITH, R. F. & ROSENnERo, R. D., The inhibition of h u m a n p l a s m i n b y h u m a n a n t i t h r o m b i n - h e p a r i n cofactor. J. biol. Chem., 1974, 249, 4335-4338. [12] KELLAWAY, C. H. & TRETHEWIG, E. R., The l i b e r a t i o n of a slow-reacting s m o o t h muscle s t i m u l a t i n g substance in a n a p h y l a x i s . Quart. J. exp. Physiol., 1940, 30, 121-145. [13] MURPHY, R. C., HAMMARSTROM, S. & SAMUWLSSON, B., Leukotriene C: a slowreacting substance from routine m a s t o c y t o m a cells. Pros. nat. Acad. Sei. (Wash.), 1979, 76, 4275-4279. [14] BORGEAT, P. & SAMUELSSON, B., Metabolism of arachidonic acid a n d homogamma-linolenic acid b y r a b b i t p o l y m o r p h o n u e l e a r leukocytes. Monoh y d r o x y acids from novel lipoxygenases. J. biol. Chem., 1979, 251, 78167820. [15] RADMARK, O., MALMSTEM, C., SAMUELSSON, B., GOTO, G., MARFAT, A. & ConE,', E. J., Leukotriene A isolation from h u m a n p o l y m o r p h o n u c l e a r leukocytes. d. biol. Chem., 1980, 255, 11828-11831. [16] FORD-HuTcHINSON, A. W., BRAY, M. A., DOIG, M. V., SHIPLEY, M. E. & SMITH, M. d. H., Leukotriene B4, a p o t e n t chemokinetic and aggregating s u b s t a n c e released from p o l y m o r p h o n u c l e a r lcukocytes. Nature (Lond.), 1980, 286, 264-265. [17] GOETZL, E. J. & PICKETT, W. C., Novel s t r u c t u r a l d e t e r m i n a n t s of the h u m a n neutrophil c h e m o t a c t i c a c t i v i t y of leukotriene B. J. exp. Med., 1981, 153, 482-487. [18] ORNING, ]_.., HAMMARSTROM, S. ~: SAMUELSSON, B., L e u k o t r i e n e D: a slowreacting substance from r a t basophilic l e u k e m i a cells. Proc. nat. Acad. Sci. (Wash.), 1980, 77, 2014-2017. [19] MICHELL, ]={. I-I. & KIRK, C. J., Trends Pharmacol. Sci., 1981, 2, 86. [20[ COCKROFT, S. & GOMPERTS, B. D., Biochem. J., 1979, 178, 681. [21] KENNERLY, D. A., SULLIVAN, T. J. & PARKER, C. W., J. Immunol., 1979, 122, 152. [22] STRANDBERG, K., SYDBOM, A. & UVNAS, B., Acla physiol, scand., 1975, 94, 54. [23] ISHIZAKA,T., HIRATA, F., ISHIZAKA,K. & AXELROD, J., Stimulation of phospholipid m e t h y l a t i o n , Ca 2+ influx and histamine release b y bridging of I g E receptors on r a t m a s t cells. Pros. nat. Aead. Sci. (Wash.), 1980, 77, 1903. [24] MOmTA, Y., CmANO, P. K. & SIRAGANIAN, In. P., Effect of inhibitors of t r a n s m e t h y l a t i o n on h i s t a m i n e release from h u m a n basophils. Biochem. Pharmaeol., 1981, 30, 785.

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IN ALLERGY

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125] CREWS, F. T., MORITA, Y., McGIVNEY, A., HIRATA, F., SIRAGANIAN, R. P. & AXELROD, J., I g E - m e d i a t e d h i s t a m i n e release in rat basophilic leukemia cells. R e c e p t o r activation, p h o s p h o l i p i d m e t h y l a t i o n , Ca 2+ flux a n d release of arachidonic acid. Arch. Biochem. Biophys., 1981, 212, 561. [26] HIRATA, F. & AXELROD, J., Phospholipid m e t h y l a t i o n and biological signal transmission. Science, 1980, 209, 1082. [27] LEwxs, R. A., HOLGATE, S. T., ROBERTS, L. J. el al., J. Immunol., 1979, 123, 1663. [28] SULLIVAN,T. J., PARKER, C. W. ~s EISEN, S. A. et al., J. Immunol., 1975, 114, 1480. [29] KALINER, M. & AUSTEN, S . F., J. Immunol., 1974, 112, 664. [30] AURBACH, G. D., R e c e p t o r - a d e n y l a t e cyclase components: a b n o r m a l i t i e s in clinical medicine. Advanc. Cycl. Nucleot. Res., 1980, 12, 1-9. [31] FOREMAN, J. C., HALLETT, M. B. d~ MONGAR, J. L., Brit. J. Pharmacol., 1977, 59, 473P. [32] BURT, D. S. & STANWORTH, D. R., The effect of ribose a n d purine-modified adenosine analogues on the secretion of histamine from r a t m a s t cells induced b y ionophore A23187. Biochem. Pharmacol., 1983, 32, 2729-2732. [33] STANWORTH, D. R., I m m e d i a t e h y p e r s e n s i t i v i t y : t h e m o l e c u l a r basis of tile allergic response, in (( F r o n t i e r s of Biology ,, vol. 28. Elsevier N o r t h H o l l a n d . Publ. Co., A m s t e r d a m , 1973. [34] STANWORTH,D. R., A p p l i c a t i o n of s y n t h e t i c peptides r e p r e s e n t a t i v e of i m m u n o globulin sequences to the delineation of receptor binding and signalling processes. Mol. Immunol., 1982, 19, 1245-1254. [35] STANWORTH, D. R., KINGS, M., RoY, P. D., MORAN, J. M. & MORAN, D. M., S y n t h e t i c p e p t i d e s comprising sequences of t h e h u m a n I g E h e a v y chain capable of releasing histamine. Biochem. J., 1979, 180, 665. 136] SALARI, H., BRAQUET, P. & BORUEAT, P., Effect of p e p t i d e mellitin on the release of arachidonic acid lipoxygenase p r o d u c t s in h u m a n leucocyte and platelet. J. tool. Pharmacol. (in press). [37] POUBELLE,P., BEAULIEU, A. D., NADEAU, M., LAVIOLETTE,M. & BORG~;AT,P., The m e t a b o l i s m of arachidonic acid t h r o u g h the l i p o x y g e n a s e p a t h w a y s in h u m a n phagocytes. Advanc. Inflammation Res. (in press). [38] GALLI, S. J., DVORAK, A. M. & DVORAK, H. F., Basophils and m a s t cells: morphologic insights into their biology, secretory p a t t e r n s and function. Progr. Allergy, 1984, 34, 1-141. [39] LEwls, R. A., HOLGATE, S. T., ROBERTS, L. J., 0ATES, J. A. & AUSTEN, K. F., Preferential generation of p r o s t a g l a n d i n D2 b y r a t and h u m a n m a s t cells, in (, B i o c h e m i s t r y of the acute allergic reactions ,, (Neacker, Simon & Austen) (p. 239-254). A. R. Liss Inc., New York, 1981. [40] LICHTENSTEIN, L. M., Mediators a n d mechanisms of release from purified h u m a n b a s o p h i l s and m a s t cells. Amer. Acad. Allergy Postgrad. Course Syllabus, 1982, 31-47. [41] PARKER, C. W., S R S - A of r a t basophil l e u k e m i a cells and r a t m a s t cells, in (( B i o c h e m i s t r y of the acute allergic reactions )) (Neacker, Simon & Austen), 23-36. A. R. Liss Inc., New York, 1981. [42] SAMUELSSON, B., O x i d a t i v e p r o d u c t s of arachidonate: leukotrienes, a new group of compounds, including slow-reacting substance of a n a p h y l a x i s (SRS-A), in (( B i o c h e m i s t r y of t h e acute allergic reactions ,, (Neacker, Simon & Austen), 23-36. A. R. Liss Inc., New York, 1981. [43] LEwis, R. A. & AUSTEN, K. F., The biologically active leukotrienes. J. clin. Invest., 1984, 73, 889-897. [44] BORGEAT, P., HAMBERG, M. & SAMUELSSON, B., T r a n s f o r m a t i o n of arachidonic acid and homo-y-linolenic acid in r a b b i t p o l y m o r p h o n u c l e a r leukocytes. M o n o h y d r o x y acids from novel lipoxygenases. J. biol. Chem., 1976, 251, 7816-7820. [45] BORGEAT, P. & SAMUELSSON, B., M e t a b o l i s m of arachidonic acid in p o l y m o r p h o n u c l e a r leukocytes. Effects of t h e ionophore A23187. Proc. nat. Acad. Sci. (Wash.), 1979, 76, 2148-2152. [46] BRAQUET, M., GARAY, R., DUCOUSSO, R., BORGEAT, P., DEFEuDIS, F. V. & BRAQUET, P., T r a n s m e m b r a n e p o t a s s i u m m o v e m e n t s and t h e arachidonic acid cascade: I. - - A s t u d y in A23187-stimulated h u m a n leucocytes, in (( P r o s t a g l a n d i n s a n d m e m b r a n e ion t r a n s p o r t )) (P. B r a q u e t et al.). R a v e n Press, New York, 1984.

:202

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[471 LEE, C. W., LEWIS, R. A., COREY, E. J., BARTON, A., OH, H., TAUBER, A. I. & AUSTEN, K. F., Proc. Nat. Acad. Sci. (Wash.), 1982, 79, 4166-4170. [48] LEE, C. W., LEwIs, R. A., TAUBER, A. I., MEHROTRA, M., COREY, E. J. & AUSTEN, K. F., J. biol. Chem., 1983, 258, 15004-15010. [49] SIROIS, P. & BORGEAT, P., Mediators of immediate hypersensitivity, in ,~ Immunopharmacology )) (P. Sirois and M. Rola-Pleszczynski) (p. 201-222), Elsevier North-Holland Publ. Go., Amsterdam, 1982. [50] BORGEAT, P. & SAMUELSSON, B., Metabolism of arachidonic acid in polymorphonuclear leukocytes: Unstable intermediate in formation of dihydroxy acids. Proc. Nat. Acad. Sci. (Wash.), 1979, 76, 3213-3217. [511 FRUTEAU de LACLOS, B., BRAQUET, P. & BORGEAT, P., Characteristics of leukotriene (LT) and hydroxy eicosatetraenoie acid (HETE) synthesis in human leukocytes in vitro: effect of arachidonic acid concentration. Proslaglandins Leukotrienes Med., 1984, 13, 47-52. [52] BORGEAT, P., RABINOVITCH,H., FRUTEAU de LAcos, B., PICARD, S., BRAQUET, P., HEBERT, J. & LAVIOLETTE, M., Eosinophil-rich human polymorphonuclear leukocyte preparations characteristically release leukotriene C4 on ionophore A23187 challenge. J. Allergy clin. Immunol., 1984, 74, 310-315. i[53] HENDERSON, W. R., HARLEY, J. B. & FAUCI, A. S., Arachidonic acid metabolism in normal and hypereosinophilic syndrome human eosinophils: generation of leukotrienes B~, C4, D4 and 15-1ipoxygenase products. I m m u n o logy, 1984, 51, 679-686. [54] ~VELLER,P. F., LEE, C. W., FOSTER, D. W., COREY, E. J., AUSTEN, K. F. & LEWIS, R. A., Generation and metabolism of 5-1ipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4. Proc. nat. Aead. Sci. (Wash.), 1983, 80, 7626-7630. [55] SHAW, R. J., CROMWELL,O. & KAY, A. B., Preferential generation of leukotriene C4 by human eosinophils. Clin. exp. Immunol., 1984, 56, 716-722. '[56] GOLDYNE, M. Eo, BURniSH, G. F., POUBELLE, P. & BORGEAT, P., Arachidonic acid metabolism among human mononuclear leukocytes: lipoxygenaserelated pathways. J. biol. Chem., 1984, 259, 8815-8819. [57] PAWLOWSEI,N. A., KAPLAN, G., HAMILL, A. L., COHN, Z. A. & SCOTT, W. A., Arachidonic acid metabolism by human monocytes: studies with plateletdepleted cultures. J. exp. Med., 1983, 158, 393-412. [58] POUBELLE, P., BEAULIEU, A. D. & BORGEAT, P., Leukotriene synthesis by synovial fluid and blood polymorphonuclear leukocytcs (PMNL) and monocyte-macrophages of rheumatoid arthritis (RA). Advanc. Inflammation Res. (in press). ~[59] FELS, A. O., PAWLOWSKI,N. A., CRAMER, E. B., KING, T. K. C., COHN, Z. A. & SCOTT, W. A., Human alveolar macrophages produce leukotriene B4. Proc. nat. Acad. Sci. (Wash.), 1982, 79, 7866-7870. [60] MACDERMOT, J., KELSEY, C. R., WADDELL, K. A., RICHMOND, R., KNIGHT, R. K., COLE, P. J., DOLLERY, C. T., LANDON, D. N. & BLAIn, I. A., Synthesis of leukotriene B4 and prostanoids by human alveolar macrophages: Analysis by gas chromatography/mass spectrometry. Prostaglandins, 1984, 27, 163-179. [61] MARTIN, T. R., ALTMAN, L. C., ALBERT, R. C. & HENDERSON, W. R., Leukotriene B 4 production by the human alveolar macrophage: a potential mechanism for amplifying inflammation in the lung. Amer. Rev. respir. Dis., 1984, 129, 106-111. [62] DAMON,M., CHAVIS,C., GODARD, Ph., MICHEL, F. B. & CRASTES de PAULET, A., Purification and mass spectrometry identification of leukotriene D4 synthesized by human alveolar macrophages. Biochem. biophys. Res. Commun., 1983, 111,518-524. [63] ROLA-PLESZCZYNKI,M., BORGEAT, P. & SIROIS, P., Leukotriene B4 induces human suppressor lymphocytes. Biochem. biophys. Res. Commun., 1982, 108, 1531-1537. [64] WEBB, D. R., NOWOWIEJSKI, I., HEALY, C. & ROGERS, T. J., Immunosuppressire properties of leukotrienes Da and E4 in vitro. Biochem. biophys. Res. Commun., 1982, 104, 1617-1622. [65] BAILEY, J. M., BRYANT, R. W., Low, C. E., PUPILLA, M. B. & VANDERHOECK, J. Y., Regulation of T-lymphocyte mitogenesis by the leucocyte product, 15-HETE. Cell. Immunol., 1982, 67, 112-120. ~66] ATLURN, D. & GOODWIN,J. S., Control of polyclonal immunoglobulin production

L E U K O T R I E N E S IN A L L E R G Y

[67] [68] [69] [70]

[71] [72] [73] [74]

[75] [76]

[77]

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L E U K O T R I E N E S AS MEDIATORS OF IMMEDIATE H Y P E R S E N S I T I V I T Y by F. A. Fitzpatrick Lipids Research Unit, The Up]ohn Company, 301, Henrietta-Street, Kalamazoo, M I 49001 USA Do leukotrienes fully account for immediate hypersensitivity? No. W h a t arguments support that answer? First, for leukotrienes to account fully for immediate hypersensitivity, one must ignore or rebut the evidence favouring other mediators such as histamine, thromboxane A2, prostaglandins H2 and F ~ and PAF-acether [1, 2]. Abandonment or dismissal of these mediators might prove inconvenient.

Second, the list of known mediators m a y be incomplete. For example, Detsouli and co-workers [3], using guineapig lung parenchymal strips superfused in vitro, have presented pharmacological evidence that antigen-dependent contractions have a myotropic component that is independent of histamine, PAF-acether, peptido-leukotrienes and prostaglandins. Third, synergism among mediators would be an uneconomical use of cellular metabolic capacities if equivalent results were possible with one. Yet instances