Immunology 1991 74 689-695

Conversion of leukotriene A4 by neutrophils and platelets from patients with atopic dermatitis R. A. HILGER, K. NEUBER & W. KONIG Institut fur Medizinische Mikrobiologie und Immunologie, AG Infektabwehr, Bochum, Germany

Acceptedfor publication 29 August 1991

SUMMARY The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.

Platelets possess a specific enzymatic capacity to transform exogenous or granulocyte-derived LTA4 to cysteinyl-containing leukotrienes. In addition to human neutrophils, LTA4 provokes aggregation, degranulation and stimulates the mobilization of free cytosolic calcium.'"3 Inflammatory mediators cause erythema and oedema as well as an inflammatory infiltrate by their chemotactic activity towards leukocytes and a hyperproliferation of the epidermis by stimulating cell growth.4 These biological activities of eicosanoids as well as the fact that there is a selective elevation of LTB4 in the skin of patients with atopic dermatitis (AD) strongly support their involvement in this disease. Interleukin 3 (IL-3) causes the release of histamine and leukotrienes by mast cells and basophils5'6 and furthermore is produced by mast cells in response to IgE receptor-mediated activation.7 Interleukin 8 (IL-8), which is secreted by human monocytes, lymphocytes, endothelial cells and dermal fibroblasts,8'9 induces the release of histamine and leukotrienes from IL-3 primed basophils.5 These results indicate a potential role of IL-3 and IL-8 for the induction of immediate and delayed allergic reactions. In previous publications no differences in mediator release between leukocytes from normal and atopic donors were detected by using non-specific stimuli, e.g. the Ca-ionophore A23187. C5a alone induces a measurable generation of lipid mediators from normal human neutrophils only in the presence of exogenous AA.'0

INTRODUCTION

The epoxide leukotriene A4 (LTA4) is generated by enzymatic transformation of arachidonic acid (AA) via 5-hydroperoxytetraenoic acid (5-HPETE) by a 5-lipoxygenase and a dehydrase. Conjugation of this compound with glutathione (GSH), leading to LTC4 formation is catalysed by a specific particulate LTC4 synthase or various unspecific, mainly cytosolic GSH S-transferases. Degradation of LTC4 leads to LTD4 and LTE4. Alternatively, LTA4 is stereospecifically converted by a 5hydrolase to the potent leukocyte activator LTB4 and via omega-oxidation to OH- and COOH-LTB4. Neutrophil granulocytes produce substantial amounts of LTB4, while LTC4 is released in minute quantities. In addition, neutrophils release LTA4 which is available for further transformation by cells of various origin in the microenvironment. Abbreviations: AA, arachidonic acid; AD, atopic dermatitis; fMLP, formyl-methionyl-leucyl-phenylalanine; GM-CSF, granulocyte/macrophage-colony-stimulating-factor; GSH, glutathione; HETE, hydroxyeicosatetraenoic acid; HSA, human serum albumin; IL-3,8, interleukin3, -8; LT, leukotriene; NaF, natrium fluoride; PBS, phosphate-buffered saline; PMN, polymorphonuclear granulocytes; RP-HPLC, reverse phase high pressure liquid chromatography. Correspondence: Professor W. Konig, Institut fur Medizinische Mikrobiologie und Immunologie, AG Infektabwehr, Universitatsstr. 150, 4630 Bochum, Germany.

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Recently, it has been shown' that receptor-specific stimuli (IL-3, IL-8, C5a and fMLP) induce the release of high amounts of LTB4 and its omega-oxidation products from neutrophils of patients with AD without any priming or in the presence of exogenous AA. This indicates that the 5-lipoxygenase of neutrophils from patients with AD may have a higher activity as compared to polymorphonuclear granulocytes (PMN) from normal donors and that neutrophils in response to receptorspecific stimuli may play a role in the pathogenesis of this disease. An involvement of platelets in the pathophysiology of asthma has been suggested.' Their role in AD is still unclear. We therefore assumed that cells from patients with AD display an increased state of activation of the 5-hydrolase or GSH S-transferases in neutrophils and platelets from patients with AD by their capacity to transform exogenous LTA4 into LTB4 or into cysteinyl-containing leukotrienes, respectively. Furthermore, we studied the role of platelets in AD and the effect of LTA4 on 12-HETE generation.

MATERIALS AND METHODS Materials The reagents used were from the following sources: Ficoll 400 was obtained from Pharmacia (Uppsala, Sweden); Macrodex (6%, wt/vol) was from Knoll (Ludwigshafen, Germany); sodium metrizoate solution (75%, wt/vol) from Nyegaard (Oslo, Norway); heparin was obtained from Sigma (Munich, Germany); acetonitrile (HPLC grade) was purchased from Baker Chemicals (Gross-Gerau, Germany); methanol, EDTA, dipotassiumhydrogenphosphate, and phosphoric acid were from Riedel de Haen (Seelze, Germany); and human serum albumin (HSA) was from Behring (Ludwigshafen, Germany). Synthetic leukotrienes LTB4, LTC4, LTD4 and LTE4 as well as the omega-oxidated products 20-OH-LTB4 and 20COOH-LTB4 and the monohydroxylated eicosatetraenoic acids (5-HETE, 12-HETE, 15-HETE) were generous gifts from Merck Frost (Pointe Claire/Dorval, Quebec, Canada). LTA4 methyl ester was synthesized in a modified way as described in ref. 13, LTA4-lithium salt (LTA4-Li) was prepared as follows: LTA4 methylester (80 pg) was dissolved in tetrahydrofurane (73 p1). This solution was degassed with argon and then treated with 7 p1 of I M aqueous lithium hydroxide. The solution was allowed to stand at 00 for 48 hr. Aliquots of the solution were evaporated with a gentle stream of argon and the residue was resuspended in phosphate-buffered saline containing 0-5% (wt/ vol) human serum albumin for further experiments. C5a, Ca-ionophore A23187 and thrombin were obtained from Sigma, and IL-3 from Genzyme (purchased from IC Chemicals, Munich, Germany). IL-3 has a specific activity of 3-5 x 105 colony forming units (CFU/pg. IL-8 was a generous gift from Dr Matsushima (Laboratory of Immunoregulation, National Institute of Health, Frederick, MD).

Blood donors The AD patients (aged 18-30 years, n = 4) suffered from active disease at the time of study and were classified according to the criteria of Hanifin and Rajka.'4 The following four basic features were present: a chronic or chronically relapsing dermatitis, flexural lichenification, pruritis and a personal or family history of atopy (asthma, rhinitis, AD). The serum IgE levels were above 500 ng/ml. The donors did not receive any steroid treatment. Healthy students served as controls (aged 19-28 years, n = 4) without any personal history of allergic diseases or atopy. The controls were matched for age and sex with the AD patients.

Preparation of the cells PMN were obtained from peripheral blood of healthy donors and from patients with AD. Platelets were isolated from platelet-rich plasma (PRP). PRP was obtained by centrifugation of peripheral blood supplemented with PBS containing 155% EDTA (1 ml) at 200g (25 min at 20°). The supernatant was mixed with an equal volume of PBS with 1 5% EDTA and centrifugated at 1285g (20 min at 4°). The pellet was washed in 10 ml PBS with 1-5% EDTA. The pellet obtained after the first centrifugation containing peripheral blood cells was resuspended in the platelet-free plasma. Cells were separated on a Ficoll-metrizoate density gradient centrifugation. The human neutrophils (PMN) were then separated from the erythrocytes by dextran sedimentation. The remaining erythrocytes were lysed by exposing the cells to hypotonic conditions. This method results in more than 97% pure PMN.'5 Cell viability was assessed microscopically by trypan blue exclusion analysis. The PMN were suspended to a final concentration of 2 x 107 cells/ml PBS.

Leukotriene A4 conversion The concentration of cells was 2 x 107/ml (PMN) and 2 x 108/ml

(platelets) respectively. The cell suspensions (500 pi) were incubated with LTA4 (5 gM or as otherwise stated in the text) in the presence of calcium (I mM) and magnesium (0 5 mM). Incubation proceeded at 37°. The reaction was terminated by the addition of 3 ml of methanol/acetonitrile (50:50, vol/vol). Cell stimulation

Buffers

PMN (I x 107/500 p1) and platelets (1 x 108/500 pi) were preincubated for 10 min with 5 pM LTA4, various stimuli (C5a, IL-3, IL-8, LTA4) as well as PBS/HSA buffer at 370 in the presence of calcium (1 mM) and magnesium (0 5 mM). Subsequently, a second stimulus was added as indicated above (CSa, IL-3, IL-8, Ca-ionophore A23187, thrombin, NaF, LTA4). The incubation time was extended for an additional 10 min. The cells were also coincubated with LTA4(5 pM) and the various stimuli (C5a, IL-3, IL-8, NaF) for 20 min at 37°. Stimulations were terminated after addition of 3 ml methanol/acetonitrile (50:50,

Unless stated otherwise, the medium used for washing the cells and for mediator release was a modified Dulbecco PBS (referred to as PBS) buffer consisting of 137 mm NaCl, 8 mm Na2HPO4, 2-7 mm KH2PO4 and 2-7 mm KCI (pH 7-4) and HSA (0-5%).

vol/vol). C5a was obtained from Sigma, and IL-3 from Genzyme. IL-3 has a specific activity of 3-5 x 105 CFU/pg. IL-8 was a generous gift from Dr Matsushima.

691

Conversion of LTA4 by neutrophils and platelets Analysis of leukotriene release The supernatants of incubated cells were deproteinized by addition of 3 ml of methanol/acetonitrile (50:50, vol/vol), overlaid with nitrogen, and frozen at -20° for 12 hr. After centrifugation at 1000 g for 15 min, the supernatants were evaporated to dryness in a freeze dryer, suspended in 600 pi of methanol/water (30:70, vol/vol), overlaid with nitrogen, and stored at - 20° for 2 hr. The samples were centrifuged (9 700 g for 2 min at room temperature), and 200 pi were then applied to reverse-phase HPLC. HPLC was performed on reverse-phase columns (4 x 250 mm) packed with Nucleosil (C18) 5-pm particles (Macherey and Nagel, Duren, Germany); chromatography was carried out with the automatic sample injection module WISP 710B (Waters Associates, Eschborn, Germany). The column temperature was maintained at 40°. The absorbance of the column effluent was monitored by using a variable UV detector adjusted to 280 nm [detection of dihydroxyeicosatetraenoic acids (DiHETEs) and cysteinyl leukotrienes] or to 235 nm [detection of monohydroxyeicosatetraenoic acids (HETES)]. The peak areas were calculated with a chromatography data system (series 3000; Nelson Analytical, Mannheim, Germany). The solvent system was a mixture of phosphate buffer (17 mm K2HPO4 containing 0-05% EDTA), acetonitrile, and methanol (50:30:20, vol/vol for the detection of dihydroxyeicosatetraenoic acids; 30:40:30, vol/vol for the detection of monohydroxyeicosatetraenoic acids) which was adjusted to pH 5-0 with phosphoric acid. The flow rate was maintained at I ml/min. All solvents were degassed before use and were constantly stirred during HPLC analysis. Identification and quantitation of leukotrienes were performed as has been described. 16 Statistical analysis Data from three different experiments with different donor cells were combined and reported as the means + standard deviation. The Student's t-test for independent means was used to provide a statistical analysis (P < 0-05 was considered as significant). RESULTS The experiments were carried out by preparing the cells from one normal and one atopic volunteer on the same day. PMN and platelets from each donor were stimulated simultaneously and compared with each other. Stimulation of PMN with receptor specific stimuli (C5a, IL-3, IL-8) in the presence of LTA4 Figure 1 shows the quantities of LTB4 and its omega-oxidation products (=total LTB4) generated after incubation of 1 x 107 PMN/650 p1 PBS buffer at 370 for 20 min with C5a, IL-3, IL-8 in the presence of LTA4. The spontaneous generation of total LTB4 as well as the leukotriene production after activation with the above stimuli in the absence of exogenous LTA4 were used as controls. As compared to PMN from normal donors the stimulated (C5a, IL-3, IL-8) and unstimulated neutrophils from atopic donors released significantly higher amounts of leukotrienes in the presence or absence of LTA4. The addition of LTA4 enhanced the generation of total LTB4 up to 2-9 + 0-8 ng (normal donors) and up to 67+0±4 ng (atopic donors) as compared to the spontaneous leukotriene synthesis (0 4 + 0 4 ng

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normal donors; 1-4+0-6 ng atopic donors). The values were additive and were composed of the sum obtained with the individual stimulus and after LTA4 addition. Stimulation of PMN with the Ca-ionophore A23187 in the presence of exogenous LTA4 Figure 2(a) represents the means + SD of three independent experiments in which PMN from normal and atopic donors were stimulated with the Ca-ionophore A23187 (5 pM) in the presence of different quantities of exogenous LTA4. Increasing amounts of LTA4 led to a significant inhibition of the Caionophore induced total LTB4 formation as compared to the control (PBA + Ca-ionophore A23 187). In this regard no significant differences were obtained when either normal or atopic PMN were studied. The ratio of the omega-oxidation products (20-COOH/20OH-LTB4) after stimulation with the Ca-ionophore A23 187 in the presence of exogenous LTA4 was analysed. A significantly higher ratio of 20-COOH/20-OH-LTB4 (0-55 and 0-52: normal PMN; 0-88 and 0-81: atopic PMN) was measured in patients with AD when lower concentrations of LTA4 (0-5 4ug, I pg) were added. At higher concentrations of LTA4 the ratios of 20COOH-/20-OH-LTB4 from both donor groups turned to similar values (Fig. 2b). Formation of the cysteinyl leukotrienes LTC4, D4 and E4 from PMN in the presence of exogenous LTA4 For the relative proportions of the cysteinyl leukotrienes LTC4, D4 and E4, no significant differences were obtained between the AD group and the normal donors either when the PMN were stimulated with the Ca-ionophore A23187 (5 pM) or when the cells were stimulated with receptor-specific stimuli (C5a, IL-3, IL-8) in the presence of exogenous LTA4 (0 5 pg up to 4 pg LTA4/1 x 107 PMN; data not shown). In contrast, a significant difference was obtained for the cysteinyl leukotriene formation in the presence of exogenous LTA4 when the PMN from normal and atopic donors were compared with each other (Fig. 3). The combined amounts of the cysteinyl leukotrienes LTC4, D4 and

K. A. Hilger, K. Neuber & W. Konig

692 250

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donors (Fig. 4b: ratio and total amounts (insert)]. At a concentration of 4-25 mm of NaF the 12-HETE release increased about fivefold in atopic patients as compared to platelets of normal donors. Figure 4(c) represents the means + SD of three independent experiments (ratio) in which platelets from both donor groups were treated with the Caionophore A23 187 at a concentration from 0-075 pM up to 5 pM. The generated amounts of 1 2-HETE from platelets of patients with atopic dermatitis were significantly higher than from normal donors (insert). Conversion of exogenous LTA4 by platelets from normal donors and from patients with AD Exogenous LTA4 was added in a dose-dependent manner (0-5 pg up to 4 pg) to platelets (I x 108/650 pi PBS) in the presence of Ca/Mg. The resulting amounts of cysteinyl leukotrienes after an incubation time of 20 min are shown in Fig. 5. Atopic platelets converted LTA4 to significantly higher amounts of the combined cysteinyl leukotrienes as compared to experiments in which cells of normal donors were used.

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platelets for one typical experiment in which one normal and one atopic volunteer are compared. After receptor-specific stimulation with thrombin [at each concentration (Fig. 4a): ratio and total amounts of 12-HETE (insert)] significantly higher amounts of 12-HETE were produced by platelets from patients with AD as compared to cells from normal donors. On average a maximal ratio was reached at a concentration of 0 06 U thrombin. Stimulation of platelets with different concentrations of the G-protein activator NaF also showed a higher state of activity in

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Stimulation of platelets Furthermore, the effects of various stimuli on the 12-HETE formation of platelets from normal donors and patients with AD were investigated. Thrombin was used for receptor-specific stimulation and NaF for direct G-protein activation. A nonligand mediated generation of 12-HETE was obtained after stimulation with the Ca-ionophore A23187. Dose-dependent are shown in Fig. 4(a-c). The experiments for each stimulus inserts demonstrate the formation of 12-HETE in ng/l x 108

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DISCUSSION Recently, it has been demonstrated that receptor-specific stimuli, e.g. IL-3, IL-8, C5a and fMLP, increased leukotriene production from neutrophils of patients with AD. " In addition, the neutrophils by themselves are potent producers of cytokines, e.g. IL-8."7 The neutrophils of patients with atopic dermatitis revealed an enhanced activation of the 5-lipoxygenase enzyme even in the unstimulated cell fraction. These data emphasize the role of neutrophils which are dependent on receptor-specific stimuli and may induce inflammatory reactions in AD.

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The epoxide leukotriene A4 is an inflammatory mediator which serves as a substrate in cell-cell interaction and is also known as a potent stimulus for rapid and transient increase in calcium influx.3 The increased production of LTB4 and LTC4 in PMN of patients with AD raised the question whether the LTA4 conversion via 5-hydrolase or GSH S-transferases by these cells is also altered. Addition of exogenous LTA4 to unstimulated PMN induced a significantly higher release of LTB4 and its omega-oxidation products as well as of the cysteinyl leukotrienes (LTC4, D4, E4) in patients with AD. These results may indicate a higher state of activity also of 5-hydrolase and of the GSH S-transferase in atopic neutrophils. After costimulation of the PMN with LTA4 and receptor-specific stimuli (IL-3, IL-8 and C5a) an increased production of total LTB4 was apparent. However, the receptorspecific stimulated conversion of exogenous LTA4 into cysteinyl leukotrienes via GSH S-transferase led to increased amounts similar in normal as well as in atopic PMN. One may suggest that additional stimulation of PMN in the presence of exogenous LTA4 leads to a substrate saturation of the enzymecomplex GSH S-transferase. On the other hand a competition of the metabolism of LTA4 via 5-hydrolase may explain this phenomenon. Non-specific stimuli, e.g. the Ca-ionophore A23187, did not induce measurable differences in leukotriene release from PMN of normal and atopic donors. 18 However, at suboptimal concentrations of the Ca-ionophore A23187 (0-15 gM) a significantly higher release of leukotrienes in allergic patients has been demonstrated.'9 Obviously, the Ca-ionophore A23187 is such a potent stimulus that slight differences in the reactions of neutrophils of normal and atopic donors cannot be detected. In the presence of exogenous LTA4 the Ca-ionophore A23187 induced an inhibition of the total LTB4 release in both donor groups. Previous data demonstrated similar inhibitory effects of exogenous LTB4 on human neutrophils after coincubation with the Ca-ionophore A23 187 or NaF in normal donors.20 These data suggested that higher amounts of cytosolic [Ca2+], leads to a decrease in LTB4 generation. However, after stimulation with the Ca-ionophore A23187 in the presence of exogenous LTA4 an increase in cysteinyl leukotriene formation

694

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by PMN to similar amounts in both donor groups was obtained (data not shown). Furthermore, the decreased ratio of 20-COOH-/20-OHLTB4 after costimulation with increasing amounts of exogenous LTA4 and the Ca-ionophore A23 187 may be due to changes in the fluidity of the membrane caused by LTA4. In addition, a characteristic feature of the LTA4 hydrolase is the suicide inactivation by its substrate LTA4.2' Interestingly, we observed a significantly higher ratio of 20-COOH-/20-OH-LTB4 in neutrophils from patients with AD when lower concentrations of LTA4 were coincubated with the Ca-ionophore A23187. The change in the ratio of omegaoxidation products may indicate a different enzymatic metabolism of LTB4 in AD. Platelets are only able to convert extracellular leukotriene A4 into LTC4.22 Unstimulated platelets from patients with AD generated significantly higher amounts of cysteinyl leukotrienes in the presence of exogenous LTA4 as compared to normal donors. The receptor-specific stimuli IL-3 and IL-8 did not modulate the conversion (data not shown). Platelet-derived 12-HETE may play a crucial role in various enzymatic pathways of the lipoxygenase cascade, on immune or inflammatory responses, cell movement, cell attachment, intracellular calcium homeostasis and cell growth.23 31 The formation of 12-HETE increases in a number of tissue injury situations, and due to its multitude of biological effects, 12-HETE has been suggested as mediator of several pathophysiological phenomena.32 The pathophysiological response of 12-HETE in inflammatory skin diseases, e.g. in psoriasis, which is a common inflammatory, proliferative skin disease, has been described.33 Similar data for AD are still not available. It was observed that unstimulated platelets from patients with AD released up to threefold higher quantities of 12-HETE. The fact that receptor-specific stimulation with thrombin as well as direct G-protein activation via NaF and non-ligand mediated generation of 12-HETE led to a significantly higher production of this inflammatory mediator, suggests also a higher activation of the 12-lipoxygenase enzyme in AD. These data may suggest a relationship between an increased formation of 12-HETE and the inflammatory reactions in AD. Since neutrophils from patients with AD produce inflammatory mediators in response to receptor-specific stimuli, e.g. cytokines or anaphylatoxins, an important role of these cells for the inflammatory reactions characteristic for this disease appears likely." The increased responsiveness of PMN from patients with AD may be due to a priming via unknown factors or by an endogenously enhanced activation of the 5-lipoxygenase as compared to non-atopic donors. The present data demonstrate that the following steps of the enzymatic cascade (5-hydrolase, GSH S-transferase) of leukotriene formation in AD are also at a higher state of activity. One may argue that not only neutrophils are important for the inflammatory reactions in AD, but also platelets due to their higher capability to convert exogenous LTA4 and to release 12-HETE. Both mediators modulate cell-cell interactions, e.g. cell attachment, chemotaxis, immune or inflammatory responses, intracellular calcium homeostasis and cell growth. Such cell-cell interactions during the inflammatory response between neutrophils and platelets or endothelial cells may play an important role in the pathophysiology of AD.

ACKNOWLEDGMENTS Supported by Deutsche Forschungsgemeinschaft (no. 427/13-7 eicosanoids to R. A. Hilger and no. 427/7-5 to W. Koning) and Bundesanstalt fur Arbeitsschutz (to K. Neuber).

REFERENCES 1. OTFLAHERTY J.T., WYKLE R.L., THOMAS M.J. & MCCALL C.E. (1984) Neutrophil degranulation responses to combinations of arachidonate metabolites and platelet-activating factor. Res. Commun. chem. Pathol. Pharmacol. 43, 3. 2. BECKMANN J.K., GAY J.C., BRASH A.R., LUKENS J.N. & OATES J.A. (1985) Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes. Biochem. biophys. Res. Commun. 133, 23. 3. LUSCINSKAs F.W., NICOLAOu K.C., WEBBER S.E., VEAKE C.A., GIMBRONE M.A., JR & SERHAN C.N. (1990) Ca2+ mobilization with leukotriene A4 and epoxytetraenes in human neutrophils. Biochem. Pharmacol. 39, 355. 4. RUZICKA T., SIMMET T., PESKAR B.A. & RING J. (1986) Skin levels of arachidonic acid-derived inflammatory mediators and histamine in atopic dermatitis and psoriasis. J. Invest. Dermatol. 86, 105. 5. DAHINDEN C.A., KURIMOTO Y., DE WECK A.L., LINDLEY I., DEWALD B. & BAGGLIOLINI M. (1989) The neutrophil-activating peptide NAF/NAP- I induces histamine and leukotriene release by interleukin 3-primed basophils. J. exp. Med. 170,1787. 6. KURIMOTO Y., DE WECK A.L. & DAHINDEN C.A. (1989) Interleukin 3-dependent mediator release in basophils triggered by C5a. J. exp. Med. 170, 467. 7. WODNAR-FILIPowicz A., HEUSSER C.H. & MORONI C. (1989) Production of the haemopoietic growth factors GM-CSF and interleukin-3 by mast cells in response to IgE receptor-mediated activation. Nature, 339, 150. 8. SCHRODER J.M., MROWIETZ U., MORITA E. & CHRISTOPHERS E. (1987) Purification and partial biochemical characterization of a

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