Immunology 1990 69 435-442
Release of leukotriene C4 (LTC4) from human eosinophils following adherence to IgE- and IgG-coated schistosomula of Schistosoma mansoni R. MOQBEL, A. J. MACDONALD,
CROMWELL & A. B. KAY Department of Allergy and Clinical Immunology, National Heart & Lung Institute, London
0.
Acceptedfor publication 14 November 1989
SUMMARY The release of leukotriene C4 (LTC4) from human low-density eosinophils following adherence to live or formalin-fixed schistosomula of Schistosoma mansoni coated with parasite-specific IgE or IgG obtained from pooled human anti-S. mansoni serum has been studied. IgE-rich fractions were obtained after fractionation of pooled immune sera on fast-protein liquid chromatography (FPLC; polyanion SI-17 column) and were identified by parasite-specific RAST. Contaminating IgG was removed by adsorption on a Staphylococcus aureus-protein A affinity column. IgG-rich FPLC fractions were identified by a specific ELISA assay. IgG-dependent activities were confirmed by protein A adsorption. Low-density eosinophils adhered to live and formalin-fixed schistosomula coated with specific antisera and released 11-7+2-7 and 16-5+3-5 pmoles of LTC4/106 cells, respectively. LTC4 release induced by A23187 (5 x 10-6 M) from the same cells was 80 + 24 pmoles/106 cells and 9-9 + 1 pmoles/106 cells in the presence of Sepharose particles (CNBr-activated 4B beads) covalently coated with normal human IgG. Fixed schistosomula coated with FPLC-purified IgE and IgG gave 7-6 + 0-4 and 6-0 + 0-1 pmoles of LTC4 per 106 low-density eosinophils, respectively. The same IgE- and IgG-rich fractions induced eosinophil-mediated cytotoxicity of live schistosomula in vitro. Removal of IgE by an anti-IgE affinity column abolished both the IgE-dependent release of LTC4 and the in vitro killing of larvae. Conversely, IgG-dependent activities were abolished by protein A, but not anti-IgE, adsorption. Normal density eosinophils generated undetectable amounts of LTC4 when incubated with IgE-coated schistosomula, whereas with IgG-coated larvae 4-6 pmoles/ 106 cells were obtained. Following preincubation with platelet-activating factor (PAF) (10-7 M) and leukotriene B4 (LTB4) (I0-7 M), normal density eosinophils released LTC4 when in contact with larvae coated with antigen-specific IgE. Lyso-PAF had no effect in any of the systems tested. The synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) had no influence on IgEdependent release of LTC4 from eosinophils. In contrast, FMLP (10-7 M) enhanced the IgGdependent LTC4 release, with PAF and LTB4 also showing a small enhancing effect. None of these agents substantially altered the release potential of low-density eosinophils in either IgE- or IgGdependent events. Thus the results presented here indicate that in an IgE-dependent system, human low-density eosinophils can be induced to adhere to and kill IgE-coated helminthic targets and release biologically relevant amounts of LTC4. This may have important implications within the context of allergic inflammation, especially as PAF and LTB4 appeared to play an amplifying role in IgEdependent activation of normal eosinophils.
INTRODUCTION Human eosinophils possess a number of surface receptors that participate in the process of adherence (Kimani, Tonnesen & Henson, 1988) and exocytosis (Spry, 1985) and, at least in vitro, mediate the killing of appropriately opsonized helminthic targets (e.g. schistosomula of Schistosoma mansoni; Butterworth, 1984). Among these receptors are those for complement (CR1 and CR3) (Fischer et al., 1986) and IgG (Fc) (Kulczycki, 1984). The majority of eosinophils from patients with hyper-
eosinophilia are of the low-density type, and appear to be more 'activated' than normal density cells (Prin et al., 1983; Winqvist et al., 1982; Parillo & Fauci, 1978). The existence of a specific receptor for IgE on human eosinophils was first alluded to by Hubscher (1975), and later described in both rat and human eosinophils by Capron et al. (1981). It was also suggested that low-density eosinophils exhibit a higher expression of this receptor (Capron & Capron, 1987), and other techniques, such as flow cytometry (Capron et al., 1985) and in vitro eosinophil-mediated cytotoxicity, were also used to confirm the presence of such receptors on human eosinophils (Capron et al., 1984). It was suggested that this receptor for IgE consisted of two polypeptide chains of 50,000
Professor A. B. Kay, Dept. of Allergy and Clinical Immunology, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, U.K.
435
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R. Moqbel et al.
and 23,000-25,000 MW (Capron et al., 1985, 1986), with a binding affinity of 1- 17 x 107 M and approximately 105 binding sites per eosinophil (Jouault et al., 1988), and designated FceRII to distinguish it from the high affinity FcR found on mast cells and basophils. Recently, a number of laboratories have questioned the validity and existence of Fc8II on human eosinophils as well as its previously described homology with CD23 found on B cells. FcRII appears to have two distinct forms: FcERIIa, present only on B cells and Fc6RIIb, which is expressed by a number of inflammatory cells, including eosinophils (Yokota et al., 1988). This area of human eosinophil biology remains controversial and is receiving further in-depth attention. A number of chemotactic factors have been shown to effect changes in the expression of membrane receptors as well as function of granulocytes (Anwar et al., 1980; Nagy et al., 1982; Moqbel et al., 1983). Platelet-activating factor (PAF) is now recognized as the most potent eosinophilotactic agent so far described (Wardlaw et al., 1986). PAF has been shown to enhance the expression of complement (C3b), Fc (IgG) and Fc (IgE) receptors on normal density eosinophils and increases antibody(IgG and IgE)- and/or complement-dependent cytotoxicity (Moqbel et al., 1987b). Human eosinophils have been shown to generate leukotriene C4 (LTC4) when stimulated (Weller et al., 1984; Shaw, Cromwell & Kay, 1984; Henderson, Harley & Fauci, 1984). This sulphidopeptide leukotriene is converted from membrane phospholipid-derived arachidonic acid through the action of a 5lipo-oxygenase enzyme. In addition to its pro-inflammatory properties, LTC4 has also been shown to be associated with immediate-type hypersensitivity reactions against worm challenge (Moqbel et al., 1986). Elevated levels of LTC4 have also been described during the process of rapid expulsion of worms from the intestine of immune rats (Moqbel et al., 1987a). Furthermore, it has been suggested that LTC4-like activity may contribute directly to worm damage by inhibiting their movement (Douch et al., 1983). A number of physiological triggers have been used for the production of LTC4 from human eosinophils. These include the stimulation of eosinophils via their IgG (Fc) receptors following contact with human IgG covalently linked to Sepharose beads (Shaw et al., 1985) or antigen-antibody complexes (Cromwell et al., 1988). Both these physiologically relevant systems utilized opsonized large particles to provide a catalytic surface for the eosinophil to adhere to and exocytose, thus mimicking the adherence of these cells to opsonized helminthic larvae observed in vitro. In this study helminthic larvae have been used as the target for low-density eosinophil adherence after coating them with specific IgE and IgG antibodies and measured the amounts of LTC4 released. IgE- and IgG-dependent eosinophil-mediated cytotoxicity were compared as well as the effect of PAF, LTB4, lyso-PAF and the synthetic chemotactic tripeptide formyl-
methionyl-leucyl-phenylalanine (FMLP) on LTC4 production, using this larval parasitic system. MATERIALS AND METHODS Regents and buffers LTC4 (as well as D4 and E4, used in HPLC analysis) in solution in distilled water were a generous gift from Dr J. Rokach (Merck Frosst Laboratories, Quebec, Canada) and was stored in borocilicate vials at - 80° until used. Aliquots were diluted in
methanol and adjusted to a concentration of 5 x 10-5 M on the basis of their molar extinction coefficients (LTC4, D4 and E4E280= 40,000).Rabbit anti-serum to LTC4 was a gift from Dr A. W. Ford-Hutchinson (Merck-Frosst Laboratories, Quebec, Canada). [3H]LTC4 was obtained from New England Nuclear/ Du Pont, Stevenage, Herts. The following were obtained as shown: analytical grade metrizamide (Nyegaard Ltd, Birmingham); Dextran 110, 6% w/v solution in 0 9% NaCl (Dextran 110; Fisons plc, Loughborough, Leics); cyanogen-activated Sepharose 4B, protein A affinity Sepharose gel (Pharmacia AB, Uppsala, Sweden); preservative-free heparin sodium, 1000 U/ml (Pain and Byrne Ltd, Greenford, Middlesex); Hanks' balanced salt solution (HBSS), RPMI-1640 with 25 mm HEPES and glutamine (Gibco Ltd, Paisley, Renfrewshire) supplemented with Ca2+ and Mg2+ (calcium chloride and magnesium chloride) (BDH, Poole, Dorset); DNase I (type A) formyl-methionylleucyl-phenylalanine (FMLP), calcium ionophore A23187, histamine dihydrochloride, sodium azide (Sigma Chemicals Co., Poole, Dorset); optiphase scintillation fluid (Pharmacia LKB, Milton Keynes, Bucks); goat anti-rabbit immunoglobulin (BioRad Laboratories Ltd, Watford, Herts); and HPLC grade methanol, water, acetic acid and phosphoric acid (Rathburn Chemicals Ltd, Walkerburn, Scotland). Synthetic PAF (C 16) and lyso-PAF (C 16) (Bachem Ltd, Saffron Waldon, Essex) were dissolved in chloroform: methanol at 9: 1 and stored at - 800 until used. PAF and lyso-PAF were always prepared in buffer containing 0-25% bovine serum albumin (BSA; Fraction IV, Sigma Chemicals); LTB4 was purchased from Miles Scientific, Slough, Berks.
Separation of eosinophils Human eosinophils were isolated from peripheral blood by the methods of Vadas et al. (1979). Normal density blood eosinophils were obtained from asthmatic subjects attending a routine allergy clinic with an eosinophilia. Between 6% and 21% and low-density cells were recovered from the blood of patients with hyper-eosinophilic syndrome, who were in the care of Professor C. J. F. Spry (St Georges Hospital, London). Blood was anticoagulated with 10 U/ml of preservative-free heparin, and mixed with 0-2 volumes of 6% dextran 110. The plasma/ leucocyte fraction was pipetted off after 30 min incubation at 370, and the cells were harvested by centrifugation 250 g for 10 min at 40, washed twice with RPMI-1640 containing DNase I, resuspended with buffer at approximately 5-7 x 107 cells/ml, and layered onto discontinuous gradients of metrizamide (18, 20, 22, 23, 24 and 24% w/v metrizamide in Tyrode buffer containing 0- 1 % gelatin). Gradients were centrifuged at 1200 g for 40 min at 200. Normal density eosinophils were recovered from their 23/24% interface (metrizamide densities 1- 123-1 129 g/ml) with a purity of > 92%, and the low-density eosinophil population from the hyper-eosinophilic patients from the 20% and 22% metrizamide layers (metrizamide 1107 and 1-118 g/ ml), with a purity ranging from 70% to 86%. with both eosinophil phenotypes, neutrophils were the major contaminant with 0 05 was considered non-significant (NS)
RESULTS Human low-density eosinophils readily adhered to larvae when mixed with either live or formalin-fixed schistosomula of S. mansoni, previously coated with immune serum. These opsonized schistosomula acted as stimuli for LTC4 generation since supernatants from these experiments had LTC4 immunoreactivity (Table 1). Opsonized live schistosomula induced the release of LTC4 from hypodense eosinophils, but the amount was slightly less compared with that elicited by fixed schistosomula. These amounts of LTC4 elicited by live and fixed schistosomula represented 14-7% and 20-6% of the total amount of LTC4 generated by hypodense eosinophils following treatment with the calcium ionophore A23187 (at 5 x 10-6 M), respectively. In addition, the amount of LTC4 released by incubating these cells with IgG-coated Sepharose beads was equivalent to that generated in the presence of opsonized live larvae. Cells adhering to unopsonized schistosomula generated negligible amounts of immunoreactive LTC4. Immune (anti-schistosome) serum was subjected to anionexchange FPLC (SI/17 polyanion column) and fractions dialysed against RPMI-1640 buffer and examined for specific IgG (by ELISA) and specific IgE (by RAST) (Fig. 1). Fractions were divided into two equal volumes: one half was left untreated and the other applied to a Sepharose-protein A affinity column and
R. Moqbel et al.
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Table 1. Eosinophil-derived LTC4 release following stimulation with opsonized schistosomula, IgG-coated beads or calcium ionophore
2-0 E 0
w
Stimulus
Immunoreactive LTC4 (pmoles/106 Adherence* cells)
Live schistosomula + immune serum Fixed schistosomula + immune serum Live schistosomula + diluent Fixed schistosomula+diluent IgG-coated beads
117 + 27 165 + 35 0 1+0 1 0 1 +0 1 99 + 10
++ ++
A23187 (5 x 10-6M)
80 0+24
-
Untreated cells
I.0 i/. 30
D5 20
S 10
-,
-
-( b)
Z/
.0
.e~ ~ ~ '
83f~-
I I
I~e~
-(c)
c
_
4 2 0
-e *C
40 -(d) 30
* 20 10
-C
lI I I
(.)
0 "'
concentrated back to the original volume. Both were tested in parallel in the assays described below (Fig. I a, b).IgE, measured by specific RAST, was detected only in relatively anionic materials (Fractions 9-13), which eluted from the column at higher salt concentrations. This activity was not influenced by protein A treatment. In contrast, two peaks of IgG were detected using specific ELISA (Fractions 1-4 and 9-13), both of which were totally removed following protein A adsorption. Each of these fractions was then used to opsonize live and fixed schistosomula (both protein A-treated and untreated). Opsonized larvae were then added to hypodense eosinophils and both cytotoxicity and LTC4 generation were measured (Fig. 1 d, e). Two peaks of both cytotoxic activity and LTC4 were observed in association with concentrated fractions containing specific IgG and IgE. Following protein A adsorption, only the IgGassociated peak of activity was removed, leaving the presumed IgE-related second peak intact. The role of the specific IgE and IgG components of immune serum in eliciting both LTC4 elaboration from, and cytotoxicity by, low-density eosinophils was assessed next. IgG-rich (1-4) and IgE-rich (9-13) fractions from FPLC analysis (Fig. 1) were pooled separately. Samples of these pooled fractions were applied to either (i) a Sepharose-protein A column, or (ii) antihuman IgE affinity column or (iii) a consecutive run of both (i) and (ii). At each stage, the concentrations of specific IgG and IgE were determined by ELISA and RAST, respectively, and are presented in Table 2. IgG present in the IgE-rich fraction pool was removed by adsorption on a protein A column. This had no effect on the IgE concentration, as judged by RAST (which showed 36% binding of '251-anti-IgE). Anti-human IgE affinity column adsorption had no effect on IgG levels in both pools but removed specific IgE antibodies from the IgE-rich pool. Consecutive runs through protein A and anti-IgE columns depleted the IgE-rich pool of both IgG and IgE. Low-density eosinophils were incubated with schistosomula which were pre-coated with various preparations of parasitespecific antibodies. These included unfractionated immune serum (used at a final dilution of 1: 20) and FPLC-fractionated IgG- and IgE-rich pools, which were either untreated or adsorbed on protein A or both protein A and anti-IgE affinity
0.0.0.%*/
g" 40
+
* Adherence ofeosinophils to schistosomula was scored thus: (-) no adherence; (+) 4-6 cells adhering; (+ +) > 6 cells adhering. The data represent the mean + SEM of seven experiments.
, . *-
0
0
.t
0
(a)
V)
8 -(e) 6
O u
E 2 0.
1 2 3 4 5 6 7 8 9 10 11 12 1314 Fraction number Figure 1. (a) FPLC (polyanion SI-17) column) fractionation of antischistosoma serum. The serum underwent a desalting step on a G-25 column. (b and c) FPLC fractions 1-4 and 9-13 were pooled (a), dialysed against RPMI-1640 buffer and examined for the presence of(b) specific IgE (by RAST) and (c) IgG (by ELISA) before (0) and after (0) adsorption on a Staphylococcus aureus-protein A affinity column (bed volume 3 5 ml). Fractions (untreated or protein A-treated) were incubated with fixed schistosomula prior to incubation with low-density eosinophils for 45 min at 37°. (d) The same fractions were used to opsonize live (3-hr-old) schistosomula and used in an in vitro cytotoxicity assay at a cell: target ratio of 2000: 1. (e) LTC4 immunoreactivity was also measured by RIA and its identity confirmed by RP-HPLC.
columns (used neat). Immune, unfractionated serum elicited the release of 15 + 4 pmoles of LTC4 from 106 low-density eosinophils, and resulted in the killing of 82 + 5% of schistosomula, in vitro. Eosinophils adhering to schistosomula coated with specific IgG generated 6-0 + 0-1 pmoles LTC4/106 cells. This activity was completely ablated (0 1 +01 pmoles LTC4/106 cells) following adsorption of the IgG fraction on a protein A column.
Similarly, IgG-dependent eosinophil-mediated cytotoxicity was drastically reduced following protein A adsorption. Hypodense eosinophils were also incubated with schistosomula coated with parasite-specific, IgE-rich pools, before and after affinity chromatography treatments. The untreated IgE-rich preparation elicited the elaboration of LTC4 (7-8 + 0-4 pmoles/106 cells) and the killing of 31 + 1% of schistosomula. This preparation, which contained substantial amounts of contaminating IgG, was applied to a protein A column and this treatment abolished 97-4% ofthe IgG present, as judged by specific ELISA assay, without affecting the levels of parasite-specific IgE. When this absorbed preparation was used to coat schistosomula, it elicited the release of 5 5 + 0 4 pmoles of LTC4 per 106 cells and 30+1-5% cytotoxicity. This was further confirmed by heating this preparation (560, 1 hr) and using it in the same assays again. No LTC4 immunoreactivity was measured, and cytotoxicity
439
Release of LTC4 from human eosinophils Table 2. The effect of various treatments of FPLC fractions of immune (anti-schistosome) serum on the levels of specific IgE and IgG antibodies and the elicitation of LTC4 release from and cytotoxicity of human low-density eosinophils. The LTC4 and cytotoxicity data represent the mean + 1 SEM of three experiments
Specific anti-S. mansoni
binding
ELISA IgG OD units
(pmoles/106 cells)
Cytotoxicity (% kill)
36
8-4
15+4
82 + 5
0
6-8
0
0-1
6-0+0-1 0-1+0-1
40+2 9-4+4
7-8 +0-4 5-5+0-4 0 2±0-2
31+1 30+1-5 8+3
RAST % IgE
Opsonin Unfractionated immune serum IgG-rich fractions: Untreated +protein A IgE-rich fractions: Untreated +protein A alone +protein A and anti-IgE
35
7-0
32 0
0-18 0-2
Table 3. Elaboration of LTC4 release from normal and low-density human eosinophils following incubation with schistosomula of S. mansoni coated with purified, parasite-specific IgG and IgE
Immunoreactive LTC4 (pmoles/106 eosinophils) Normal density
Treatment Diluent PAF 10-7 M
Lyso-PAF 10-7 M LTB4 10-7 M FMLP 10-7 M
Low density
IgG-rich
IgE-rich
IgG-rich
IgE-rich
5-3 68
0-4 4-4
7-1 8-9
63 79
4-0 65 10-8
0-6 3-6 1.1
6-2 7-2
58 68 68
10-3
Eosinophil functional assays
Cells were either preincubated with diluent or an optimal dose of various chemotactic agonists. IgG-coated beads elicited the release of 4-6 and 7-3 pM of LTC4/106 normal and low-density eosinophils, respectively. The results represent the average of two experiments (in triplicate).
values were reduced to baseline values (6%). This suggests that the LTC4 generation and larval killing by the low-density eosinophils was mainly due to the presence of parasite-specific IgE and not to the contaminating IgG. Consecutive absorption on protein A and anti-IgE columns resulted in the total ablation of LTC4 elaboration and eosinophil cytotoxicity. The identity of LTC4, released in these experiments, was confirmed by HPLC analysis. The elution time (14-5 min) of LTC4 immunoreactivity was compared with that of synthetic markers for LTC4, LTD4 and LTE4. The majority of the immunoreactivity obtained was accounted for by LTC4 (data not shown). The effect of PAF, lyso-PAF, LTB4 and FMLP on influencing the ability of normal and low-density eosinophils to produce LTC4 in IgE- and IgG-dependent systems was compared. Mediators were used at doses of 10-7 M, shown previously to be optimal. Cells were preincubated with mediators for 30 min
LTC4 release
prior to mixing them with opsonized schistosomula (45 min). Due to the complex nature of this experiment, the results of two triplicate experiments (average values) are reported (Table 3). Normal density eosinophils, in the absence of treatment with any of the chemotactic factors, elaborated immunoreactive LTC4 when in contact with IgG- but not IgE-coated larvae. In contrast, hypodense eosinophils elaborated LTC4 in the presence of specific IgE- and IgG-coated schistosomula. IgGdependent release by normal density cells was slightly enhanced by PAF and LTB4 (30% and 18%, respectively) with FMLP having a greater effect (102%). Some of the mediators produced small increases in LTC4 generation by hypodense eosinophils under both IgG and IgE stimuli. Lyso-PAF did not induce any enhancement of generation from either cell type. Normal density eosinophils, stimulated with PAF (10-7 M), released 4-4 pmoles of immunoreactive LTC4 per 106 cells, when incubated with IgE-coated schistosomula, a 10-fold increase. LTB4 also enhanced IgE-dependent LTC4 release from these cells to 3-6 pmoles/106 cells. In contrast to PAF and LTB4, FMLP showed only a slight increase (to -1 pmoles/106 cells) under IgE stimulus. IgG-coated beads were used as positive controls and in this system hypodense eosinophils elaborated 7-3 pmoles of LTC4 compared with 4-4 pmoles per 106 normodense cells (Table 3).
DISCUSSION Inflammatory cells recruited to the relevant site of allergic reaction are thought to contribute to subsequent tissue damage, via a number of mechanisms, including the elaboration of an array of mediators and enzymes. These cellular products are either released from cytoplasmic stores or granules or synthesized de novo from membrane phospholipids. Many triggers for mediator release have been studied. These range from the calcium ionophore A23187 (Weller et al., 1984; Shaw et al., 1984) to physiological models involving IgG-, complement- or immune complex-dependent mechanisms using appropriately coated particles (Shaw et al., 1985; Cromwell et al., 1988). In this study, opsonized schistosomula were used as insoluble large targets to stimulate the eosinophil to elaborate LTC4.
440
R. Moqbel et al.
Eosinophils are known to adhere to and kill schistosomula, at least in vitro, as part of their putative function in the protection of the host against invading parasitic worms (Butterworth, 1984). Thus, in these respects the model described here is assumed to be pathophysiologically relevant. The results of this study indicated that low-density human eosinophils adhered to IgG- and IgE-coated schistosomula. Adherence of eosinophils to live schistosomula resulted in in vitro killing of larvae coated with purified, parasite-specific IgG and IgE. These results indicate that exocytosis, secretion of mediators, as well as cytotoxicity, are causally related (Gleich & Adolphson, 1986). The concentration of immunoreactive LTC4 elaborated by low-density eosinophils following interaction with fixed schistosomula was biologically relevant, representing approximately 26% of the total capacity of these cells to elaborate LTC4 compared with A23187-induced release. In a previous study, 21% of the total LTC4 capacity was obtained using IgG-coated Sepharose beads (Shaw et al., 1985). These results also confirm the observation that low-density eosinophils are involved functionally as effector inflammatory cells in IgE-dependent mechanisms (Capron et al., 1984, 1986). To reach this conclusion, it had to be ensured that the pool of immune sera from patients used in this study (containing both specific IgG and IgE) was carefully analysed and purified into IgG- and IgE-rich fractions. In addition to FPLC analysis, the IgE- and IgG-dependent events were further dissected by selective depletion of each component by affinity chromatography. Adsorption on protein A was employed either to deplete IgG (from the IgG-rich fractions) or remove contaminating IgG from IgE-rich pools without influencing the levels of IgE. Similarly, an anti-human IgE affinity column was used to abolish IgE-dependent activities in fractions previously depleted of IgG by protein A adsorption. Furthermore, in order to ascertain that LTC4 release (and cytotoxicity) induced by the IgE-purified pool was not due to any post-protein A residual IgG contamination, these fractions were heated (56°, I hr) to remove the IgE and used in the same assays. Both LTC4 release and cytotoxicity were reduced (data not shown), suggesting that these activities were not due to the presence of any traces of IgG. Thus specific IgE, as measured by RAST, appears to have been involved in binding of human low-density eosinophils to schistosomula larval killing, as well as LTC4 release. There was an interesting difference in the amounts of LTC4 generated from eosinophils following interaction with live compared with formalin-fixed, opsonized schistosomula (Table 1). The higher amounts of immunoreactive LTC4 elicited by dead schistosomula may have been due to the better accessibility of this immobile large surface compared with motile live larvae. It is also tempting to postulate that live larvae may generate substances which either inhibit, antagonize or modulate sulphidopeptide leukotrienes as a defence mechanism against the spasmogenic and contractile properties of these molecules. This is worthy of further investigation, bearing in mind the suggested inhibitory action of SRS-A-like activity against the motility of larvae of Haemonchus contortus (an ovine helminth), which was described in the mucus of immune sheep (Douch et al., 1983). Unfractionated immune serum used intact (i.e. unheated) to opsonize schistosomula (Table 2) contained complement, in addition to parasite-specific IgG and IgE. Activation of complement, which is known to occur via the alternative pathway as a
consequence of contact with helminthic larvae (Anwar et al., 1979), results in the deposition of C3b and C3bi onto the surface of schistosomula, in vitro. These two complement products are potent opsonins that can mediate adherence and killing by eosinophils via CRI and CR3 on their surface membrane, respectively. Complement therefore appears to augment eosinophil cytotoxicity and mediator release obtained with unfractionated serum, beyond the observed effect elicited by the purified IgG and IgE elements of that serum. Our study employed low-density eosinophils from eosinophilic patients, since they appear to be more metabolically 'activated' than normal density eosinophils, and are thought to have a higher expression of membrane receptors. The only contaminating cell seen in these preparations, after metrizamide purification, was the neutrophil. Since this study was interested in measuring LTC4, a predominantly eosinophil product (Weller et al., 1984; Shaw et al., 1984), the presence of contaminating neutrophils presented no major obstacle. With larval killing, eosinophils, especially of the hypodense type, are more proficient as cytotoxic cells than neutrophils, especially in antibody-dependent systems. It is noteworthy that pretreatment of low-density eosinophils with PAF prior to incubation with IgG- and IgE-coated schistosomula did not alter the amount of LTC4 elaborated (Table 3). This confirms previous data which suggested that these cells may already be activated (Shaw et al., 1985; Fitzharris et al., 1987) and cannot be further up-regulated. In contrast, normal density cells did not release LTC4 in the IgE-dependent system only. However, they elaborated significant amounts of LTC4 in the IgE-dependent system following stimulation with PAF. LTB4 also enhanced the IgE-dependent release, but to a considerably lesser extent than PAF. This indicates that the ability of resting cells to elaborate mediators may be related to the up-regulation of their receptor expression (cell activation) as a consequence of the action of secretagogues (Moqbel et al., 1987b). Of particular interest was the observation that FMLP was able to up-regulate IgG- but not IgE-dependent eosinophil functions. This is in agreement with similar results obtained using various assays to measure eosinophil receptors and cytotoxicity (Moqbel et al., 1987b; Walsh et al., 1987, 1989). These results indicate clearly that there was a significant functional activity of human low-density eosinophils mediated against larvae coated with purified, parasite-specific IgE. It is arguable that this activity, which was heat-labile, may be complement-dependent. However, the complex manipulations used in the various purification steps of IgE-rich fractions would certainly have destroyed any biologically relevant C components (i.e. C3b and iC3b), or at least diluted them beyond any viable concentration. Whether these IgE-dependent activities of human eosinophils (of low-density cells or PAF-stimulated eosinophils) operate via an IgE-specific receptor on these cells e.g. FcRIIb, or through the stimulation of other receptor ligands, is still unclear. It is now thought that the putative receptor for IgE on eosinophils may be totally distinct from FcRII found on B cells (i.e. CD23) (Yokota et al., 1988). This may be relevant in regard to unsuccessful attempts to identify CD23+ human eosinophils (of both low and normal density) using a considerable panel of anti-CD23 monoclonal antibodies and FACS analysis (Hartnell et al., 1989). Of course the possibility that IgE-dependent functions of the human eosinophil may operate via other, hitherto unidentified, receptors, or
Release of LTC4 from human eosinophils that such effects may be triggered by utilizing other existing receptors, such as CR3, can not be excluded. These aspects require further probing and are the subject of a separate study.
ACKNOWLEDGMENTS We wish to thank Miss Jane Lillywhite, Imperial College, London, for her help with the specific ELISA assays and Mrs Fiona Hackett, National Institute for Medical Research, Mill Hill, London, for the supply of S. mansoni-infected snails. We are also grateful to Dr Christine Harvey for helpful discussions. This work was supported by the Medical Research Council (U.K.).
REFERENCES ANWAR A.R.E., MCKEAN J.R., SMITHERS S.R. & KAY A.B. (1980) Human eosinophil- and neutrophil-mediated killing of schistosomula of Schistosoma mansoni in vitro. I. Enhancement of complementdependent damage by mast cell-derived mediators and formylmethionyl peptides. J. Immunol. 124, 1122. ANWAR A.R.E., SMITHERS S.R. & KAY A.B. (1979) Killing of schistosomula of Schistosoma mansoni coated with antibody and/or complement by human leucocytes in vitro: requirements for complement in preferential killing by eosinophils. J. Immunol. 122, 628. BUTTERWORTH A.E. (1984) Cell-mediated damage to helminths. Adv. Parasitol. 23, 143. CAPRON M. & CAPRON A. (1987) The IgE receptor of human eosinophils. In: Allergy and Inflammation (ed. A. B. Kay), p. 151. Academic Press, London. CAPRON M., CAPRON A., DESSAINT J.P., TORPIER G., JOHANSSON S.G.O. & PRIN L. (1981) Fc receptors for IgE on human and rat eosinophils. J. Immunol. 126, 2087. CAPRON M., JOUAULT T., PRIN L., JOSEPH M., AMEISEN J.-C., BUTTERWORTH A.E., PAPIN J.-P., KUSNIERZ J.-P. & CAPRON A. (1986) Functional study of a monoclonal antibody to IgE Fc receptor (FcE R2) of eosinophils, platelets and macrophages. J. exp. Med. 164, 72. CAPRON M., KUSNIERZ J.P., PRIN L., SPIEGELBERG H.L., OVLAGUE G., GOSSETT P., TONNEL A.B. & CAPRON A. (1985) Cytophilic IgE on human blood and tissue eosinophils: detection by flow microfluorometry. J. Immunol. 134, 3013. CAPRON M., SPIEGELBERG H.L., PRIN L., BENNICH H., BUTTERWORTH A.E., PIERCE A.E., OUAISSI M.A. & CAPRON A. (1984) Role of IgE receptors in effector function of human eosinophils. J. Immunol. 132, 462. CROMWELL O., MOQBEL R., FITZHARRIS P., KURLAK L., HARVEY C., WALSH G.M., SHAW R.J. & KAY A.B. (1988) Leukotriene C4 generation from human eosinophils stimulated with IgG-Aspergillus fumigatus antigen immune complex. J. Allergy Clin. Immunol. 85,535. CROMWELL O., SHAW R.J., WALSH G.M., MALLET A.I. & KAY A.B. (1985) Inhibition of leukotriene C4 and B4 generation by human eosinophils and neutrophils with lipoxygenase pathway inhibitors U60257 and BW755C. Int. J. Immunopharmacol. 7, 775. DE SAVIGNY D. & VOLLER A. (1980) The communication of ELISA data from laboratory to clinician. J. Immunoassay, 1, 105. DOUCH P.G.C., HARRISON G.B.L., BUCHANAN L.L. & GREER K.C. (1983) In vitro bioassay of sheep gastro-intestinal mucus for nematode paralysing activity mediated by substance with some properties characteristic of SRS-A. Int. J. Parasitol. 13, 207. FISCHER E., CAPRON M., PRIN L., KUSNIERZ J.P. & KAZATCHKINE M.D. (1986) Eosinophils express CRI and CR3 complement receptors for cleavage fragments of C3. Cell Immunol. 97, 297. FITZHARRIS P., MOQBEL R., THORNE K.J.I., RICHARDSON B.A., HARTNELL A., CROMWELL O. & KAY A.B. (1987) The effects of eosinophil
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activating factor on IgG-dependent sulphidopeptide leukotriene generation by human eosinophils. Immunology, 61, 449. GLEICH G.J. & ADOLPHSON C.R. (1986) The eosinophilic leukocyte: structure and function. Adv. Immunol. 39, 177. HARTNELL A., WALSH G.M., MOQBEL R. & KAY A.B. (1989) Phenotypic markers on human eosinophils. J. Allergy Clin. Immunol. 83,192 (abs 81). HARVEY C., SHAW R.J. & LONGBOTTOM J.L. (1987) Diagnostic specificity of a sandwich ELISA for Aspergillus-related diseases. J. Allergy Clin. Immunol. 79, 324. HENDERSON W.R., HARLEY J.B. & FAUCI A.S. (1984) Arachidonic acid metabolism in normal and hypereosinophilic syndrome human eosinophils: generation of leukotrienes B4, C4, D4 and 15-lipoxygenase products. Immunology, 51, 679. HUBSCHER T. (1975) Role of the eosinophil in allergic reactions. I. EDI, an eosinophil-derived inhibitor of histamine release. J. Immunol. 114, 1379. JOUAULT T., CAPRON M., BALLOUL J.M., AMEISEN J.C. & CAPRON A. (1988) Quantitative and qualitative analysis ofthe Fc receptor for IgE (FcRII) on human eosinophils. Eur. J. Immunol. 18, 237. KIMANI G., TONNESEN M.G., & HENSON P.M. (1988) Stimulation of eosinophil adherence to human vascular endothelial cells in vitro by platelet-activating factor. J. Immunol. 140, 3161. KULCZYCKI A. JR. (1984) Human neutrophils and eosinophils have structurally distinct Fc receptors. J. Immunol. 133, 849. MOQBEL R., KING S.J., MACDONALD A.J., MILLER H.R.P., CROMWELL O., SHAW R.J. & KAY A.B. (1986) Enteral and systemic release of leukotrienes during anaphylaxis of Nippostrongylus brasiliensis in primed rats. J. Immunol. 137, 296. MOQBEL R., SAss-KUHN S.P., GOETZL E.J. & KAY A.B. (1983) Enhancement of neutrophil- and eosinophil-mediated complementdependent killing of schistosomula of Schistosoma mansoni, in vitro, by leukotriene B4. Clin. exp. Immunol. 52, 519. MOQBEL R., WAKELIN D., MACDONALD A.J., KING S.J., GRENCIS R.K. & KAY A.B. (1987a) Release of leukotrienes during rapid expulsion of Trichinella spiralis from immune rats. Immunology, 60, 425. MOQBEL R., WALSH G.M., MACDONALD A.J., WARDLAW A.J. & KAY A.B. (1987b) Activation of human eosinophils by PAF-acether and other inflammatory mediators. Thorax, 42, 221 (abs). NAGY L., LEE T.H., GOETZL E.J., PICKETT W.C. & KAY A.B. (1982) Complement receptor enhancement and chemotaxis of human neutrophils and eosinophils by leukotrienes and other lipotygenase products. Clin. exp. Immunol. 47, 541. PARILLO J.E. & FAUCI A.S. (1987) Human eosinophils. Purification and cytotoxic capability of eosinophils from patients with the hypereosinophilic syndrome. Blood, 51, 457. PRIN L., CAPRON M., TONNEL A.B., BLETRY 0. & CAPRON A. (1983) Heterogeneity of human eosinophils; variability in cell density and cytotoxic ability in relation to the level and the origin of hypereosinophilia. Int. Arch. Allergy Appl. Immunol. 72, 336. RAMALHO-PINTO F.J., GAZZINELLI G., HOWELLS R.E., MOTA-SANTOS T.A., FIGUIREDO E.A. & PELLEGRINO J. (1974) Schistosoma mansoni: defined system for stepwise transformation of cercaria to schistosomula in vitro. Exp. Parasitol. 36, 360. SHAW R.J., CROMWELL 0. & KAY A.B. (1984) Preferential generation of leukotriene C4 by human eosinophils. Clin. exp. Immunol. 56, 716. SHAW R.J., WALSH G.M., CROMWELL O., MOQBEL R., SPRY C.J.F. & KAY A.B. (1985) Activated human eosinophils generate SRS-A leukotrienes after physiological (IgG-dependent) stimulation. Nature (Lond.), 316, 150. SPRY C.J.F. (1985) Synthesis and secretion of eosinophil granule substances. Immunol. Today, 6, 332. VADAS M.A., DAVID J.R., BUTTERWORTH A.E., PISANI N.T. & SIONGOK T.A. (1979) A new method for the purification of human eosinophils and neutrophils and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni. J. Immunol. 122, 1228.
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WALSH G.M., MOQBEL R., NAGAKURA T., IIKURA Y. & KAY A.B. (1989) Enhancement of the expression of eosinophil IgE receptor (Fc6 RII) and its function. J. Lipid Mediators (in press). WALSH G.M., MOQBEL R., WARDLAW A.J. & KAY A.B. (1987) In vitro effects of chemotactic factors on eosinophil Fc and complement receptor expression. Acta Paediat. Jap. 29, 672. WARDLAW A.J., MOQBEL R., CROMWELL 0. & KAY A.B. (1986) Platelet activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. J. clin. Invest. 78, 1701. WELLER P.F.C., LEE C.W., FOSTER D.W., COREY E.J., AUSTEN K.F. &
LEWIS R.A. (1984) Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4. Proc. natl. Acad. Sci. U.S.A. 80, 7626. WINQVIST I., OLOFSSON T., OLSSON I., PERSSON A.M. & HALLBERG T. (1982) Altered density, metabolism and surface receptors of eosinophils in eosinophilia. Immunology, 47, 531. YOKOTA A., KIKUTANI H., TANAKA T., SATO R., BARSUMIAN E.L., SuEMuRA M. & KISHIMOTO T. (1988) Two species of human Fc, receptor II (Fcc RII/CD23): tissue-species and IL-4-specific regulation of gene expression. Cell, 55, 611.
Release of leukotriene C4 (LTC4) from human eosinophils following adherence to IgE- and IgG-coated schistosomula of Schistosoma mansoni R. MOQBEL, A. J. MACDONALD,
CROMWELL & A. B. KAY Department of Allergy and Clinical Immunology, National Heart & Lung Institute, London
0.
Acceptedfor publication 14 November 1989
SUMMARY The release of leukotriene C4 (LTC4) from human low-density eosinophils following adherence to live or formalin-fixed schistosomula of Schistosoma mansoni coated with parasite-specific IgE or IgG obtained from pooled human anti-S. mansoni serum has been studied. IgE-rich fractions were obtained after fractionation of pooled immune sera on fast-protein liquid chromatography (FPLC; polyanion SI-17 column) and were identified by parasite-specific RAST. Contaminating IgG was removed by adsorption on a Staphylococcus aureus-protein A affinity column. IgG-rich FPLC fractions were identified by a specific ELISA assay. IgG-dependent activities were confirmed by protein A adsorption. Low-density eosinophils adhered to live and formalin-fixed schistosomula coated with specific antisera and released 11-7+2-7 and 16-5+3-5 pmoles of LTC4/106 cells, respectively. LTC4 release induced by A23187 (5 x 10-6 M) from the same cells was 80 + 24 pmoles/106 cells and 9-9 + 1 pmoles/106 cells in the presence of Sepharose particles (CNBr-activated 4B beads) covalently coated with normal human IgG. Fixed schistosomula coated with FPLC-purified IgE and IgG gave 7-6 + 0-4 and 6-0 + 0-1 pmoles of LTC4 per 106 low-density eosinophils, respectively. The same IgE- and IgG-rich fractions induced eosinophil-mediated cytotoxicity of live schistosomula in vitro. Removal of IgE by an anti-IgE affinity column abolished both the IgE-dependent release of LTC4 and the in vitro killing of larvae. Conversely, IgG-dependent activities were abolished by protein A, but not anti-IgE, adsorption. Normal density eosinophils generated undetectable amounts of LTC4 when incubated with IgE-coated schistosomula, whereas with IgG-coated larvae 4-6 pmoles/ 106 cells were obtained. Following preincubation with platelet-activating factor (PAF) (10-7 M) and leukotriene B4 (LTB4) (I0-7 M), normal density eosinophils released LTC4 when in contact with larvae coated with antigen-specific IgE. Lyso-PAF had no effect in any of the systems tested. The synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) had no influence on IgEdependent release of LTC4 from eosinophils. In contrast, FMLP (10-7 M) enhanced the IgGdependent LTC4 release, with PAF and LTB4 also showing a small enhancing effect. None of these agents substantially altered the release potential of low-density eosinophils in either IgE- or IgGdependent events. Thus the results presented here indicate that in an IgE-dependent system, human low-density eosinophils can be induced to adhere to and kill IgE-coated helminthic targets and release biologically relevant amounts of LTC4. This may have important implications within the context of allergic inflammation, especially as PAF and LTB4 appeared to play an amplifying role in IgEdependent activation of normal eosinophils.
INTRODUCTION Human eosinophils possess a number of surface receptors that participate in the process of adherence (Kimani, Tonnesen & Henson, 1988) and exocytosis (Spry, 1985) and, at least in vitro, mediate the killing of appropriately opsonized helminthic targets (e.g. schistosomula of Schistosoma mansoni; Butterworth, 1984). Among these receptors are those for complement (CR1 and CR3) (Fischer et al., 1986) and IgG (Fc) (Kulczycki, 1984). The majority of eosinophils from patients with hyper-
eosinophilia are of the low-density type, and appear to be more 'activated' than normal density cells (Prin et al., 1983; Winqvist et al., 1982; Parillo & Fauci, 1978). The existence of a specific receptor for IgE on human eosinophils was first alluded to by Hubscher (1975), and later described in both rat and human eosinophils by Capron et al. (1981). It was also suggested that low-density eosinophils exhibit a higher expression of this receptor (Capron & Capron, 1987), and other techniques, such as flow cytometry (Capron et al., 1985) and in vitro eosinophil-mediated cytotoxicity, were also used to confirm the presence of such receptors on human eosinophils (Capron et al., 1984). It was suggested that this receptor for IgE consisted of two polypeptide chains of 50,000
Professor A. B. Kay, Dept. of Allergy and Clinical Immunology, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, U.K.
435
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R. Moqbel et al.
and 23,000-25,000 MW (Capron et al., 1985, 1986), with a binding affinity of 1- 17 x 107 M and approximately 105 binding sites per eosinophil (Jouault et al., 1988), and designated FceRII to distinguish it from the high affinity FcR found on mast cells and basophils. Recently, a number of laboratories have questioned the validity and existence of Fc8II on human eosinophils as well as its previously described homology with CD23 found on B cells. FcRII appears to have two distinct forms: FcERIIa, present only on B cells and Fc6RIIb, which is expressed by a number of inflammatory cells, including eosinophils (Yokota et al., 1988). This area of human eosinophil biology remains controversial and is receiving further in-depth attention. A number of chemotactic factors have been shown to effect changes in the expression of membrane receptors as well as function of granulocytes (Anwar et al., 1980; Nagy et al., 1982; Moqbel et al., 1983). Platelet-activating factor (PAF) is now recognized as the most potent eosinophilotactic agent so far described (Wardlaw et al., 1986). PAF has been shown to enhance the expression of complement (C3b), Fc (IgG) and Fc (IgE) receptors on normal density eosinophils and increases antibody(IgG and IgE)- and/or complement-dependent cytotoxicity (Moqbel et al., 1987b). Human eosinophils have been shown to generate leukotriene C4 (LTC4) when stimulated (Weller et al., 1984; Shaw, Cromwell & Kay, 1984; Henderson, Harley & Fauci, 1984). This sulphidopeptide leukotriene is converted from membrane phospholipid-derived arachidonic acid through the action of a 5lipo-oxygenase enzyme. In addition to its pro-inflammatory properties, LTC4 has also been shown to be associated with immediate-type hypersensitivity reactions against worm challenge (Moqbel et al., 1986). Elevated levels of LTC4 have also been described during the process of rapid expulsion of worms from the intestine of immune rats (Moqbel et al., 1987a). Furthermore, it has been suggested that LTC4-like activity may contribute directly to worm damage by inhibiting their movement (Douch et al., 1983). A number of physiological triggers have been used for the production of LTC4 from human eosinophils. These include the stimulation of eosinophils via their IgG (Fc) receptors following contact with human IgG covalently linked to Sepharose beads (Shaw et al., 1985) or antigen-antibody complexes (Cromwell et al., 1988). Both these physiologically relevant systems utilized opsonized large particles to provide a catalytic surface for the eosinophil to adhere to and exocytose, thus mimicking the adherence of these cells to opsonized helminthic larvae observed in vitro. In this study helminthic larvae have been used as the target for low-density eosinophil adherence after coating them with specific IgE and IgG antibodies and measured the amounts of LTC4 released. IgE- and IgG-dependent eosinophil-mediated cytotoxicity were compared as well as the effect of PAF, LTB4, lyso-PAF and the synthetic chemotactic tripeptide formyl-
methionyl-leucyl-phenylalanine (FMLP) on LTC4 production, using this larval parasitic system. MATERIALS AND METHODS Regents and buffers LTC4 (as well as D4 and E4, used in HPLC analysis) in solution in distilled water were a generous gift from Dr J. Rokach (Merck Frosst Laboratories, Quebec, Canada) and was stored in borocilicate vials at - 80° until used. Aliquots were diluted in
methanol and adjusted to a concentration of 5 x 10-5 M on the basis of their molar extinction coefficients (LTC4, D4 and E4E280= 40,000).Rabbit anti-serum to LTC4 was a gift from Dr A. W. Ford-Hutchinson (Merck-Frosst Laboratories, Quebec, Canada). [3H]LTC4 was obtained from New England Nuclear/ Du Pont, Stevenage, Herts. The following were obtained as shown: analytical grade metrizamide (Nyegaard Ltd, Birmingham); Dextran 110, 6% w/v solution in 0 9% NaCl (Dextran 110; Fisons plc, Loughborough, Leics); cyanogen-activated Sepharose 4B, protein A affinity Sepharose gel (Pharmacia AB, Uppsala, Sweden); preservative-free heparin sodium, 1000 U/ml (Pain and Byrne Ltd, Greenford, Middlesex); Hanks' balanced salt solution (HBSS), RPMI-1640 with 25 mm HEPES and glutamine (Gibco Ltd, Paisley, Renfrewshire) supplemented with Ca2+ and Mg2+ (calcium chloride and magnesium chloride) (BDH, Poole, Dorset); DNase I (type A) formyl-methionylleucyl-phenylalanine (FMLP), calcium ionophore A23187, histamine dihydrochloride, sodium azide (Sigma Chemicals Co., Poole, Dorset); optiphase scintillation fluid (Pharmacia LKB, Milton Keynes, Bucks); goat anti-rabbit immunoglobulin (BioRad Laboratories Ltd, Watford, Herts); and HPLC grade methanol, water, acetic acid and phosphoric acid (Rathburn Chemicals Ltd, Walkerburn, Scotland). Synthetic PAF (C 16) and lyso-PAF (C 16) (Bachem Ltd, Saffron Waldon, Essex) were dissolved in chloroform: methanol at 9: 1 and stored at - 800 until used. PAF and lyso-PAF were always prepared in buffer containing 0-25% bovine serum albumin (BSA; Fraction IV, Sigma Chemicals); LTB4 was purchased from Miles Scientific, Slough, Berks.
Separation of eosinophils Human eosinophils were isolated from peripheral blood by the methods of Vadas et al. (1979). Normal density blood eosinophils were obtained from asthmatic subjects attending a routine allergy clinic with an eosinophilia. Between 6% and 21% and low-density cells were recovered from the blood of patients with hyper-eosinophilic syndrome, who were in the care of Professor C. J. F. Spry (St Georges Hospital, London). Blood was anticoagulated with 10 U/ml of preservative-free heparin, and mixed with 0-2 volumes of 6% dextran 110. The plasma/ leucocyte fraction was pipetted off after 30 min incubation at 370, and the cells were harvested by centrifugation 250 g for 10 min at 40, washed twice with RPMI-1640 containing DNase I, resuspended with buffer at approximately 5-7 x 107 cells/ml, and layered onto discontinuous gradients of metrizamide (18, 20, 22, 23, 24 and 24% w/v metrizamide in Tyrode buffer containing 0- 1 % gelatin). Gradients were centrifuged at 1200 g for 40 min at 200. Normal density eosinophils were recovered from their 23/24% interface (metrizamide densities 1- 123-1 129 g/ml) with a purity of > 92%, and the low-density eosinophil population from the hyper-eosinophilic patients from the 20% and 22% metrizamide layers (metrizamide 1107 and 1-118 g/ ml), with a purity ranging from 70% to 86%. with both eosinophil phenotypes, neutrophils were the major contaminant with 0 05 was considered non-significant (NS)
RESULTS Human low-density eosinophils readily adhered to larvae when mixed with either live or formalin-fixed schistosomula of S. mansoni, previously coated with immune serum. These opsonized schistosomula acted as stimuli for LTC4 generation since supernatants from these experiments had LTC4 immunoreactivity (Table 1). Opsonized live schistosomula induced the release of LTC4 from hypodense eosinophils, but the amount was slightly less compared with that elicited by fixed schistosomula. These amounts of LTC4 elicited by live and fixed schistosomula represented 14-7% and 20-6% of the total amount of LTC4 generated by hypodense eosinophils following treatment with the calcium ionophore A23187 (at 5 x 10-6 M), respectively. In addition, the amount of LTC4 released by incubating these cells with IgG-coated Sepharose beads was equivalent to that generated in the presence of opsonized live larvae. Cells adhering to unopsonized schistosomula generated negligible amounts of immunoreactive LTC4. Immune (anti-schistosome) serum was subjected to anionexchange FPLC (SI/17 polyanion column) and fractions dialysed against RPMI-1640 buffer and examined for specific IgG (by ELISA) and specific IgE (by RAST) (Fig. 1). Fractions were divided into two equal volumes: one half was left untreated and the other applied to a Sepharose-protein A affinity column and
R. Moqbel et al.
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Table 1. Eosinophil-derived LTC4 release following stimulation with opsonized schistosomula, IgG-coated beads or calcium ionophore
2-0 E 0
w
Stimulus
Immunoreactive LTC4 (pmoles/106 Adherence* cells)
Live schistosomula + immune serum Fixed schistosomula + immune serum Live schistosomula + diluent Fixed schistosomula+diluent IgG-coated beads
117 + 27 165 + 35 0 1+0 1 0 1 +0 1 99 + 10
++ ++
A23187 (5 x 10-6M)
80 0+24
-
Untreated cells
I.0 i/. 30
D5 20
S 10
-,
-
-( b)
Z/
.0
.e~ ~ ~ '
83f~-
I I
I~e~
-(c)
c
_
4 2 0
-e *C
40 -(d) 30
* 20 10
-C
lI I I
(.)
0 "'
concentrated back to the original volume. Both were tested in parallel in the assays described below (Fig. I a, b).IgE, measured by specific RAST, was detected only in relatively anionic materials (Fractions 9-13), which eluted from the column at higher salt concentrations. This activity was not influenced by protein A treatment. In contrast, two peaks of IgG were detected using specific ELISA (Fractions 1-4 and 9-13), both of which were totally removed following protein A adsorption. Each of these fractions was then used to opsonize live and fixed schistosomula (both protein A-treated and untreated). Opsonized larvae were then added to hypodense eosinophils and both cytotoxicity and LTC4 generation were measured (Fig. 1 d, e). Two peaks of both cytotoxic activity and LTC4 were observed in association with concentrated fractions containing specific IgG and IgE. Following protein A adsorption, only the IgGassociated peak of activity was removed, leaving the presumed IgE-related second peak intact. The role of the specific IgE and IgG components of immune serum in eliciting both LTC4 elaboration from, and cytotoxicity by, low-density eosinophils was assessed next. IgG-rich (1-4) and IgE-rich (9-13) fractions from FPLC analysis (Fig. 1) were pooled separately. Samples of these pooled fractions were applied to either (i) a Sepharose-protein A column, or (ii) antihuman IgE affinity column or (iii) a consecutive run of both (i) and (ii). At each stage, the concentrations of specific IgG and IgE were determined by ELISA and RAST, respectively, and are presented in Table 2. IgG present in the IgE-rich fraction pool was removed by adsorption on a protein A column. This had no effect on the IgE concentration, as judged by RAST (which showed 36% binding of '251-anti-IgE). Anti-human IgE affinity column adsorption had no effect on IgG levels in both pools but removed specific IgE antibodies from the IgE-rich pool. Consecutive runs through protein A and anti-IgE columns depleted the IgE-rich pool of both IgG and IgE. Low-density eosinophils were incubated with schistosomula which were pre-coated with various preparations of parasitespecific antibodies. These included unfractionated immune serum (used at a final dilution of 1: 20) and FPLC-fractionated IgG- and IgE-rich pools, which were either untreated or adsorbed on protein A or both protein A and anti-IgE affinity
0.0.0.%*/
g" 40
+
* Adherence ofeosinophils to schistosomula was scored thus: (-) no adherence; (+) 4-6 cells adhering; (+ +) > 6 cells adhering. The data represent the mean + SEM of seven experiments.
, . *-
0
0
.t
0
(a)
V)
8 -(e) 6
O u
E 2 0.
1 2 3 4 5 6 7 8 9 10 11 12 1314 Fraction number Figure 1. (a) FPLC (polyanion SI-17) column) fractionation of antischistosoma serum. The serum underwent a desalting step on a G-25 column. (b and c) FPLC fractions 1-4 and 9-13 were pooled (a), dialysed against RPMI-1640 buffer and examined for the presence of(b) specific IgE (by RAST) and (c) IgG (by ELISA) before (0) and after (0) adsorption on a Staphylococcus aureus-protein A affinity column (bed volume 3 5 ml). Fractions (untreated or protein A-treated) were incubated with fixed schistosomula prior to incubation with low-density eosinophils for 45 min at 37°. (d) The same fractions were used to opsonize live (3-hr-old) schistosomula and used in an in vitro cytotoxicity assay at a cell: target ratio of 2000: 1. (e) LTC4 immunoreactivity was also measured by RIA and its identity confirmed by RP-HPLC.
columns (used neat). Immune, unfractionated serum elicited the release of 15 + 4 pmoles of LTC4 from 106 low-density eosinophils, and resulted in the killing of 82 + 5% of schistosomula, in vitro. Eosinophils adhering to schistosomula coated with specific IgG generated 6-0 + 0-1 pmoles LTC4/106 cells. This activity was completely ablated (0 1 +01 pmoles LTC4/106 cells) following adsorption of the IgG fraction on a protein A column.
Similarly, IgG-dependent eosinophil-mediated cytotoxicity was drastically reduced following protein A adsorption. Hypodense eosinophils were also incubated with schistosomula coated with parasite-specific, IgE-rich pools, before and after affinity chromatography treatments. The untreated IgE-rich preparation elicited the elaboration of LTC4 (7-8 + 0-4 pmoles/106 cells) and the killing of 31 + 1% of schistosomula. This preparation, which contained substantial amounts of contaminating IgG, was applied to a protein A column and this treatment abolished 97-4% ofthe IgG present, as judged by specific ELISA assay, without affecting the levels of parasite-specific IgE. When this absorbed preparation was used to coat schistosomula, it elicited the release of 5 5 + 0 4 pmoles of LTC4 per 106 cells and 30+1-5% cytotoxicity. This was further confirmed by heating this preparation (560, 1 hr) and using it in the same assays again. No LTC4 immunoreactivity was measured, and cytotoxicity
439
Release of LTC4 from human eosinophils Table 2. The effect of various treatments of FPLC fractions of immune (anti-schistosome) serum on the levels of specific IgE and IgG antibodies and the elicitation of LTC4 release from and cytotoxicity of human low-density eosinophils. The LTC4 and cytotoxicity data represent the mean + 1 SEM of three experiments
Specific anti-S. mansoni
binding
ELISA IgG OD units
(pmoles/106 cells)
Cytotoxicity (% kill)
36
8-4
15+4
82 + 5
0
6-8
0
0-1
6-0+0-1 0-1+0-1
40+2 9-4+4
7-8 +0-4 5-5+0-4 0 2±0-2
31+1 30+1-5 8+3
RAST % IgE
Opsonin Unfractionated immune serum IgG-rich fractions: Untreated +protein A IgE-rich fractions: Untreated +protein A alone +protein A and anti-IgE
35
7-0
32 0
0-18 0-2
Table 3. Elaboration of LTC4 release from normal and low-density human eosinophils following incubation with schistosomula of S. mansoni coated with purified, parasite-specific IgG and IgE
Immunoreactive LTC4 (pmoles/106 eosinophils) Normal density
Treatment Diluent PAF 10-7 M
Lyso-PAF 10-7 M LTB4 10-7 M FMLP 10-7 M
Low density
IgG-rich
IgE-rich
IgG-rich
IgE-rich
5-3 68
0-4 4-4
7-1 8-9
63 79
4-0 65 10-8
0-6 3-6 1.1
6-2 7-2
58 68 68
10-3
Eosinophil functional assays
Cells were either preincubated with diluent or an optimal dose of various chemotactic agonists. IgG-coated beads elicited the release of 4-6 and 7-3 pM of LTC4/106 normal and low-density eosinophils, respectively. The results represent the average of two experiments (in triplicate).
values were reduced to baseline values (6%). This suggests that the LTC4 generation and larval killing by the low-density eosinophils was mainly due to the presence of parasite-specific IgE and not to the contaminating IgG. Consecutive absorption on protein A and anti-IgE columns resulted in the total ablation of LTC4 elaboration and eosinophil cytotoxicity. The identity of LTC4, released in these experiments, was confirmed by HPLC analysis. The elution time (14-5 min) of LTC4 immunoreactivity was compared with that of synthetic markers for LTC4, LTD4 and LTE4. The majority of the immunoreactivity obtained was accounted for by LTC4 (data not shown). The effect of PAF, lyso-PAF, LTB4 and FMLP on influencing the ability of normal and low-density eosinophils to produce LTC4 in IgE- and IgG-dependent systems was compared. Mediators were used at doses of 10-7 M, shown previously to be optimal. Cells were preincubated with mediators for 30 min
LTC4 release
prior to mixing them with opsonized schistosomula (45 min). Due to the complex nature of this experiment, the results of two triplicate experiments (average values) are reported (Table 3). Normal density eosinophils, in the absence of treatment with any of the chemotactic factors, elaborated immunoreactive LTC4 when in contact with IgG- but not IgE-coated larvae. In contrast, hypodense eosinophils elaborated LTC4 in the presence of specific IgE- and IgG-coated schistosomula. IgGdependent release by normal density cells was slightly enhanced by PAF and LTB4 (30% and 18%, respectively) with FMLP having a greater effect (102%). Some of the mediators produced small increases in LTC4 generation by hypodense eosinophils under both IgG and IgE stimuli. Lyso-PAF did not induce any enhancement of generation from either cell type. Normal density eosinophils, stimulated with PAF (10-7 M), released 4-4 pmoles of immunoreactive LTC4 per 106 cells, when incubated with IgE-coated schistosomula, a 10-fold increase. LTB4 also enhanced IgE-dependent LTC4 release from these cells to 3-6 pmoles/106 cells. In contrast to PAF and LTB4, FMLP showed only a slight increase (to -1 pmoles/106 cells) under IgE stimulus. IgG-coated beads were used as positive controls and in this system hypodense eosinophils elaborated 7-3 pmoles of LTC4 compared with 4-4 pmoles per 106 normodense cells (Table 3).
DISCUSSION Inflammatory cells recruited to the relevant site of allergic reaction are thought to contribute to subsequent tissue damage, via a number of mechanisms, including the elaboration of an array of mediators and enzymes. These cellular products are either released from cytoplasmic stores or granules or synthesized de novo from membrane phospholipids. Many triggers for mediator release have been studied. These range from the calcium ionophore A23187 (Weller et al., 1984; Shaw et al., 1984) to physiological models involving IgG-, complement- or immune complex-dependent mechanisms using appropriately coated particles (Shaw et al., 1985; Cromwell et al., 1988). In this study, opsonized schistosomula were used as insoluble large targets to stimulate the eosinophil to elaborate LTC4.
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Eosinophils are known to adhere to and kill schistosomula, at least in vitro, as part of their putative function in the protection of the host against invading parasitic worms (Butterworth, 1984). Thus, in these respects the model described here is assumed to be pathophysiologically relevant. The results of this study indicated that low-density human eosinophils adhered to IgG- and IgE-coated schistosomula. Adherence of eosinophils to live schistosomula resulted in in vitro killing of larvae coated with purified, parasite-specific IgG and IgE. These results indicate that exocytosis, secretion of mediators, as well as cytotoxicity, are causally related (Gleich & Adolphson, 1986). The concentration of immunoreactive LTC4 elaborated by low-density eosinophils following interaction with fixed schistosomula was biologically relevant, representing approximately 26% of the total capacity of these cells to elaborate LTC4 compared with A23187-induced release. In a previous study, 21% of the total LTC4 capacity was obtained using IgG-coated Sepharose beads (Shaw et al., 1985). These results also confirm the observation that low-density eosinophils are involved functionally as effector inflammatory cells in IgE-dependent mechanisms (Capron et al., 1984, 1986). To reach this conclusion, it had to be ensured that the pool of immune sera from patients used in this study (containing both specific IgG and IgE) was carefully analysed and purified into IgG- and IgE-rich fractions. In addition to FPLC analysis, the IgE- and IgG-dependent events were further dissected by selective depletion of each component by affinity chromatography. Adsorption on protein A was employed either to deplete IgG (from the IgG-rich fractions) or remove contaminating IgG from IgE-rich pools without influencing the levels of IgE. Similarly, an anti-human IgE affinity column was used to abolish IgE-dependent activities in fractions previously depleted of IgG by protein A adsorption. Furthermore, in order to ascertain that LTC4 release (and cytotoxicity) induced by the IgE-purified pool was not due to any post-protein A residual IgG contamination, these fractions were heated (56°, I hr) to remove the IgE and used in the same assays. Both LTC4 release and cytotoxicity were reduced (data not shown), suggesting that these activities were not due to the presence of any traces of IgG. Thus specific IgE, as measured by RAST, appears to have been involved in binding of human low-density eosinophils to schistosomula larval killing, as well as LTC4 release. There was an interesting difference in the amounts of LTC4 generated from eosinophils following interaction with live compared with formalin-fixed, opsonized schistosomula (Table 1). The higher amounts of immunoreactive LTC4 elicited by dead schistosomula may have been due to the better accessibility of this immobile large surface compared with motile live larvae. It is also tempting to postulate that live larvae may generate substances which either inhibit, antagonize or modulate sulphidopeptide leukotrienes as a defence mechanism against the spasmogenic and contractile properties of these molecules. This is worthy of further investigation, bearing in mind the suggested inhibitory action of SRS-A-like activity against the motility of larvae of Haemonchus contortus (an ovine helminth), which was described in the mucus of immune sheep (Douch et al., 1983). Unfractionated immune serum used intact (i.e. unheated) to opsonize schistosomula (Table 2) contained complement, in addition to parasite-specific IgG and IgE. Activation of complement, which is known to occur via the alternative pathway as a
consequence of contact with helminthic larvae (Anwar et al., 1979), results in the deposition of C3b and C3bi onto the surface of schistosomula, in vitro. These two complement products are potent opsonins that can mediate adherence and killing by eosinophils via CRI and CR3 on their surface membrane, respectively. Complement therefore appears to augment eosinophil cytotoxicity and mediator release obtained with unfractionated serum, beyond the observed effect elicited by the purified IgG and IgE elements of that serum. Our study employed low-density eosinophils from eosinophilic patients, since they appear to be more metabolically 'activated' than normal density eosinophils, and are thought to have a higher expression of membrane receptors. The only contaminating cell seen in these preparations, after metrizamide purification, was the neutrophil. Since this study was interested in measuring LTC4, a predominantly eosinophil product (Weller et al., 1984; Shaw et al., 1984), the presence of contaminating neutrophils presented no major obstacle. With larval killing, eosinophils, especially of the hypodense type, are more proficient as cytotoxic cells than neutrophils, especially in antibody-dependent systems. It is noteworthy that pretreatment of low-density eosinophils with PAF prior to incubation with IgG- and IgE-coated schistosomula did not alter the amount of LTC4 elaborated (Table 3). This confirms previous data which suggested that these cells may already be activated (Shaw et al., 1985; Fitzharris et al., 1987) and cannot be further up-regulated. In contrast, normal density cells did not release LTC4 in the IgE-dependent system only. However, they elaborated significant amounts of LTC4 in the IgE-dependent system following stimulation with PAF. LTB4 also enhanced the IgE-dependent release, but to a considerably lesser extent than PAF. This indicates that the ability of resting cells to elaborate mediators may be related to the up-regulation of their receptor expression (cell activation) as a consequence of the action of secretagogues (Moqbel et al., 1987b). Of particular interest was the observation that FMLP was able to up-regulate IgG- but not IgE-dependent eosinophil functions. This is in agreement with similar results obtained using various assays to measure eosinophil receptors and cytotoxicity (Moqbel et al., 1987b; Walsh et al., 1987, 1989). These results indicate clearly that there was a significant functional activity of human low-density eosinophils mediated against larvae coated with purified, parasite-specific IgE. It is arguable that this activity, which was heat-labile, may be complement-dependent. However, the complex manipulations used in the various purification steps of IgE-rich fractions would certainly have destroyed any biologically relevant C components (i.e. C3b and iC3b), or at least diluted them beyond any viable concentration. Whether these IgE-dependent activities of human eosinophils (of low-density cells or PAF-stimulated eosinophils) operate via an IgE-specific receptor on these cells e.g. FcRIIb, or through the stimulation of other receptor ligands, is still unclear. It is now thought that the putative receptor for IgE on eosinophils may be totally distinct from FcRII found on B cells (i.e. CD23) (Yokota et al., 1988). This may be relevant in regard to unsuccessful attempts to identify CD23+ human eosinophils (of both low and normal density) using a considerable panel of anti-CD23 monoclonal antibodies and FACS analysis (Hartnell et al., 1989). Of course the possibility that IgE-dependent functions of the human eosinophil may operate via other, hitherto unidentified, receptors, or
Release of LTC4 from human eosinophils that such effects may be triggered by utilizing other existing receptors, such as CR3, can not be excluded. These aspects require further probing and are the subject of a separate study.
ACKNOWLEDGMENTS We wish to thank Miss Jane Lillywhite, Imperial College, London, for her help with the specific ELISA assays and Mrs Fiona Hackett, National Institute for Medical Research, Mill Hill, London, for the supply of S. mansoni-infected snails. We are also grateful to Dr Christine Harvey for helpful discussions. This work was supported by the Medical Research Council (U.K.).
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