Br. J. Pharmacol. (1990), 99, 798-802

.Iv,'-. Macmillan Press Ltd, 1990

Inhibition of neutrophil and eosinophil induced chemotaxis by nedocromil sodium and sodium cromoglycate 'P.L.B. Bruijnzeel, R.A.J. Warringa, P.T.M. Kok & J. Kreukniet Department of Pulmonary Disease, State University Hospital Utrecht, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands 1 Neutrophils and eosinophils infiltrate the airways in association with the allergen-induced late phase asthmatic reaction. Mobilization of these cells takes place via lipid-like and protein-like chemotactic factors. In this study platelet-activating factor (PAF), leukotriene B4 (LTB4), zymosan-activated serum (ZAS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as illustrative examples of both groups. Chemotaxis was studied in human neutrophils and eosinophils. The inhibitory effects of nedocromil sodium and sodium cromoglycate were evaluated. 2 All chemotactic factors tested attracted neutrophils with the following rank order of activity: ZAS > PAF FMLP -LTB4. Eosinophils were only mobilized by PAF, LTB4 and ZAS with the following rank order of activity: ZAS > PAF > LTB4 .

3 Nedocromil sodium and sodium cromoglycate were equally active as the PAF antagonist BN 52021 in inhibiting the PAF-induced chemotaxis of neutrophils (IC50,o 10-8M). Both drugs were also equally active in inhibiting the chemotaxis of neutrophils induced by ZAS (IC50 10- 7-10-6 M), FMLP (IC50 10- 7M) and LTB4 (IC50 10- 6M). 4 Nedocromil sodium significantly inhibited the chemotaxis of eosinophils induced by PAF (IC50 10- 6M) and LTB4 (IC50 ; 10- 7M). The inhibitory potency of BN 52021 was similar to that of nedocromil sodium on the PAF-induced chemotaxis of eosinophils. Sodium cromoglycate was incapable of eliciting significant inhibition of these chemotactic responses. However, sodium cromoglycate significantly inhibited the chemotaxis of eosinophils induced by ZAS (IC50 10-7M), whereas nedocromil sodium was ineffective. -

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Introduction When allergic asthmatic individuals are challenged with allergen an early phase asthmatic reaction (within 30min of challenge) and a late phase reaction (4-6h after challenge) may develop. The cellular events taking place during this late phase reaction are considered highly relevant to the pathogenesis of asthma (Durham & Kay, 1985; de Monchy, 1986). Bronchoalveolar lavage (BAL) studies of the cellular changes during this response have demonstrated the presence of inflammatory cells (e.g. eosinophils and neutrophils) (de Monchy et al., 1985; Metzger et al., 1985; Diaz et al., 1987), indicating active migration of these cells into the bronchial lumen. Inflammatory cells may be mobilized by chemotactic agents of protein- or lipid-nature such as neutrophil chemotactic factor, eosinophil chemotactic factor, complement fragments such as C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF) (Shaw et al., 1981; Atkins & Wasserman, 1983). Although none of these factors is specifically chemotactic for either neutrophils or eosinophils, evidence has been put forward that PAF is a more selective chemotactic agent for eosinophils (Wardlaw et al., 1986). Most studies comparing the chemotactic activity of neutrophils and eosinophils have used eosinophils from hypereosinophilic donors (Uden et al., 1983; Wardlaw et al., 1986). Since it has been shown that these eosinophils have an altered metabolic activity they may also show a different chemotactic behaviour (Pincus et al., 1981; Uden et al., 1983; Prin et al., 1984; Wardlaw et al., 1986). Recently we have developed an isolation procedure for eosinophils from the blood of healthy volunteers which gives relatively high recovery and purity (Koenderman et al., 1988). This enabled us to compare the chemotactic response of neutrophils and eosinophils from the same healthy volunteers to the following chemotactic com1 Author for correspondence at present address: Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH 7270 Davos, Switzerland.

pounds: PAF, zymosan-activated serum (ZAS), N-formylmethionyl-leucyl-phenylalanine (FMLP) and LTB4. In our hands ZAS was the most powerful chemoattractant for neutrophils and eosinophils. With the exception of FMLP which showed hardly any chemoattractant activity towards eosinophils, the other chemotaxins had lower activity than ZAS but were approximately equiactive with each other. The neutrophils always showed a much stronger chemotactic response than the eosinophils. In addition, we evaluated the effects of nedocromil sodium and sodium cromoglycate on migratory responses elicited by optimum concentrations of the chemotaxins.

Methods Purification of granulocytes Granulocytes were isolated as follows. Platelet-rich plasma was removed after centrifugation of citrated blood (15min, 275 g,,,, room temperature). After collection of the buffy coat, granulocytes were separated from lymphocytes and monocytes by centrifugation on Ficoll-Paque (20 min, 1000 , room temperature). The remaining erythrocytes were removed by ammonium chloride lysis at 0°C and subsequent centrifugation. Thereafter the cells were allowed to recover for 30 min at 37°C in minimal essential medium containing 10% foetal calf serum. After centrifugation the mixed granulocyte preparation was resuspended in buffer A (composition: phosphate

buffered saline/trisodium citrate (38gl')/human pasteurised plasma protein (protein: 40 gP1; sodium caprylate: 4mmol 1- ; sodium: 130}1606mmolP1) = 80/10/10, v/v/v) at a concentration of 75 x 106 cells ml

Purification of neutrophils One millilitre of the mixed granulocytes (containing 75 x 106 cells) was carefully layered on 4 ml of an isotonic Percoll solution (density: 1.082 g ml -1) and centrifuged (10 min, 1000 g..,

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INHIBITION OF EOSINOPHIL AND NEUTROPHIL CHEMOTAXIS

room temperature). Neutrophils (about 98% pure, recovery > 85%), separated at the top of the 1.082gml-1 Percoll solution. These neutrophils were collected, washed in buffer A and resuspended in Gey's buffer (composition: Gey's balanced salt solution/human serum albumin (5%, w/v)/ heparin (10 iu ml `)/glucose (1 mgml 1)).

Purification of eosinophils The remainder of the mixed granulocyte preparation (containing 75 x 106 cellsmlP1 in buffer A) was first incubated with FMLP (10nM) in buffer A for 10min at 370C before 1 ml of cell suspension was layered on 4 ml of an isotonic Percoll solution (density: 1.082 g ml'). To prevent contamination of the eosinophils with cell debris and remaining erythrocytes, 1 ml of Percoll (density: 1.100gmlP') was brought under the Percoll layer of 1.082 g ml -. After centrifugation (10min, 1000g,,,x, room temperature) eosinophils with a purity of over 85% (recovery > 50%) were present at the interface of both Percoll layers. These eosinophils were collected, washed with buffer A and resuspended in Gey's buffer. As described elsewhere (Koenderman et al., 1988), the eosinophils isolated in this way are not 'primed' or 'activated'. The cell viability of both neutrophils and eosinophils was > 99%.

Chemotaxis assay Chemotaxis was measured with a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD, U.S.A.). Chemoattractants or Gey's buffer (30 pl) were placed in the lower wells. Two filters were placed between the upper and lower compartment. The lower filter had a pore width of 0.45 pm (Millipore) and the upper filter had a pore width of 8 pm (Sartorius). The filters had previously been soaked in Gey's buffer. Purified neutrophils and eosinophils were placed in the upper compartment of the control and test wells (105 cells in 25 M1 for each well). Concentration ranges of the chemotactic agents PAF, lyso-PAF, ZAS, FMLP and LTB4 were tested using the buffer as a control. At optimal concentrations of these chemotactic agents, dose-response inhibition curves for nedocromil sodium and sodium cromoglycate were constructed. In the case of PAF-induced chemotaxis, doseresponse inhibition curves for BN 52021 were also included. The cells were preincubated for 30min with the drugs before the chemotaxis assay was carried out. The chambers were incubated for 1.5h at 37°C for neutrophils and for 2.5h at 370C for eosinophils. Thereafter, the filters were removed, fixed in butanol/ethanol (20/80, v/v) for 10min and stained with Weigerts solution (composition: 1% haematoxylin (v/v) in 95% ethanol (v/v) and an acidic FeCl3-solution (70mM) mixed in a volume ratio of 1:1). The filters were dehydrated with ethanol, made transparent with 100% xylene (v/v) and fixed upside down. The number of cells per high power field (hpf) was determined with light microscopy (magnification 400 x ). In this way, the number of cells that had passed the upper filter was determined.

formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4) were purchased from Sigma (St. Louis, MO, U.S.A.). Synthetic LTB4 was also obtained from Dr J. Rokach (Merck-Frosst Laboratories, Pointe Claire-Dorval, Quebec, Canada). The purity of both LTB4 products was checked (>98%) by means of reversed phase h.p.l.c. as described by Verhagen et al. (1984). Zymosan activated serum (ZAS) was prepared as described previously (Goldstein et al., 1975). Ficoll-Paque (1.077gmlP ) and Percoll (1.129gmlP1) were obtained from Pharmacia (Uppsala, Sweden). Nedocromil sodium and sodium cromoglycate were provided by Fisons plc (Loughborough, Leicestershire). BN 52021 was a kind gift of Dr P. Braquet (Institut Henri Beaufour, Le Plessis Robinson, France). All other materials were reagent grade. Human blood was obtained from healthy volunteers at the Red Cross Bloodbank Foundation (Utrecht).

Results Dose-response curvesfor chemotaxis of neutrophils and eosinophils The concentration-dependency of PAF-, lyso-PAF-, ZAS-, FMLP- and LTB4-induced chemotaxis of isolated neutrophils and eosinophils is depicted in Figures 1-4. In the case of neutrophils all compounds except lyso-PAF induced significant chemotaxis. In the case of eosinophils, all compounds with the exception of lyso-PAF and FMLP induced significant chemotaxis. The optimal chemotactic concentrations for neutrophils were for PAF: 1 gm, for ZAS: a dilution of 1/3, for FMLP: 10 nm and for LTB4: 1 pM. With eosinophils the optimal chemotactic concentrations were for PAF: 1 gm, for ZAS: a dilution of 1/3 and for LTB4: 1 ym. For neutrophils the order of activity determined by the maximum response to the tested chemotactic compounds was: ZAS > PAF -FMLP _ LTB4. For eosinophils the order of activity was: ZAS > PAF > LTB4. In our first series of experiments ZAS was a very powerful chemoattractant for eosinophils, whereas in later experiments it had a much weaker chemotactic potency (see Figures 2 and 6). Since the shape of the ZAS concentration curve with both ZAS batches was similar, we used all data obtained. This however resulted in large standard errors of the mean (see Figure 2). The dose-response curve for the FMLPinduced chemotaxis of eosinophils (see Figure 3) suggests significant chemotaxis of eosinophils. This measured chemotaxis however can be completely attributed to neutrophil contamination of the eosinophil cell preparation. It is noteworthy that all chemotactic agents tested attracted neutrophils more powerfully than eosinophils. At the optimal chemotactic concentration this ratio was generally 2-3. b

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Statistical analysis All data are presented as mean + s.e.mean. Before statistical analysis the buffer controls were subtracted from the data.

Thereafter statistical analysis was performed using the Student's t test for paired samples. P values < 0.05 were considered significant. Drug IC50 values were calculated with a polynomial fit programme.

Materials

1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glycero-3-phosphoryl(PAF) and 1-0-hexadecyl-sn-glycero-3-phosphorylcholine (lyso-PAF) were purchased from Bachem

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(Bubendorf, Switzerland) and were stored at -20°C in a toluene/ethanol (1:1, v/v) solution under N2 .Zymosan A, N-

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Figure 1 PAF- (0) and lyso-PAF- (0) induced chemotaxis of neutrophils (a) and eosinophils (b) with buffer used as a control (n = 12; * P < 0.05). The cell purities were 98 ± 1% for neutrophils and 90 ± 2% for eosinophils. In this and subsequent figures, hpf = high power field.

P.L.B. BRUIJNZEEL et al.

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The inhibition of the PAF-induced chemotactic response of neutrophils and eosinophils by nedocromil sodium, sodium cromoglycate and BN 52021 is shown in Figure 5. All drugs tested significantly inhibited the PAF-induced chemotactic response of neutrophils. The IC50 for nedocromil sodium was 7.3 x 10-9M, for sodium cromoglycate 1.8 x 10-8M and for

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BN 52021 9.0 x 10-9M. In the case of eosinophils, sodium cromoglycate hardly inhibited the PAF-induced chemotaxis. The IC50 for nedocromil sodium was 1.3 x 10-6M, for BN 52021 1.3 x 10 6M.

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i/81 1/27 1/9 1/3 1 ZAS dilution Figure 2 Zymosan-activated serum (ZAS)-induced chemotaxis of neutrophils (a) and eosinophils (b) with buffer used as a control (n = 12; * P < 0.05). The cell purities were 98 ± 2% for neutrophils and 89 + 2% for eosinophils. In cases where heat-inactivated serum was used instead of buffer, similar control values were obtained. 0

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Inhibition experiments with nedocromil sodium and sodium cromoglycate Inhibition with PAF as chemotaxin In further experiments we investigated the inhibitory activities of sodium cromoglycate and nedocromil sodium against optimum concentrations of the leucocyte chemotaxins. In addition, the effect of the PAF antagonist BN 52021 was also evaluated. b

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Inhibition with zymosan-activated serum as chemotaxin The inhibition of the ZAS-induced chemotaxis of neutrophils and eosinophils by nedocromil sodium and sodium cromoglycate is shown in Figure 6. Both drugs significantly inhibited the ZAS-induced chemotactic response of neutrophils. The IC50 for nedocromil sodium was 7.8 x 10-8M for sodium cromoglycate 1.2 x 10-6M. Only sodium cromoglycate inhibited the ZAS-induced chemotaxis of eosinophils significantly. The IC5o for sodium cromoglycate was 1.5 x 10-7M. Inhibition with FMLP as chemotaxin The inhibition of the FMLP-induced neutrophil and eosinophil chemotaxis by nedocromil sodium was 2.4 x 10 -7M, and for sodium croFigure 7. Both drugs significantly inhibited the FMLPinduced chemotactic response of neutrophils. The IC50 for nedocromil sodium was 2.4 x 10iM, and for sodium cromoglycate 2.5 x 10- 7M. In the case of eosinophils the FMLPinduced chemotactic response could be accounted for by the presence of contaminating neutrophils.

Inhibition with leukotriene LTB4 as chemotaxin The inhibition of the LTB4-induced chemotaxis of neutrophils and eosinophils by nedocromil sodium and sodium cromoglycate is shown in Figure 8. Both drugs significantly inhibited the LTB4-induced chemotactic response of neutrophils. The IC50 for nedocromil sodium was 1.1 x 10-6M and for sodium cromoglycate 7.3 x 10 7 M. With eosinophils there was little inhibition of LTB4-induced chemotaxis by sodium cromoglycate,

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-log M Figure 3 N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis of neutrophils (a) and eosinophils (b) with buffer used as a control (n = 12; * P 0.05). The cell purities were 98 + 1% for neutrophils and 90 + 2% for eosinophils.

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Inhibition of neutrophil and eosinophil induced chemotaxis by nedocromil sodium and sodium cromoglycate.

1. Neutrophils and eosinophils infiltrate the airways in association with the allergen-induced late phase asthmatic reaction. Mobilization of these ce...
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