European Journal of Clinical Investigation ( 1 992) 22, 732-739

Leukotriene B4 level in stimulated blood neutrophils and alveolar macrophages from healthy and asthmatic subjects. Effect of beta-2 agonist therapy Y. PACHECO*, R. HOSNIt, B. CHABANNESt, F. CORMAND*, P. MOLIEREt, M. GROSCLAUDE*, D. PIPERNO*, M. LAGARDEt & M. PERRIN-FAYOLLE* *Department of lung medicine, Centre Hospital0 Universitaire Lyon Sud and tUnite Inserm U352 INSA Lyon, France Received 24 October 1991 and in revised form 24 June 1992; accepted 8 July 1992

Abstract. Leukotriene B4 levels were measured after stimulation by calcium ionophore A23 187: (i) in peripheral neutrophils (PMN) from allergic asthmatics, rhinitis and healthy subjects; (ii) in macrophages collected by bronchoalveolar lavage. LTB4 levels in PMNs were significantly higher in non-treated allergic asthmatics and non-treated subjects with rhinitis compared to controls. Beta-2 agonist-treated asthmatics showed a significantly decreased LTB4 production which was not different from those of controls. In "ifro, LTB4 production decreased significantly after PMN incubation with Salbutamol mol I-'). LTB4 produced by AM collected by BAL was measured in non-treated ( n = 5 ) and treated ( n = 1 1 ) asthmatics with inhaled beta-2 agonist. AM collected from all controls and non-treated asthmatics produced LTB4. By contrast, no production of LTB4 was observed in the treated group. LTB4 production decreased when normal AM were incubated in tiitro with Salbutamol (lo-* mol I-'). These results suggest that biochemical differences occur in PMN and macrophages from subjects treated with beta-2 agonist, presumably in changing the 5-lipoxygenase pathway. Keywords. Alveolar macrophage, Anti-asthmatic drug, Asthma, blood PMN, leukotriene B4. Abbreviations PBS = Phosphate buffered saline; LTB4, C4, D4 = leukotriene B4, C4, D4; PMN = Polymorphonuclear neutrophils; AM =Alveolar macrophage; BAL= Bronchoalveolar lavage; AA = Arachidonic Acid; 12 HPETE: 12 hydroxyperoxy eicosatetraenoic acid; ILI, IL4, ILB:interleukin 1,4,8; PGB2, PGD2, PGF2, = prostaglandin B2, D2, F2a; GMCSF: granulocyte macrophage colony stimulating factor; RP-HPLC = Reverse Phase-High Performance Liquid Chromatography. Introduction The importance of inflammation in asthma has been recognized for a long time and recently proved in man Correspondence: Dr Y . Pacheco, Service de Pneumologie. Centre Hospitalier Lyon-Sud, 693 10 Pierre-Benite, France.

732

and animal models. Among inflammatory cells, neutrophils (PMN) and alveolar macrophages (AM) are probably involved in exacerbation of asthma. Leukotriene B4 (LTB4) is one of the important mediators produced by these cells. LTB4 is a membrane derived lipid mediator, produced from arachidonic acid via the 5 lipoxygenase pathway. This leukotriene is produced in vitro by neutrophils, monocytes, macrophages, eosinophils and mast cells [I]. LTB4 appears to have relatively weak and indirect bronchoconstrictor effects but it is an extremely powerful chemotactic agent for neutrophils and eosinophils [2]. Stimulated human alveolar macrophages produce large amounts of LTB4 in normal subjects [3]. LTB4plays a role in host defence by its capacity to recruit neutrophils and lymphocytes in the lung [4]. Tobacco smoke inhibits this production [5]. This physiological mechanism can be overpassed and lead to amplify inflammation in the lung [6,7,8]. LTB4 appears as a candidate among the mediators implied in asthmatic late phase reaction [9]. Nevertheless conflicting results have been observed for LTB4 production by AM in asthmatic patients. On one hand, Damon et al. showed an increased generation of the arachidonic acid metabolites LTB4 and 5-HETE by human alveolar macrophages in patients with asthma [ 101. Wardlaw e f al. observed increased concentrations of LTB4 in alveolar fluid collected in asthmatic subjects [ 1 I]. But on the other hand, Balter et al. did not observe any difference between asthmatics and controls [ 121. The finding that peripheral PMNs of asthmatic subjects generate more LTB4 than normal PMNs has previously been demonstrated in PMN homogenates [13]. in isolated PMN preparations [I41 and in whole blood [ 151. In a recent paper we have reported higher production of LTB4 by blood PMN in non-treated allergic subjects presenting asthma or rhinitis [I 61. Uotila et al. [ 171 showed increased levels in serum from allergic asthmatic subjects. Various anti-asthmatic drugs can modulate the production of inflammatory mediators [ 181. Beta receptors are present in a variety of inflammatory cells and beta-2 agonist may have the potential to modulate inflammatory processes. Nielson et al. [ I91 demonstrated an in vitro modulation of the polymorphonuc-

LTB4 IN BETA-2 AGONIST TREATED ASTHMATICS

non smokers and they did not take any drug for at least 2 months. All subjects gave us their informed consent.

lear leukocyte respiratory burst and of LTB4 production by beta-adrenergic agonist. Nevertheless, beta-2 agonist are until a recent past considered as a symptomatic treatment only with little or no effect on the inflammatory process that underlies the late asthmatic response. Twentyman et al. [20] in a clinical study, recently showed that inhaled Albuterol protected against the allergen-induced late asthmatic response. No information concerning leukotriene B4 during beta-2 agonist therapy in asthma is available. This paper reports an ex uiiio study on LTB4 production by blood PMN and alveolar macrophages in various groups of patients treated or not with a betaadrenergic agonist, as well as an in citro study on the beta-2 agonist action on LTB4 production by both populations.

2. Preparation of blood cell suspensions and stirnulation conditions. All subjects were fasted for at least 12 h before venepuncture. Thirty minutes elapsed between the last inhaled dose of Salbutamol and venepuncture in asthmatic treated group. Venous blood (50 ml) was drawn, collected into polypropylen tubes containing 5 ml of 3.8% sodium citrate solution, and was immediately centrifuged at 1 2 0 x g for 20 min at room temperature. The upper phase (platelet-rich plasma) was removed and the lower phase (remaining blood cells) was treated as described below. Neutrophils were purified by the method of Boyum [21]. Briefly, the remaining blood cells (lower phase) were mixed with a 4.5% Dextran (PM 100.000-200.000) solution (Serva Feinbiochemica GMBH and Co., Heidelberg, Germany) at a lower phase/Dextran solution ratio of 5: 1 and sedimented for 30 min at 37°C. The upper neutrophil-rich fraction (6 ml) was diluted twice with phosphate buffered saline solution (PBS, pH 7.4, of the following mmol-l composition: NaCl 136.8, KCI 2.7, Na2HP048.0 and KHzP04 1.5) and layered onto 5 ml Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) and centrifuged at 600 x g for 20 min. The erythrocytes of the neutrophil containing pellets were lysed by Vortexing for 30 s with sterile water. Then 0.9% NaCl was added and the mixture centrifuged in the same conditions. The resulting neutrophil pellets were washed twice with PBS containing CaClz (8 mmol-I) and MgClz (2 mmol-I) and cell concentration was adjusted at about 4 x lo7cells per ml. Aliquot of the final neutrophil preparation was stained with May-Griinwald Giesma (Farmitalia Carlo Erba) for differential cell counting. The neutrophil count and viability were greater than 96%. Neutrophil contamination by platelets measured by phase contrast microscopy (Leitz, Wetzlar, Germany) was about 0.5 to 1 platelet per neutrophil. Eighty minutes elapsed between venepuncture and PMNs stimulation. In 3 ml polypropylen tubes, 0.05 ml of neutrophil suspension (2 x IO6cells)was preincubated for 5 min at 37°C. Then 0.10 ml PBS and 0.05 ml ionophore A23 187 (Sigma Chemical Co., St Louis, MO, USA) (final concentration 1.5 x 10-6mol I - ' ) in Dimethylsulfoxide (DMSO) (the final concentration of DMSO in the buffer did not exceed 0.2%) were added and the mixture

Materials and methods Ex-uiuo study on LTB4 production by blood P M N 1. Studied population. Blood PMN were purified from venous blood collected from 1 1 controls and 22 allergic asthmatic patients, 1 1 patients presenting allergic rhinitis. All allergic patients hospitalized in our department of rcspiratory diseases had a story of atopic disorder, elevated IgE blood levels, positive skin tests and specific IgE for house dust mite or graminae pollen allergens. In the group of patients allergic to pollinic allergens, blood was collected during the exposition season, between March and July in our region. Allergic asthmatics were classified into two groups according to the presence or absence of a beta adrenergic treatment. Group I was composed of 1 1 patients, with recently diagnosed mild asthma, and was non-treated. In group 11, 1 1 patients regularly checked in our department for mild asthma, were treated with Salbutamol spray alone (6 Puffs of 100 pg day-l) for 1 0 k 3 years. We matched these two groups in terms of age, sex, respiratory volumes, bronchial hyperreactivity, blood IgE levels and allergen types (Table I). For comparison, we studied 1 1 patients who presented allergic rhinitis, who were not treated (mean age 3 3 f 3 years) and 1 1 healthy volunteers (mean age 23 f3 years) with negative personal and family history of atopy, and no clinical evidence of atopic or other diseases. Asthmatics and subjects with rhinithis were

Table 1: Studied population foresr~icostudyofleukotrieneBqproduction by bloodneutrophils.Comparativecharacteristics of asthmatic groups treated by Beta agonist or not treated (mean +SEM) Sex Age (years)

F

M

Treatment duration (years)

Non-treated group ( n = 11) 5 6 33+3 Beta agonist treated group ( n = I I ) 5 6 10k3 27+3

FEVI/FVC (XI predicted)

PD20 Carbacholine (pg)

(Iu ml- ' )

Allergen type mite pollens

95+5

76+3

284 & 67

1386k647

7 4

look3

80+2

310k75

701k220

6 5

Baseline FEVl (YOpredicted)

733

Serum IgE

734

Y. PACHECO et al.

was incubated for another 5 rnin at 3 7 T . The final concentrations of CaClz and MgC12 in the incubation medium were 2 mmol 1-' and 0.5 mmol I-', respectively. The incubation was terminated by the addition of 0.2 ml cooling methanol and stored at -20°C until analysis. After centrifugation at 1500 x g and 4°C for 10 min, the supernatant was analysed by HPLC. Modulation by Salbutamol of LTBd production from PMN: in vitro study

I . Studied population. Blood PM N have been prepared from venous blood collected in six non-treated allergic asthmatic patients and seven normal subjects. Preparation of blood cell suspensions was performed as described above. 2. Incubations. Purified neutrophils were incubated with various concentrations mol I-' to lop6mol 1 I ) of Salbutamol, for two incubation times (10 and 30 min) before the stimulation by the ionophore A23 187 for LTB4 production. Ex tivo study of LTB4 production by alveolar macrophages

I . Studied population. Alveolar macrophages were collected by bronchoalveolar lavage BAL in 16 allergic asthmatic patients, 16 controls (mean age 23 f3 years) and nine healthy smokers (mean age 41 f5 years). We obtained informed consent for alveolar lavage from all subjects. The asthmatic patients were all allergic to house dust mite. Eleven of these patients (mean age 37 k 2 years) were treated daily with the inhaled beta-2 agonist Salbutamol (6 Puffs of 100 pg day-') and did not stop their treatment before BAL. In five other patients (mean age 25 f3 years) three were strictly not treated and two had stopped their beta-2 agonist treatment 1 week before BAL. In five of the regularly treated subjects, AM were collected by BAL 5 rnin after a local specific allergenic stimulation by dermatophagoides pteronyssinus. In these five patients, locally stimulated with specific antigen, we observed a transient bronchoconstriction during fibroscopy. BAL was performed after this stimulation in the same bronchus. Clinical characteristics of these patients are presented in Table 2. We included in this study healthy smokers according to the knowledge of decreased LTB4 production by AM, reported by some authors [5,24].

2. Bronchoalveolar lavage ( B AL ) technique. To obtain cells from the lower respiratory tract, BAL was performed in patients and control subjects using a total of 150 ml of 0.9% saline solution as previously described [22]. For specific stimulation during fibroscopy, 10 ml physiological saline containing 200 ng ml- D. pteronyssinus allergen (laboratoire Stallergene, France) were instilled according to the technique presented by A.B. Tonne1 and colleagues [23]. 3. Macrophage preparation. Macrophages were separated from lavage fluid by centrifugation for 5 min at 20°C. The pellet was resuspended in 5 ml PBS. Evaluation of cell viability by exclusion of trypan blue dye regularly showed more than 90% living cells. Differential cell counts of macrophages, lymphocytes, eosinophils and neutrophils by May Griinwald Giemsa were systematically performed. 4 . Macrophage stimulation and incubation with Salbutamol. Alveolar macrophages (4 x lo6 cells ml I ) in PBS were preincubated for 10 min at 37°C with or without Salbutamol (lo-' to l o p 9mol I-'). Stimulation was initiated by addition of calcium ionophore A23187 (1.5 pmol I-') and were carried out at 37 C for 10 min according to the method of Laviolette at al. [24]. The final concentrations of CaCI2 and MgClz in the incubation medium were 2 mmol I-' and 0.5 mmol I-'. respectively. All incubations were terminated by the addition of 1 volume of methanol, then centrifuged for 10 rnin at 1500 x g . The supernatant was kept frozen at - 20°C until analysis by RP-HPLC. Purijication and assay of Ieukotrienes

Leukotrienes were purified and assayed by reversed phase high performance liquid chromatography (RP-HPLC). A Kontron HPLC system (Miinchen, Germany) consisting of a 420 solvent delivery pump, a 430 UV absorbance detector and a 450 data system was used. Separation of all eicosatetraenoic acids was achieved on a reversed phase spherisorb ~ column ( 1 :22 cm, internal ODs-2 C I microbore diameter: 0.4 cm, particle size 5 pm) (Interchim, Monthcon, France). The column was isocratically pumped with the mobile phase acetonitrile-methanol-water-acetic acid (250: 200: 500: 4) adjusted to pH 5.6 with N H 4 0 H and at the flow rate of 1.2 ml

Table 2. Studied population for PS c?imstudy of leukotriene Bq production by alveolar macrophages. Comparative characteristics of asthmatic groups treated by Beta agonist or not treated (mean SD)

*

Sex Age (years)

F

M

Treatment duration (years)

Non-treated group ( n = 5 ) I 4 25+3 Beta agonist-treated group ( n = I I ) 5 15+2 6 31k2

Baseline FEV I ' X predicted)

FEVliFVC (Yo predicted)

PD20 Carbacholine (pg)

Serum IgE (IU ml ')

96k6

76+5

255

* 66

750 283

84+6

79*4

193+83

526 f230

LTB4 I N BETA-2 AGONIST TREATED ASTHMATICS

735

LTB., pmol cells 5 min-' min-I. All solvents were HPLC grade (Farmitalia Carlo Erba) and the eluent was monitored at P < 0.02 270 nm. Routinely, 0.1 ml aliquot of supernatant 7 were injected directly without prior extraction or '0°1 P < O O 1 P