Increased Oxygen Species Generation in Blood Monocytes of Asthmatic Patlents'?

ISABELLE VACHIER, MARCELLE DAMON, CHRISTIAN LE DOUCEN, ANDRE CRASTES DE PAULET, PASCAL CHANEZ, FRANCOIS BERNARD MICHEL, and PHILIPE GODARD

Introduction

Bronchial asthma has been defined as a reversible airway obstruction induced by the association of smooth muscle spasms and airway inflammation. The inflammatory process is characterized by local infiltration of inflammatory cells in the bronchi, principally eosinophils and mononuclear cells, such as lymphocytes and mononuclear phagocytic cells (1), as assessed by histologic investigations of airway lesions in patients who died of asthma or in patients with mild asthma (2). Mononuclear phagocytic cells include alveolar macrophages (AM) and circulating blood monocytes (3). When these cells are stimulated in vitro either by phagocytosis or by specificor nonspecific stimuli, they generate oxygen speciesduring the "respiratory burst" (4, 5). Oxygen is reduced by NADPH oxidase (6), leading to generation of the superoxide anion (02 ";) which is further transformed by superoxide dismutase (SOD) into hydrogen peroxide (H 202 ) (7). H 202 is then utilized by peroxidases, such as myeloperoxidase, which produces hypochlorous acid (HOCI) (8). H 202 could also be the source of hydroxyl radical (0H· ) through a Fe2 +-catalyzed reaction. These oxygen species generate chemiluminescence with the release of photons (9). They were shown to be involved in the pathogenesis of many pulmonary disorders, such as acute respiratory distress syndrome (10), lower respiratory tract infection in cigarette smokers (11), and interstitial lung disease in children (12). They could also be involved in the pathogenesis of asthma. In a previous study (13), we showed that AM known to derive from monocytes (14)wereactivated in asthmatic patients. The capacity of stimulated AM to release oxygen species was correlated with the severity of asthma (15). Moreover, we showed that AM from asthmatic patients without recent episodes of

SUMMARY Besides eoslnophlls, Inflammatory processes In asthma are characterized by an Infiltration of Inflammatory cells, Including mononuclear phagocytes, such as alveolar macrophages (AM) and blood monocytes, In the airways. Monocyte activation has been observed In the blood after exercise or allergen-Induced asthma. Stimulated AM In chronic and stable asthmatic patients have been shown to release oxygen species. We thus Investigated the Intensity of the activation of monocytes from 18 asthmatic patients compared with 18 healthy subjects. Oxygen species release was analyzed for monocytes In suspension by chemiluminescence using a lumlnometer and for monocytes maintained In adherence using conventional assay and video Imaging camera. Circulating blood monocytes In suspension from asthmatic patients and control subjects showed the same baseline free radical release. Monocytes In suspension from asthmatic patients were more stimulatable by PMA: specifically, monocytes release more H20 and peaks of O2;" are sooner; moreover, peaks of total free radical release are higher, and this plateau is sustained. Compared with monocytes from control subjects, those from asthmatic patients evaluated after adherence show a higher baseline for O2:- and higher total free radical release. Monocytes from asthmatic patients spontaneously release more O2 :- over time In nonstlmulated cells and release more O 2 :- with PMA stimulation; they show the same peak level total free radical release as those from control subjects after stimulation. SOD activity analysis on adherent monocytes was lower In asthmatic compared with control subjects. These data show that monocytes from asthmatic patients were activated compared with control monocytes. The difference between the suspension and adherence procedures Indicated that monocyte margination and adherence on endothelial cells and bronchoalveolar walls could be associated with Increased oxygen species release In asthma. AM REV RESPIR DIS 1992; 146:1161-1166

asthma were activated compared with those from healthy subjects (16). Monocyte activation was also observed after exerciseor allergen-induced asthma (17). Thus, we hypothesized that monocytes could be activated in the bloodstream in asthmatic patients in the chronic stage of the disease, even in very mild asthma, as described for AM, and that this activation could differ if the cells adhere or do not adhere to a surface. Our objective was to study monocyte activation through the release of oxygenspecies and superoxide dismutase activity. The study was conducted using conventional assays, chemiluminescence methods, that is, Iuminometer or video imaging analysis for monoeytes in suspension or in adherence, respectively. Methods Subjects The study included 18control subjects ranging in age from 20 to 57 yr (mean 34 yr) without detectable pulmonary lesions or evidence

of abnormalities of the airways and 18 asthmatic patients ranging in age from 18 to 57 yr (mean 36 yr). All were nonsmokers. and none had been on medication for at least I month before the study. Informed consent was obtained from all patients in accordance with the Ethics Committee of the University of Montpellier. Asthma were diagnosed according to the American Thoracic Society statement (I8). Patients classified as having asthma were selected on the basis of reversible airway obstruction with FEV! > 20% {forced expired

(Receivedin originalform November 19, 1991 and in revised form May 6, 1992) 1 From INSERM Unite 58 and the Clinique des Maladies Respiratoires de l' Aiguelongue, Montpellier, France. 2 Supported in part by a grant from the Comite Departemental de la lutte contre les Maladies Respiratoires, Montpellier, France. 3 Correspondence and requests for reprints should be addressed to Isabelle Vachier, INSERM Unite 58,60 rue de Navacelles, 34090 Montpellier, France.

1161

1162

volume in one second) of predicted values. The staging of asthma was performed according to a clinicalscoredefined as Aas (19), combining the frequency and duration of asthma attacks with or without bronchial obstruction and chronic restriction of functions and ranging from 1 to 5. Allergy was assessed by the history of asthma, the presence of at least three positive skin tests with common aeroallergens, and a high immunoglobulin E (lgE) level in the blood.

VACHIER, DAMOM, LE DOUCEN, de PAULET, CHANEZ. MICHEL. AND GODARD

results were compared to protein quantities measured by Lowry's method (20). The viability of the cells determined using either the trypan blue exclusion test or lactate dehydrogenase (LDH) determination was always greater than 95%.

Oxygen Species Analysis Monocytes were studied either in suspension using a luminometer or in adherence using a conventional spectrophotometer or a video imaging camera. Reactive oxygen species reMaterials lease was induced by PMA, which is the best Percell" was from Pharmacia France (St. agent for testing protein kinase C (PKC) acQuentin en Yveline,France); nutritive medi- tivity, which is known to take an important um (RPMI 1640and Medium 199), fetal calf part in NADPH oxidase activation (21). serum (FCS), and phosphate-buffered saline Chemiluminescence (CL) assay:luminom(PBS) were purchased from GIBCO Chemi- eter analysis: After scraping and removing cal (Glasgow, Scotland). Petri dishes (35 x monocytes in RPMI 1640 medium, the cell 10mm) and multi well dishes were from Bec- suspension was adjusted to 5 x 105 cells per ton Dickinson (Grenoble, France). 800 ul medium. PMA was dissolved as a stock The two luminescent probes, lucigenin solution of 1 mg/ml in DMSO maintained (10,10'-dimethyl-9,'Y-biacridinium dinitrate) at - 70° C. Before use, the stock solution was and luminol (5-amino-2,3-dihydro-1,4-phtal- diluted in saline solution to be used at 10-7 M azidinedione), the enzymes superoxide dis- (final concentration). CL was monitored in mutase (Grade IV), horseradish peroxidase a Model 1251 luminometer LKB Wallac (Wal(HRP), and xanthine oxidase, and phorbol lac Co., Turku, Finland) connected to an Apmyristate acetate (PMA), ferricytochrome c ple computer. Two different luminescent (Type VI), xanthine, HEPES, bovine serum probes were used. albumin (BSA), glycine buffer, ethylenediLucigenin enhanced the luminescence inaminetetraacetic acid (EDTA), sodium salicy- duced by O 2 ,; production. Lucigenin (5 mg) late, and dimethylsulfoxide (DMSO)werefrom was dissolved in 100ml Medium 199containSigma Chemical Company (St. Louis, MO). ing 476 mg HEPES and 100 mg gelatin with heating to obtain a 10--4 M concentration. The Preparation of Human Blood Monocytes final concentration was 10-5 M. Peripheral blood was obtained by venipuncLuminol enhanced the luminescence inture using heparin (25 Vlml). Mononuclear duced by the whole set of reactive oxygen specells wereisolated by centrifugation of blood cies. Luminol (18 mg) was suspended in a 1% samples over isotonic Percoll solutions with solution of BSA in 100 ml PBS (pH 7.4) to densities of 1.097and 1.086glml at 400 x g obtain a 10-3 M concentration. The final concentration was 10-4 M. for 20 min at room temperature. Mononuclear cell suspension in Percoll was All procedures wereperformed in the dark. washed once with an equal volume of RPMI Medium (800 ul) containing 5 x 105 purified 1640 medium. Contaminating erythrocytes monocytes was placed in luminometer vials were lysed by incubation for 10min in a solu- at 37° C with continuous stirring. First, 100 tion of 130 mM NH 4CI, 10 mM Tris, and 16 ul luminescent probe was added, a first valmM K2C02 and recovered by centrifugation. ue was determined, and 100ul PMA was addThe mononuclear cells were washed with ed (9), and then measurements were taken RPM I 1640medium and counted in the ini- every minute for 20 or 30 min. Experimental tial cell suspension with neutral red stain spe- controls were performed with monocytes cific for monocytes (neutral red cell suspen- incubated without PMA. A kinetic study sion 3:1). Cells were then incubated in petri of PMA-stimulated monocyte responses was dishes with 2 ml RPMI 1640medium and 20% carried out. The difference between asthmafetal calf serum for 2 h at 37° C in a humid tic and control (18 subjects in each group) atmosphere of 95070 air and 5% CO 2 , The was determined at CL peak (maximal value monolayers werethen washed three times with obtained after stimulation) and up to the warm medium to remove non-adherent cells, time when the peak was reached. Results such as lymphocytes. were expressed as millivolts per 5 x 105 cells Adherent isolated monocytes were then (mean ± SEM). characterized with neutral red staining, and H 202 release was evaluated using peroxiMay-Grunwald-Giemsa staining was carried dase-catalyzed CL oxidation of luminol. out to certify that no other cells were present. Horseradish peroxidase was added to opThe purity was> 95%. They were then used timize the reaction between luminol and H 202 either in adherence or in homogeneous sus- (22). Luminometer measurements were carpension obtained by gentle scraping with a ried out as described earlier. HRP (100 ul) rubber policeman into RPMI 1640. When was added to the reaction mixture at 1 Vlml monocytes were tested in suspension, results (final concentration). From each population werecompared with the last count after scrap- 18 subjects were studied. Results were exing; when they were tested in adherence, pressed as mVI 5 X 105 cells (mean ± SEM).

Assay for superoxide anion (0 2 ':) generation (conventional method). O 2,; generation was measured by SOD-inhibitable reduction of ferricytochrome C, according to Johnston and colleagues (23). Adherent cells (106 cells) were incubated in 1 ml of a 80 J,lM solution of ferricytochrome c in RPMI 1640 medium and then stimulated by PMA, which was added 5, 10, 15,20,30,45, or 60 min before the measurement. These incubations were performed at 37° C in a humid atmosphere of 95% air and 5% CO 2 • Experimental controls included incubation of monocytes with cytochrome c without PMA and cytochrome c and PMA without monocytes. At the end of incubation, the supernatants were collected and centrifuged at 800 x g for 10minutes. Optical density was then determined with absorbance at 550 nm (spectrophotometer; Philips, France). Each reaction was run in duplicate. To assess the specificity of cytochrome c reduction, superoxidedismutase was included in parallel assaymixtures at 30 ug/ml (final concentration), in addition to PMA. These assays were performed on monocytes from six control subjects and six asthmatic patients. Results were expressed as umol/mg protein (mean ± SEM). Video imaging camera. O2,; or the whole set of reactive oxygen species were measured with lucigenin or luminol on 5 x lOS adherent monocytes in multiwell dishes with 800 ul RPM I 1640medium. Photons werecounted by an ultrasensitive photon-counting imaging camera monitored by a computer-assisted image processor (Argus'" 100; Hamamatsu Photonics, Japan) (24). The experimental conditions werethe same as for the luminometer CL measurements. Briefly, 100 ul lucigenin or luminol was added (final concentration of 10-5 or 10-4 M, respectively). A first measurement was taken before stimulation. PMA (100 ul) was added (10-7 M final concentration). The procedure was performed in the dark at room temperature. Measurements werecarried out before and after stimulation every minute until CL peak was reached. Six subjects in each population werestudied. The results were expressed as photon numberl 5 x 105 cells (mean ± SEM). SOD Determination: CL Analysis SOD activity was measured by a CL method described by Corbisier and coworkers (25). O2 ,; was generated by the xanthine/xanthine oxidase system. Briefly,monocytes (106 ) maintained in adherence werestimulated by PMA (10-7 M final concentration) and sonicated for SOD activity analysis. CL assays were performed in a 0.1 M glycine NaOH buffer, pH 9.0, containing 1 mM EDTA and 1 mM sodium salicylate. The reaction was started by the addition of 0.2 ml xanthine solution at a final concentration of 2.5 x 10-5 M to 0.2 ml xanthine oxidase (final concentration 4 mVI sample) in the presenceof lucigenin(fmal concentration 0.1 mM). Light intensity was measured 30 s after initiation of the reaction by xanthine. SOD quantities in samples werecal-

1163

ACTIVATION OF HUMAN BLOOD MONOCYTES IN ASTHMA

TABLE 1 DESCRIPTION OF PATIENTS Age SUbjects

FEV, (%)

Gravity

51 57 55 55 56 35 39

102 93 90 129 93 80 112 60 70 47

1 1 1 1 2 2 2 2 2 3

31 31 24 20 26 18 23 19

126 126 85 94 106 71 50 67

1 1 1 2 2 2 3 3

Sex

(yr)

1 2 3 4 5 6 7 8 9 10

F F M F M M F F M M

35 42

11 12 13 14 15 16 17 18

M F M M F M M M

AP (nM)

45

M

Definition of abbreviations: nAA = nonallergic asthma; AA = allergic asthma; AP = asthmatic patient.

culated by comparing their inhibitory effect on O 2 ; formation with the effect of known increased concentrations of purified SOD, using heat-inactivated SOD as 00/0 inhibition. The measurements were performed before stimulation and after stimulation, when the lucigenin-dependent CL peak was reached. Six subjects in each group were studied. The results wereexpressedas ng SOD/mg proteins (mean ± SEM).

Statistical Analysis The nonparametric Kruskal-Wallis test was used for statistical analysis of the results obtained in the kinetic study. For the H 202 generation measurement, the chi-square test had to be used to compare the distribution of valuesbetweenthe three groups, because some values were overloaded. The CL peak (maximal value) and the time of its appearance (T peak) were analyzed using the Kruskal-Wallis test or the Mann-Whitney U test, depending on the number of samples.

tient with score 3. The value of FEV l of control subjects was always up to 100070.

Analysis on Suspended Cells /h02 analysis. Adding HRP to the reaction mixture, the optimum capacity for cells to release H 202 was evaluated. Values obtained under stimulation with PMA were significantly greater in asthmatic than in control subjects. In monocytes from asthmatic patients, the intensity of the response provided overloaded values. Thus, to study differences between both groups of subjects, we used a chi-square test with a distribution in three groups depending on the production ofH202 : weak responders « 20,000 mV/S x 105 cells), mild responders (> 20,000 to < 100,000 mV/S x 105 cells), and high responders 100,000mV/S x 105 cells). The results in table 2 showed that there

c-

was a significant difference (P < O.OS) between the two populations relative to the distribution of values. In particular, no control subjects werein the high-responder group, whereas 33070 of asthmatic patients were in this group. Of the control subjects 7S070 were in the low-responder group whereas only 16070 of asthmatic patients were in this group. Lucigenin-enhanced CL. Values obtained before stimulation did not differ significantly between the two populations (18 ± 2 versus 17 ± 3 mV/S x 105 cells, respectively). However, kinetic curves showed that O2 ; formation was higher in PMA-stimulated monocytes from asthmatic than in those from control subjects. The curve obtained for control subjects showed a slow increase until the peak value, whereas the curve for asthmatic subjects showed a faster increase and then a slow decrease. The statistical analysis at each time showed significant differences between the two groups at the beginning of the stimulation (4 to 8 min with p < O.OS). After 25 min, both groups produced the same quantities of O2 ; (figure 1).The CL peak was higher (699 ± 122 versus 597 ± 91 mV/5 x lOS cells) and the T peak was earlier (15 ± 1.38 versus 26 ± 1.96 min) in monocytes from asthmatic than in those from control subjects, but the differences were not significant. Luminol-enhanced CL. Values obtained before stimulation were not significantly different (160 ± 53 versus 96 ± 34 mV/S X 105 cells from asthmatic and control subjects, respectively). The kinetic curves showed that CL values obtained for control stimulated monocytes increased from 0 to 4 min and then decreased until 20 min. In asthma, CL increased from 0 to 6 min and then a plateau was observed. The formation of oxygen species was greater in monocytes from asthmatic patients than in those

Results

Subjects Morphologic characteristics of the asthmatic patients are described in table 1. Among the 18 asthmatic patients 10 were allergic asthmatic and 8 were nonallergic, The mean value of FEV 1 was 88 ± 6% and ranged from SO to 129%. The Aas score was 1 in 7 patients, 2 in 8, and 3 in 3. Just before the study, 3 had a mild nocturnal asthma crisis that did not require ~2 spray. In each experiment with 6 patients wechose 3 patients with a score of 1, 2 patients with score 2, and 1 pa-

TABLE 2 ANALYSIS OF H202 PRODUCTION" Percentage of SUbjects in Each Group Study Group Healthy Asthmatic

< 20,000

20,000-100,000

> 100,000 Total

(mV/5 x 105 cells)

75 16.7

25 50

o 33.3

100 100

* H202 generation using luminometer analysis. Luminol (10'" M) was used as enhancer and HAP (1 U/ml) to optimize the reaction for the CL analysis. Monocytes were purified by adherence, scraped, and resuspended in medium (5 x 1Q5 cells/ml). Luminol and HAP were added, and a first measurement was carried out. PMA (10- 7 M) was then added and measurements were taken every minute until the CL peak (maximal value) was reached. A distribution in three groups depending on the level of prodl1ctlon was used. The results showed a significant difference in this distribution between the two poputions. Chi-square analysis showed that this difference was significant, p < 0.05.

1164

VACHIER, DAMON, LE DOUCEN, de PAULET, CHANEZ. MICHEL, AND GODARD

800

= 1II

~

1ft

600

0 P"4

lIS

11

1

400

a

200

Fig. 1. Kinetic study of superoxide anion generation using luminometer analysis. Lucigenin (10-5 M) was used as enhancer for the CL analysis. Monocytes were purified by adherence, scraped, and resuspended in medium (5 x 105 cellslml). Lucigenin was added, and a first measurement was taken. PMA (10-7 M) was then added, and measurements were carried out every minute for 30 minutes. Controls were performed with monocytes incubated without PMA. Each measure was run in duplicate. The study was performed on monocytes from asthmatic patients and control subjects. The results were expressed as mV/5 x 105cells (mean ± SEM). Statistical analysis showed a significant difference between the two populations from 4 to 8 min, p < 0.05. Closed circles = control subjects; open circles = asthmatic subjects.

from control subjects. Responses obtained here were two- to sixfold lower than results obtained with HRP in the same conditions in asthmatic as well as in control subjects. Significant differences were observed for each time during the kinetic study (figure 2). The CL peak was twofold higher in monocytes from asthmatic than from control subjects (11,751 ± 1,612 versus 5,371 ± 1,115 mV/5 x 105 cells, p < 0.005). Moreover, the CL peak was reached twofold later in asthmatic than in control subjects (8.2 ± 1.74versus 3.9 ± 0.42 min, p < 0.001).

Analysis on Adherent Cells Ferricytochrome c reduction assays. Nonstimulated monocytes from control subjects did not produce O 2 "; during the kinetic study (from 5 to 60 min), whereas monocytes from asthmatic patients released increasing quantities of O 2 "; from Fig. 2. Kinetic study of the whole set of o to 72 ± 24 umol/mg protein at 60 min. reactive oxygen species using luminom12000 eter analysis. Luminol (10-" M) was used Moreover, a lag phase (5 min) was obas enhancer for the CL analysis. Monoserved after medium containing fercytes were purified by adherence, ~u 10000 ricytochrome c was added. scraped. and resuspended in medium As observed in the CL experiments, (5 x 105cellslml). Luminol was added, '& 8000 and a first measurement was taken evPMA-stimulated monocytes from asthery minute for 20 min. Controls were permatic patients released higher quantities formed with monocytes incubated with6000 of O 2 "; than those from control subjects. out PMA. Each measurement was run The kinetic curves were almost identical in duplicate. The study was performed d 4000 in shape, with a plateau from 40 to 60 on monocytes from 18 asthmatic patients and 18 control subjects. Results min. The differences between asthmatic were expressed as mV/5 x 105 cells 2000 and control subjects were significant (mean ± SEM). Statistical analysis from 15 to 60 min after stimulation showed a significant difference between (p < 0.05) (figure 3). In individual analy20 the two populations at each time, p < 10 15 5 0.01.Closed circles = control subjects; sis we observed that there was a differTime: min open circles = asthmatic subjects. ence in kinetic shape between some patients; this could be explained by the analysis of the nearness of asthma attack (data not shown) . .l5O (hI Lucigenin video imaginganalysis. The (a) I of O 2 "; (CL peak) was greater formation 300 90 in stimulated monocytes from asthmatA. 250 ic than from control subjects (247,238 ± 70 129,927 versus 51,697 ± 9,337 photon 200 number/5 x 105 cells, p < 0.01), as shown SO 150 in table 3. Moreover, values obtained beiI ..= 30 fore the stimulation were also higher in 100 ." than in control subjects (8,882 asthmatic "R 10 ± 4,602 versus 2,126 ± 1,121 photon E 50 A. number/5 x 105 cells, p < 0.01). The cI 0 -10 differences between asthmatic and con0 10 20 30 40 50 60 50 60 40 0 10 20 30 Time: min Time: min trol subjects obtained on adherent monot NIdioa .umue cytesweremore significant than those obtained on suspended monocytes both beFig. 3. Comparison of superoxide anion generation in nonstimulated adherent monocytes from six asthmatic pafore and after stimulation. tients and six control subjects. 02~ accumulation was measured using SOD-inhibitable reduction of ferricytochrome c. (a) Measurements were taken just after fresh medium containing ferricytochrome c was added to the cell monolayLuminol video imaging analysis. ers. After a 5-min lag phase. 02~ accumulation was observed only in monocytes from asthmatic patients. (b) 02~ Values obtained before stimulation were accumulation was evaluated before stimulation at various intervals after PMA (10-7 M) was added. Each measuresignificantly higher in asthmatic than in ment was carried out in duplicate. Results were expressed as I!mol/mg protein (mean ± SEM). Statistical analysis control subjects (283,791 ± 81,744 vershowed significant differences between the two populations. p < 0.05. Closed circles = control subjects; open circles = asthmatic subjects. sus 7,113 ± 2,243 photon numbers/5 x 5

...

l

1

I

I I II

10

15 20 Time: min

25

30

1165

ACTIVATION OF HUMAN BLOOD MONOCYTES IN ASTHMA

tion mixture showed that monocytes from asthmatic patients wereable to produce higher quantities of H 202 than in 5 CL: Photon Number/5 x 10 Cells (mean ± SEM) Study monocytes from control subjects. MoreBefore Stimulation Group After Stimulation over, SOD quantities were significantly lower in monocytes from asthmatic than 2,126 ± 1,212 Controls 51,697 ± 9,337 8,882 ± 4,602 Asthmatics 247,238 ± 129,927 in those from control subjects. These results suggest that monocytes • Lucigenin (10- M) was used as enhancer for the CL assays. Monocytes maintained in adherence were washed with warm medium, and luminescent probe was added. A first measurement was taken, from patients with chronic stable asthand then PMA (10- M) was added. Photons were counted using an ultrasensitive camera monitored ma were more stimulable than control by a computer-assisted image processor. Measurements were run in duplicate and taken every minute monocytes. Since the adherence process until CL peak (maximal value) was obtained. In each group six subjects were studied. Statistical results showed a significant difference between the two populations before and after stimulation, p < 0.01. was always identical for both populations, wethought that there is hyperreacTABLE 4 tivity of rnonocytes from asthmatic patients. This was particularly evident with VIDEO IMAGING ANALYSIS OF H202 • respect to oxygen species from the CL: Photon Number/5 x 105 Cells (mean ± SEM) myeloperoxidase-Hioj-halide system, Study Before Stimulation Group After Stimulation which is known to be involved in stimulated monocytes (26). Moreover, when 7,113 ± 2,243 1,167,556 ± 279,635 Controls monocytes werestudied in adherence, the 283,791 ± 81,744 Asthmatics 975,048 ± 275,508 increased accumulation of O 2 "; and the • Luminol (10-" M) was used as enhancer for the CL assays. Monocytes maintained in adherence greater quantities of the whole set of reacwere washed with warm medium, and luminescent probe was added; a first measurement was carried out, and then PMA (10- M) was added. Photons were counted using an ultrasensitive camera monitive oxygen species observed in adherent tored by a computer-assisted image processor. Measurements were run in duplicate and taken every monocytes in asthmatic patients without minute until CL peak (maximal value) was obtained. In each group six subjects were studied. Statistical exogenous stimulation could be exanalysis showed a significant difference between the two populations before and after stimulation, p < 0.Q1. plained by adherence on petri dishes considered physical stimulation of cells (27) and could represent the first step of in 105 cells with p < 0.01), as shown in table tients the response was different because vivo transformation of monocytes into 4. In these experiments, the same obser- they had experienced recent asthma at- AM (14). Similarly, luminol-enhanced vation as for lucigenin could be made tacks; O 2 "; accumulation increased when CL showed that before stimulation the related to the increase in significant patients had an attack 12 h before the release of oxygen species was greater in differences between two populations study, whereas a plateau was observed asthmatic than in control subjects. Afwhen monocytes were in adherence. On in monocytes from asthmatic patients in ter stimulation, however, CL values obthe other hand, after stimulation the CL a stable state. Significant differences be- tained for asthmatic adherent monocytes peaks wereidentical in both groups. Max- tween asthmatic and control groups were were quite identical to those of control. imal capacity was obtained here for both also observed when monocytes were As described for polymorphonuclear leupopulations. maintained in adherence, as shown by kocyte (PMN) (28), adherence, which is video imaging camera analysis and fer- regarded as a costimulus that primes cells Superoxide Dismutase CL Assays ricytochrome c assays. Values obtained for an increased oxidative burst in reThese results were obtained on adherent before stimulation were higher in adher- sponse to soluble stimuli, promoted monocytes. Before stimulation, SOD ac- ent monocytes from asthmatic compared greater activation of monocytes from tivity was significantly lower in mono- with control subjects. The same differ- asthmatic patients. Thus, after stimulacytes from asthmatic patients than in ences were observed in analysis of the tion they seemed to have reached their those from control subjects (0.33 ± 0.04 whole set of reactive oxygen species. maximal capacity to release oxygen speversus 1.12 ± 0.09 ng/mg proteins, p < Monocytes from asthma released higher cies, whereas monocytes from control 0.001). Significant differences were also quantities of these compounds than con- subjects wereless activated and more senobserved in stimulated monocytes (0.40 trol monocytes. When monocytes were sitive to PMA. Moreover, the difference ± 0.09 and 1.13 ± 0.15 ng/mg proteins studied in suspension, values obtained observed between cellsin suspension and from asthmatic and control subjects, before stimulation wereidentical in asth- in adherence indicated that monocyte respectively; p < 0.006). No difference matic and control subjects. On the con- margination in vessels and the bronchoalwas observed before and after stimula- trary, in adherent monocytes, these values veolar wall could be associated with oxtion in asthmatic or in control subjects. were significantly higher in asthmatic ygen species release, particularly OH·. than in control subjects. In our ex- The low SOD activity, which explains the Discussion perimental conditions we did not observe increased accumulation of O 2 "; in asthThis study demonstrated that monocytes significant differences in the percentage ma, was described as favoring the forfrom asthmatic patients were able to re- of adherent monocytes between asthmat- mation of OH· from O 2 "; and H 202 in lease oxygen species in higher amounts ic and control subjects. However, we ob- the presence of iron complexes (29). This than those from control subjects. O 2 "; ac- served some individual variations in both increased release of toxic oxygen species cumulation was greater in PMA-stimu- populations. After stimulation, values could explain the fibrosis around the lated monocytes from asthmatic than in were similar in asthmatic and control bronchi observed in asthma (30, 31). those from control subjects. In a few pa- groups. The addition of HRP to the reacThese data suggest that monocytes TABLE 3

VIDEO IMAGING ANALYSIS OF

5

7

7

02~·

1166

VACHIER, DAMON, LE DOUCEN, de PAULET, CHANEZ, MICHEL, AND GODARD

from asthmatic patients are primed in vivo. Since PMA is the most effective stimulus used to demonstrate that priming the AM results in an enhanced respiratory burst (32), the enhanced capacity of PMA-stimulated monocytes from asthmatic patients to release oxygen species could be considered evidence of monocyte priming in asthma. This is in agreement with results obtained upon allergen or exercise challenge, which showed that circulating monocytes become activated or primed, shown by the enhanced expression of complement receptor (CRl) and IgG Fc receptors on the cell surface (33,34) and the increased cytotoxic capacity (35). Among the cytokines involved in the pathogenesis of bronchial asthma, interferon-y is known to be able to prime monocytes in vitro to enhance fungicidal or bactericidal activities (36) and to promote the release of increasing amounts of oxygen species when these cells are stimulated by PMA or FMLP, priming considered the in vitro analog of monocyte activation (37). Moreover, it has also been shown that human monocytes express high-affinity receptors for human interferon-y (38). It is known that interferon-y is one of the lymphokines produced by T lymphocytes (CD4+ or CD8+), which were found in bronchoalveolar lavage and peripheral blood during asthma after challenge by antigen (1, 39). This could explain the enhanced capacity of monocytes to produce reactive oxygen species, particularly considering the increasing release of 02~ in asthmatic patients after asthma attacks. Conversely, the release of these derivatives by monocytes from stable asthmatic patients was not related to the gravity of the asthma. The current study showed that monocytes are activated in chronic asthma and presented an enhanced capacity to produce reactive oxygen species. This suggests that monocytes play an important role not only by their maturation into AM but also by their own behavior through their ability to release mediators known to participate in inflammation in asthma. Acknowledgment We thank Mrs. P. Peray (Departement d'Information Medicale, CHR Caremeau, Nimes, France) for her help with the statistics. References 1. Corrigan JC, Kay AB. T lymphocytes and their products in chronic asthma: allergic inflammatory mediators and bronchial hyperresponsiveness. Immunol Allergy Clin North Am 1990; 10:319-28. 2. Jeffrey PK, Wardlaw AJ, Nelson FC, Collins

JV, Kay AB. Bronchial biopsies in asthma. Am Rev Respir Dis 1989; 140:1745-53. 3. Martin TR, Goodman RB. The role of lung mononuclear cells in asthma: allergic inflammatory mediators and bronchial hyperresponsiveness. Immunol Allergy Clin North Am 1990; 10:295-308. 4. Adams DO, Hamilton TA. Phagocytic cells: cytotoxic activities of macrophages. In: Gallin JL, Goldstein 1M, Snyderman R, eds. Inflammation, basic principles and clinical correlates. New York: Raven Press, 1988; 471-92. 5. Chanez P, Damon M, Loubatiere J, et al. Superoxide anion release by alveolar macrophages stimulated by FMLP from healthy subjects and asthmatics (abstract). Am Rev Respir Dis 1987; 135:393a. 6. Nakamura M, Baxter CR, Masters BSS. Simultaneous demonstration of phagocytosis-connected oxygen consumption and corresponding NAD(p)H oxidase activity: direct evidence for NADPH as the predominant electron donor to oxygen in phagocytozing human neutrophils. Biochem Biophys Res Commun 1981; 98:743-51. 7. Babior BM. The respiratory burst of phagocytes. J Clin Invest 1984; 73:599-601. 8. Klebanoff SJ. Phagocytic cells: products of oxygen metabolism. In: Gallin JL, Goldstein 1M, Snyderman R, eds. Inflammation, basic principles and clinical correlates. New York: Raven Press, 1988; 391-444. 9. Allen RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 1986; 133:449-93. 10. Wallaert B, Lassalle P, Fortin F, et al. Superoxide anion generation by alveolar inflammatory cells in simple pneumoconiosis and in progressive massive fibrosis of nonsmoking coal workers. Am Rev Respir Dis 1990; 141:129-33. 11. Richter AM, Abboud RJ, Johal SS, Fera TA. Acute effect of smoking on superoxide production by pulmonary alveolar macrophages. Lung 1986; 164:233-42. 12. Clement A, Chadelat K, Mashah J, et al. A controlled study of oxygen metabolite release by alveolar macrophages from children with interstitial lung disease. Am Rev Respir Dis 1987; 136:1424-8. 13. Damon M, Cluzel M, Chanez P, Godard P. Phagocytosis induction of chemiluminescence and chemoattractant increased superoxide anion release from activated human alveolar macrophages in asthma. J Biolumin Chemilumin 1989; 4:279-86. 14. Evans MJ, Shami SG. Lung cell kinetics. In: Massaro D, ed. Lung cell biology. New York: Dekker, 1983; 17-27. 15. Cluzel M, Damon M, Chanez P, et al. Enhanced alveolar cell luminol-dependent chemiluminescence in asthma. J Allergy Clin Immunol 1987; 80:195-201. 16. Damon M, Chavis C, Daures JP, Crastes de Paulet A, Michel FE, Godard P. Increased generation of the arachidonic metabolites LTB4 and 5-HETE by human alveolar macrophages in patients with asthma: effect in vitro of nedocromil sodium. Eur Respir J 1989; 2:202-9. 17. Durham SR, Carroll M, Walsh GM, Kay AB. Leukocyte activation in allergen-induced late-phase asthmatic reactions. N Engl J Med 1984; 311: 1398-402. 18. American Thoracic Society. Standard for diagnosis and care of patients with chronic obstructive disease (COPD) and asthma. Am Rev Respir Dis 1987; 136:225-45. 19. Aas K. Heterogeneity of bronchial asthma: subpopulation or different stages of the disease. Allergy 1981; 36:3-14. 20. LowryOH, RosenburgNJ, Farr AL, Randall

RJ. Protein measurement with the folin phenol red reagent. J BioI Chern 1951; 193:165-75. 21. Heyworth PG, Segal AW.Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils. Biochern J 1986; 239:723-31. 22. Thorpe GHG, Kricka LJ. Enhanced chemiluminescent reactions catalysed by horse radish peroxidase. Methods Enzymol 1986; 133:331-53. 23. Johnston RB, Godzik CA, Cohn ZA. Increased superoxide anion production by immunologically activated and chemically ellicited macrophages. J Exp Med 1978; 148:115-27. 24. Damon M, Vachier I, Le Doucen C, Godard P, Nicolas J C. Video-imaging of blood monocytes chemiluminescence: application in asthma. In: Stanley PE, Kricka LJ, eds. Bioluminescence and chemiluminescence: current status. Chichester: John Wiley and Sons, 1990; 345-8. 25. Corbisier P, Houbion A, Remacle J. A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence. Anal Biochem 1987; 164:240-7. 26. Weissler JC, Lipscomb MF, Lem VM, Toews GB. Thmor killing by human alveolar macrophages and blood monocytes. Am Rev Respir Dis 1986; 134:532-7. 27. Berton G, Gordon S. Superoxide release by peritoneal and bone marrow-derived mouse macrophages: modulation by adherence and cell activation. Immunology 1983; 49:693-704. 28. Kownatzki E, Uhrich S. Adherence-induced enhancement of the oxidative burst of human neutrophilic granulocytes: effects of the surface coat and of divalent cations. Agents Actions 1991; 32:41-5. 29. Bannister JV, Bannister WH, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. Critical Review Clinical 1987; 22:111-80. 30. Roche WR, Williams JH, Beasley R, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989; 2:520-3. 31. Strausz J, Muller-Quernheim J, Steppling H, Ferlinz R. Oxygen radical production by alveolar inflammatory cells in idiopathic pulmonary fibrosis. Am Rev Respir Dis 1990; 141:124-8. 32. Johnston RB, Kitagawa S. Molecular basis for the enhanced respiratory burst of activated macrophages, Fed Proc 1985; 44:2927-32. 33. Gerberick GF, Jaffe HA, Willoughby JB, Willoughby WF. Relationships between pulmonary inflammation, plasma transduction, and oxygen metabolic secretion by alveolar macrophages. J Immunol 1986; 137:114-21. 34. Durham SR, Carroll M, Walsh GM, Kay AB. Leucocyte activation in allergen-induced late-phase asthmatic reaction. N Engl J Med 1984; 311: 1398-402. 35. Gin W, Shaw R, Kay AB. Airways reversibility after prednisolone therapy is chronic asthma associated with alterations in leucocyte function. Am Rev Respir Dis 1985; 132:1199-203. 36. Johnson KJ, Ward PA. Inflammation and active oxygen species. In: Oberley LW, ed. SOD, Vol. III. Boca Raton, FL: CRC Press, 1985; 129-42. 37. Vecchiarelli A, Todisco T, Puliti M, Dottorini M, Bistoni F. Modulation of anti-candida activity of human alveolar macrophages by interferongamma or interleukin-l-alpha. Am J Respir Cell Mol BioI 1989; 1:49-55. 38. Finbloom DS. The interferon gamma receptor on human monocytes, monocyte-like cell lines and polymorphonuclear leucocytes. Biochem Soc Trans 1990; 18:222-4. 39. Corrigan CJ, Hartnell A, Kay AB. 'l-lymphocyte activation in acute severe asthma. Lancet 1988; 1:1129-34.