Tumor Necrosis Factor Alpha and Interleukin-6 Production by Human Mononuclear Phagocytes from Allergic Asthmatics after IgE-dependent Stimulation 1, 2

PHILIPPE GOSSET, ANNE TSICOPOUlOS, BENOIT WAllAERT, MICHEL JOSEPH, ANDRE CAPRON, and ANDRE-BERNARD TONNEl

Introduction

If the onset of immediate bronchoconstriction in allergic asthmatics is usually considered as linked to the rapid release of mast-ceIl-associated mediators (1), other cells such as macrophages (2), eosinophils (3), and even platelets (4) have to be taken into account in the late asthmatic reaction (LAR). These so-called inflammatory cells express on their surface a second class of receptors for IgE (FcERII) with a lower affinity that can be stimulated by IgE-dependent mechanisms. In humans, alveolar macrophages (AM) from asthmatic patients or from control subjects after previous sensitization with allergic sera release in the presence of anti-IgE or the related allergen, lysosomal enzymes such as p-glucuronidase, superoxide anion, platelet-activating factor (PAF) (5), leukotriene B4 (LTB4 ) (6), and a chemotactic factor for neutrophils and eosinophils (7). Rat AM stimulated by IgE immune-complexes also produce LTC4 (8). Moreover, intrabronchial instillation of the relevant allergen in asthmatic patients rapidly provokes AM stimulation in situ, arguing for an in vivo AM activation during the first step of the provocation test (9). The LAR is accompanied by an increased responsiveness to inhaled histamine and methacholine (10). If the combined release of bronchoactive agents explains the development of the immediate bronchoconstriction, in contrast, precise mechanisms underlying the LAR are not clearly understood. The rapid influx of inflammatory cells (11) and the efficiency of corticosteroid treatment (12) strongly suggest the participation of proinflammatory mediators during the development of the LAR, among which the secretion of cytokines appears as an interesting hypothesis. Recently,it has been shown that IgE receptor cross-linking from rat lung tissue induces tumor necro768

SUMMARY We have previously demonstrated the production of tumor necrosis factor a (TNF) and interleukln-6 (IL-6) by alveolar macrophages (AM) from allergic asthmatics developing a late asthmatlc reaction after bronchial allergen challenge. In order to explal n the modalities of this monokine synthesis, we tested in vitro the effect of an IgE-dependent stimulation on blood monocytes (8M) and AM from control and asthmatic subjects. TNF and IL·6 secretions were evaluated in 24-h supernatants by radioimmunoassay and by the nD1 cell proliferation test, respectively. AM from allergic asthmatics secreted spontaneously higher concentrations of TNF and IL-6 than did 8M or AM from control subjects. 8M from asthmatics also produced spontaneously Increased levels of TNF, but at a lesser degree than did AM. The addition of antl-lgE Induced a significant Increase of TNF and IL-6 secretions by mononuclear phagocytes from control subjects only after previous sensitization with IgE·rlch medium. In contrast, the direct stimulation by allergen or antl-lgE of AM and 8M from asthmatics enhanced significantly the production of TNF and IL-6 when compared with cells eultured in medium alone. In these conditions, IgE-dependent activation of cells from allergic asthmat· Ics compared with those from control subjects Increased monoklne production in a similar manner. Costlmulatlon by recombinant human Interferon yand IgE-dependent triggering had a synergistic effect on TNF production, but It had only an additive action on IL-6 synthesis (respective Increase index: 9.8 compared with 2.9 and 9.8 compared with 2.1, respectively, for 8M from control and asthmatic subjects). In order to avoid the Influence of a potential contamination by low endotoxin concentration In the pneumoallergens and the antibody used In this stUdy, we evaluated In the 8M from patients presenting with drug hypersensitivity (amoxicillin, rifamycin, or myorelaxants) the production of cytokines after addition of pharmaceutically pure molecules. Drug addition to 8M of such patients induced a low but significant increase of the secretion of TNF and IL-6 concentrations (p < 0.05 in both cases). In conclusion, we demonstrated that IgE-dependent activation enhances the TNF and IL-6 production by AM and 8M. Moreover, the AM from asthmatics had an enhanced capacity to produce TNF and IL-6. AM REV RESPIR DIS 1992; 146:768-n4

sis factor alpha (TNF) production (13). AM are potent producers of TNF, IL-6, and, to a lesser degree, IL-lP, which all possess a broad range of proinflammatory properties (14). Moreover,the monokine production by AM has been evaluated after bronchial allergenic challenge, and TNF and IL-6 production has been demonstrated only in patients exhibiting an LAR (15). In contrast, no IL-IP secretion has been noted. In order to explain modalities of mono kine synthesis observed after bronchial allergen challenge, we decided to test in vitro the effect of an IgE-dependent stimulation on mononuclear phagocytes: blood monocytes (BM) and AM. We have previously studied IL-l synthesis: BM produce IL-IP after IgE triggering (16), but at the level of lung paren-

chyma, the situation appears to be more complex: allergen or anti-IgE addition to AM cultures does not increase the secretion of IL-l p but augments the release of an IL-l inhibitory factor (16). In the present study, the synthesis of TNF and IL-6 was measured after allergen or anti-IgE addition, respectively, by BM and AM recovered from healthy subjects,

(Received in original form July 31, 1991 and in revised form February 28, 1992) 1 From CJF No. 90-06 and CIBP, Unite mixte INSERM 167- CNRS 624, Institut Pasteur, and the Servicede Pneumologie et Immunoallergologie, Hopital Calmette, CHR, Lille, France. 2 Correspondence and requests for reprints should be addressed to P. Gosset, CIBP, Institut Pasteur, BP 245; 59019 Lille, France.

TNF AND IL-6 PRODUCTION BY MONONUCLEAR PHAGOCYTES AFTER IgE-DEPENDENT STIMULATION

allergic asthmatics, and patients with IgE-mediated drug hypersensitivity. The reported data demonstrate that allergen or anti-IgE induce a slight but significant secretion of TNF and IL-6. Moreover, AM from asthmatics produce significantly more TNF and IL-6 either spontaneously or after anti-IgE stimulation than do those from control subjects. Methods Patients Twenty-eight asthmatic patients, nine with drug hypersensitivity, and 19 asymptomatic control subjects were included in this study. These subjects werenonsmokers. All asthmatic subjects responded to the usual criteria of allergicasthma: history of personal or familial atopy, mild asthma at distance from any acute phase, positive cutaneous prick tests (number of subjects with positive cutaneous prick tests: 18 with Dermatophagoides pteronyssinus [Dpt] and 13 with grass pollen), elevated levels of serum IgE (378 ± 52 KUIIL) and of specific IgE antibodies related to the allergen tested. The additional criteria used to select asthmatic patients were: minimal baseline FEV 1 greater than 70070 of the predicted value and stable pulmonary functions for 1 month before bronchoalveolar lavage (BAL). Airway reversibility obstruction was evaluated after inhalation of two puffs of salbutamol spray: all patients exhibited a reversible obstructive disorder as judged by ~ 15% change in FEV 1 postbronchodilatation. BAL was carried out after corticosteroids, and 132-adrenergic drugs were discontinued for at least 1 wk. Theophylline and cromolyn sodium were also stopped at least 1 day before the lavage. Nine patients exhibiting an IgE-dependent sensitivity to therapeutic agents were selected on the basis of a history of an anaphylactic shock, positive cutaneous test, and serum IgE directed against the corresponding agent. The geometric mean of serum IgE concentrations was 170 ± 46 KUIIL. This group comprised four patients sensitiveto myorelaxants (succinylcholine; Kabivitrum, Noisy Ie Grand, France), three to amoxicillin (Lab. Lepetit, Neuilly sur Seine, France), and two to rifamycin (Lab. Allard, Paris, France). All patients were tested at least 1 month after the anaphylactic shock. Nineteen normal subjects were selected on the basis of the following criteria: normal serum IgE (55 ± 17KUIIL), no history of asthma or drug intolerance, absence of atopy, and normal pulmonary function tests. The study was approved by the hospital ethical committee, and written informed consent was obtained from both allergic and normal donors. For ethical and technical reasons, it was impossible to systemicallytest both BM and AM.

Bronchoalveolar Lavage BAL was performed in 12 asthmatics and 11 control subjects as described previously (5)

under fiberoptic bronchoscopy by successive instillations of one 20-ml aliquot followed by five 50-ml aliquots of physiologic saline in a subsegmental bronchus. The first 20-ml aliquot corresponded to the so-called "bronchial lavage." The further five 50-ml aliquots represented the BAL itself; only cells recovered from this second part of the lavage were cultured and tested for cytokine production. Although BAL has been proved safe in mild asthma, it was always performed after careful evaluation of pulmonary function tests. In these conditions BAL was well tolerated, leading to bronchospasm in only one case, which was rapidly relieved by inhalation of two puffs of a 132-adrenergic drug.

Mononuclear Phagocyte Isolation BM were separated from heparinized venous blood from 14 asthmatics (collected simultaneously to the BAL for one patient) and 14 normal subjects (collected simultaneously to the BAL for six patients) by centrifugation on a gradient of Ficoll Paque (Pharmacia, Uppsala, Sweden) as previously reported (17).Mononuclear cells were washed three times and resuspended in RPMI 1640plus 5% heat-inactivated fetal calf serum (FCS) and 2 mM of L-glutamine (GIBCO, Cergy Pontoise, France) at a cell concentration of 5 x 106/ml. Endotoxin contamination of medium was quantitated by limulus amoebocyte test (Coatest; Kabevitrum, Vienna, Austria) and was below 50 pg/ml. This quantity was insufficient to increase TNF mRNA expression and secretion (data not shown). Cellswere allowed to adhere to plastic petri dishes (2 ml in a well 35 mm in diameter) for 2 h at 37° C. The nonadherent cells were removed by three washings with RPMI. Adherent cells contained 97 ± 3% of BM (defined by nonspecific-esterase staining) and 3 ± 2% of T-Iymphocytes (determined by immunofluorescence with anti-CD3 antibody [Becton Dickinson, Rungis, France]). AM were isolated as previously described (15). Briefly, the lavage fluid was filtered through sterile surgical gauze and centrifuged at 400 g for 10min at 4° C. After three washings, the pellet was resuspended at a cell concentration of 1.5 x 106/ml in RPM I 1640containing 5% heat-inactivated FCS and 2 mM of L-glutamine (GIBCO). Differential cell counts showed the following results in adherent cells from control and asthmatic subjects: respectively, 99.3 and 96.8% macrophages, 0.4 and 2.6% lymphocytes, 0.1 and 0.2% neutrophils, 0.2 and 0.4% eosinophils, and no detectable basophiloid cells(by toluidine blue staining). Mononuclear Phagocyte Activation Monokine production was evaluated after in vitro IgE-dependent activation. Cells from normal subjects were passively sensitized by a 30-min incubation at 37° C with sera from asthmatic patients (20% vol/vol) or in some experiments with purified human myeloma IgE (a generous gift of Dr. Bennich). IgE was

769

purified by passage through a column of antiIgE Sepharose (Pharmacia, St. Quentin en Yvelines, France). This preparation contained more than 90% IgE as determined by a RIST (Pharmacia). After five washings, antihuman IgE (sheep IgG fraction, 15 ul/ml; Cappel, Cooper Biomedical, Malvern, PA) or the allergen corresponding to the sensitivity of the serum donor (Dpt or grass pollen, 100 reactivity index units/ml; Laboratoire des Stallergenes, Fresnes, France), used at a final dilution of 10-5 , were added to the cultures. AM and BM from asthmatic patients were directly triggered by anti-IgE or the relevant allergen. Positive controls were obtained by addition of 10 ug/ml of lipopolysaccharide (LPS type 055B5; Difco, Detroit, MI). In three additional experiments, mononuclear phagocyteswereactivated by addition of IgE-coated Sepharose beads (50 III per well of a one to two dilution; Pharmacia) prepared in endotoxin-free conditions with purified myeloma IgE as defined by the manufacturer. Becauseantibody and allergen preparations contained detectable amounts of endotoxin, it was removed by passage through a column of Detoxi-gel (Pierce, Rockford, IL). After this step, Dpt, grass pollen, and anti-IgE solution contained, respectively, 255, 25, and 130 pg/ml of endotoxin. In the case of patients sensitive to drugs, BM were exposed to the relevant allergen used at physiologic concentrations: amoxicillin, succinylcholine, and rifamycin (these preparations contained no detectable endotoxin). Because preliminary data demonstrated that IgE-dependent activation induced a low level of monokine production, the effect of interferon-y (IFN-y), which is known as able to increase mRNA expression for mono kines, was evaluated on the TNF and IL-6 production induced after FCERII cross-linking. Addition of recombinant human (rh) IFN-y (250 U/ml; Boerhinger Mannheim, Mannheim, Germany; specific activity: 2 x 107 U/mg; endotoxin level < 10EU/mg) was performed just before stimulation. After a 24-h incubation at 37° C in humidified air with 5% CO 2, culture supernatants were harvested and filtered through 0.45-llm filters (Millipore, Molsheim, France).

Monokine Assay TNF concentration was evaluated sequentially by L929 cytotoxic test and radioimmunoassay. Biologic activity was estimated from a standard curve as the amount necessary to kill 50% of actinomycin-treated L929 mouse fibroblasts after 18h of culture. Subsequently, TNF level was confirmed by the use of an immunoradiometric assay (IRE-Medgenix, Fleurus, Belgium) after adequate dilution. A highly significant correlation was found between both methods (r = 0.9; p < 0.001).The TNF concentration was finally expressed in pg/ml as defined by the standards provided by the manufacturer. IL-6 was assayed as described by Van Snick and coworkers (18)with some modifications.

770

Briefly, the hybridoma cells 7TDI (a generous gift of Dr. Van Snick, Institut Ludwig, Brussels, Belgium) were incubated with serial dilutions of cell supernatants in microtiter plates. After 4 days of incubation, the number of cells was evaluated by a colorimetric method. A 4-mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma eo, St. Louis, MO) was added at 10070. After a 4-h incubation, the supernatants were discarded, and 0.2 ml of 0.04N HCI-isopropanol solution was added to each wellto solubilize the reduced MTT precipitate. After homogenization, the optical density was read in a multiwell spectrophotometer at 570 nm with a reference at 650 nm. All analyses wereperformed in duplicate, and concentrations were calculated by a probit analysis in comparison with a standard of recombinant human IL-6 (Boehringer Mannheim). The results were expressed in units/ml. The specificity of the test was controlled by the inhibition of cell proliferation after addition of a neutralizing anti-IL-6 rabbit antibody (Genzyme, Boston, MA). In all cases, specific antibody addition to AM supernatants induced an inhibition greater than 90%.

Northern Blot Analysis of TNF mRNA Mononuclear phagocyte stimulation was performed as outlined above. After a 3-hr culture, total cellular RNA was isolated by a guanidium isothiocyanatemethod with cesium chloride modification (19). Equal amounts of RNA, determined by absorbance at 260 nm, were denatured at 50° e for 1 h in glyoxal buffer and then fractionated byelectrophoresis through 1.1% agarose gel. RNA transfer to nitrocellulose membranes was accomplished by capillary blotting for 18 h. After transfer, membranes were dried and baked at 80° C in vacuum, and the RNA was visualized by methylene blue staining to determine the position of 18Sand 28S rRNA bands, to assess the integrity of RNA, and to check that equal amounts have been loaded in all wells after transfer to nitrocellulose. Prehybridization was performed at 63° C for 16 to 24 h in buffer containing 50070 formamide, 50 mM phosphate buffer, 5 x standard saline citrate (SSC), 2 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 2.5 x Denhardt solution (all from Sigma). Labeled RNA probes were obtained by transcription of Pst 1 linear fragment (0.3 Kb TNF fragment length in pRSI plasmid, a generous gift of P. Vassali, Geneva). The probe was labeled with a specific activity of 5 x 106 cpm/ng by using 32p dUTP (Amersham, Buckinghamshire, UK). Hybridization was performed for 18 h at 65° e in prehybridization buffer with 2 x 106 cpm/ml of labeled pro be. The membrane was washed twice with 2 x sse, 0.1% SDS at 65° C, and then with 0.2 x sse and 0.1% SDS for 15 min at 73° C. Statistical Analysis Results were expressed as mean ± SEM.

GOSSET, TSICOPOULOS, WALLAERT, JOSEPH, CAPRON, AND TONNEL

Statistical analysis wasperformed using Wilcoxon's test or the Mann-Whitney U test.

Results

Spontaneous Production ofMonokines by Mononuclear Phagocytes Collected from Control or Allergic Asthmatic Subjects Concerning spontaneous TNF production, AM and BM from allergic patients secreted higher mono kine concentrations than did those from control subjects (p < 0.05 in both cases) (table 1). Moreover, AM from asthmatic patients spontaneously released significantly more IL-6 than did those from control subjects (p < 0.01). In contrast, no difference was observed between IL-6 levels found in BM supernatants from control and asthmatic subjects. Production of TNF and IL-6 by Mononuclear Phagocytes from Control Subjects after IgE-dependent Activation The stimulation of mononuclear phagocytes from control subjects by anti-IgE or allergen did not increase the secretion of monokines (data not shown, n = 5 for blood monocytes and n = 6 for AM). For this reason, these cells werepreviously sensitized by allergic sera or IgE myeloma before addition of anti-IgE antibody. In these conditions, we detected in AM supernatants a significant increase of TNF and IL-6 concentrations (P < 0.05 in both cases) (figure 1). Similar results were obtained with BM (p < 0.05 in both

0

8000

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6000

:::E C)

D.

4000

u. Z

I-

..J

:::E

TNF (pg/m/)

Control

BM AM

139 ± 36 235 ± 38

Asthmatic

BM AM

327 ± 89* 1,060 ± 306*

IL-6 (Ulm/) 245 ± 84 203 ± 80 246 ± 56 467 ± 120t

• p < 0.05 compared with the results of cells from control subjects. t p < 0.01 compared with results of cells from control subjects.

cases). Moreover, the costimulation of the IgE-sensitized AM and BM with 250 Vlml of rhlFN-y enhanced significantly the TNF and IL-6 production (P < 0.01 in all cases); rhlFN-y addition did not modify significantly the spontaneous TNF and IL-6 production. However, rhlFN-y seemed to increase IL-6 production to a lesser degree than that of TNF with AM (increase index between cells stimulated by anti-IgE plus rhlFN-y and anti-IgE alone for TNF and IL-6 was, respectively, 4.2 and 1.3) as with BM (respectively, 9.8 and 2.9). Even after costimulation with rhIFN-y, the amount of cytokine produced after IgE-dependent stimulation was relatively low when compared with data obtained after LPS activation of AM and BM: respectively, 271,450 ± 52,255 and 48,543 ± 15,174

13

•

MEDIUM FN

• ~

ANTI-IGE ANTI-IGE + IFN

Fig. 1. Production of TNF (A) and IL-6 (B) by alveolar macrophages (AM) and blood monocytes (BM) from control subjects spontaneously and stimulated by IFN-y or anti-lgE alone or the combination of both. Mononuclear phagocytes were previously sensitized with IgE-rich medium. * p < 0.05 in comparison with unstimulated cells; § p < 0.01 in comparison with cells activated byanti-lgE.

AM

3000

2000

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CD

=

Cells

Subjects

2000

8M

@

TABLE 1 SPONTANEOUS PRODUCTION OF TNF AND IL-6 BY HUMAN MONONUCLEAR PHAGOCYTES FROM CONTROL SUBJECTS OR ALLERGIC ASTHMATICS

1000

8M

AM

13

•

MEDIUM FN

• ~

ANTI-IGE ANTI-IGE + IFN

771

TNF AND IL·6 PRODUCTION BY MONONUCLEAR PHAGOCYTES AFTER IgE-DEPENDENT STIMULATION

0

8000

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6000

0.28, and r = 0.63, p = 0.07, respectively, for TNF production by AM and by BM. •

:::E

~

•

4000

fZI

o

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Fig. 2. Production of TNF (A) and IL-6 (B) by alveolar macrophages (AM) and blood monocytes (BM) from asthmatic patients spontaneously and stimulated by IFN-y, anti-lgE, or allergen alone and by the combination of IFN-y with either anti-lgE or allergen. * p < 0.Q1 in comparison with unstimulated cells; § p < 0.05 in comparison with unstimulated cells; ** p < 0.05in comparison with cells triggered by addition of anti-lgE.

Z I-

2000

Ell

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2000

ALLERGEN + IFN

MEDIUM

G

IFN

•

ANTIIGE

o

AI..L.ERGEN

m ANTI·IGE + IFN

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=

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AI..L.ERGEN

3000 •

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AM

8M

®

t.£OlUM

rJFN

m AlLERGEN + IFN

1000

AM

8M

pglml of TNF or, respectively, 54,446 ± 13,222 and 38,895 ± 12,254 Vlml of IL-6.

Production of TNF and IL-6 by Mononuclear Phagocytes from Patients with Allergic Asthma after IgE-dependent Activation Cells from patients with allergic asthma were directly exposed to the relevant allergen or anti-IgE antibody. Addition of anti-IgE induced a significant increase of TNF and IL-6 production by AM (p < 0.01 in both cases), whereas allergen stimulation did not significantly modify these concentrations (figure 2). In most cases, the effect of rhlFN-y could not be tested because of the limited number of cells isolated from the BAL. Concerning BM from the same patients, addition of allergen or anti-IgE increased significantly TNF production (p < 0.05 in both cases) (figure 2A) and IL-6 production (p < 0.01 in both cases) (figure 2B). As previously shown with mononuclear phagocytes from control subjects, costimulation by rhlFN-y amplified significantly both monokine production by BM. However, the increase index between cells stimulated by FceRII cross-linking plus rhlFN-y and FceRII cross-linking alone was higher for TNF than for IL-6 (9.8 versus 2.1 and 2.9 versus 1.8, respectively, in coactivation with anti-IgE or allergen). In the presence of LPS, BM from these patients secreted similar levels of monokines compared with cells from control subjects (data not

shown). Moreover, with BM recovered from three patients, the effect of the costimulation between IFN-y and either anti-IgE or IgE-coated Sepharose beads was evaluated. The results showed that IgE-coated Sepharose beads induce, either alone or with IFN-y, TNF and IL-6 production higher than that obtained with anti-IgE in the respective situations (p = NS) (table 2). However, the increase index between cellsstimulated or not with IFN-y and either IgE-coated Sepharose beads or anti-IgE were quite similar (respectively, 6.2 and 5.7 for TNF production and 3.2 and 3.1 for IL-6 production). We have evaluated the relationship between IgE serum levels of asthmatic patients and the anti-IgE-induced production of TNF and IL-6 by their mononuclear phagocytes. The results suggested that higher serum IgE levels are associated with an enhanced production of monokines; however, no significant correlations are found: r = 0.4, p =

Production of TNF and IL-6 by Monocytes from Patients with IgE-related Sensitivity to Drugs In order to avoid stimulation caused by potential contamination by endotoxin of the pneumoallergens used in the in vitro experiments, weevaluated the production of cytokines by pharmaceutically pure molecules. Some patients are known to present with IgE-dependent drug hypersensitivity caused by antibiotics (penicillin, rifamycin) or myore1axants(succinylcholine). The drugs used in these conditions contained no detectable levels of endotoxin. As shown in figure 3, drug addition at physiologic concentration to BM of such sensitized patients induced a low but significant increase of the secretion of TNF and IL-6 concentrations (p < 0.05 in both cases). Although costimulation by rhlFN-y had a synergistic effect on the TNF production (p < 0.01), it induced only an additive effect on IL-6 synthesis even if the difference is still significant (p < 0.05). Indeed, the augmentation index between cells stimulated by drug plus rhlFN-y and drug alone is 2.6 and 1.3, respectively, for the production of TNF and IL-6. Expression of mRNA for TNF by Mononuclear Phagocytes after IgE-dependent Activation The mRNA expression for TNF was evaluated with AM from control and asthmatic subjects after addition of anti19B.Methylene blue staining demonstrated that equal amounts of RNA wereloaded in each well (figure 4). A net increase oftnRNA expression for TNF was observed after addition of anti-IgE for both types of AM. In the presence of rhlFN-y alone, AM from control subjects augmented their mRNA expression for TNF,

TABLE 2 PRODUCTION OF TNF AND IL-6 BY BLOOD MONOCYTES FROM THREE ASTHMATIC PATIENTS STIMULATED BY IFN-y WITH OR WITHOUT EITHER IgE-COATED SEPHAROSE OR ANTI-lgE ANTIBODY Addition of rh-Interferon-y (250 Vlm/) Without IFN-y

TNF, pg/ml IL·6, U/ml

With IFN-y

Medium

IgE-coated Sepharose

Anti-lgE

Medium

IgE-coated Sepharose

Anti-lgE

482 ± 159 262 ± 84

1,436 ± 756 553 ± 138

657 ± 120 403 ± 110

1,055 ± 410 519 ± 232

8,897 ± 2,238 1,793 ± 719

3,733 ± 865 1,256 ± 346

772

GOSSET, TSICOPOULOS, WALLAERT, JOSEPH, CAPRON, AND TONNEL

allergenic preparations can directly trigger the TNF and IL-6 secretion. Nevertheless, endotoxin in our experimental design can be disregarded be2000 • MEDIUM cause (1) the addition of the same aller~ IFN oe, o AUEAGEN gen or anti-IgE preparations to unsensim AllERGEN + IFN u, 1000 Z tized cells from control subjects did not IFig. 3. Production of TNF (A) and IL-6 induce monokine production, and (2) (B) by blood monocytes (BM) from paBM from patients presenting with drug tients with IgE-related sensitivity to 8M drugs. The BM are unstimulated or actihypersensitivity secreted after exposure vated by addition of IFN-yor drug-related to endotoxin-free allergenic preparations allergen (amoxicillin, succinylcholine, concentrations of TNF and IL-6 similar and rifamycin) and the combination of to those of BM exposed to an environboth. * P < 0.05 in comparison with un800 mental allergen. Moreover, the bronchistimulated cells; § p < 0.05 and ** p < 0.01 in comparison with BM activated by al tree from allergic patients is naturally addition of allergen. 600 exposed to allergens that are certainly not • MEDIUM endotoxin-free preparations. o IFN :::J 400 So, IgE-dependent stimulation triggers o AlJ..EA(?£N EI AllERGEN + IFN the release of TNF and IL-6 by mono:::! 200 nuclear phagocytes, and their respective concentrations in supernatants are well correlated, as previously described in an8M other model of AM activation (21). After a 3-h stimulation with LPS, the TNF production by AM, expressed as the raand costimulation by both activators had TNF production. IgE-dependent TNF tio of the 24-h level (22%), was above a synergistic effect on this parameter. production by human blood monocytes that of IL-6 (130/0, data not shown), sugSimilar results were obtained with BM has been recently described using IgE- gesting that TNF is able to stimulate the (data not shown). Moreover, in culture immune complexes (20). The TNF levels IL-6 synthesis by AM, as recently shown with medium alone, the mRNA expres- in supernatants from BM at baseline or with chondrocytes (22) and after TNF insion for TNF was higher in AM from after IgE-dependent activation reported fusion in humans (23). In addition, alasthmatic subjects than in those from in that study are comparable with those though TNF (24) and IL-6 (data not control subjects. found in our study. The specificity of the shown) production by AM exposed to IgE-dependent stimulation (as performed LPS was significantly higher in comparDiscussion in our study) has been previously demon- ison with that of BM, no difference can In this study, we demonstrated that IgE- strated on several occasions (5, 7), and be found between the monokine secredependent stimulation of AM and BM it has been confirmed by the use of IgE- tion of AM and that of BM from coninduces a significant increase of TNF and coated Sepharose beads. Moreover, the trol subjects after IgE-dependent stimuIL-6 production. However, the percent- synergism between the effect of these lation, suggesting that the activation age of enhancement compared with base- beads and IFN-y for monokine produc- mechanisms are different. line values was between 100 and 200070, tion demonstrated that this mechanism When we compared the response to whereas the addition of LPS provoked is mediated by the FCERII receptor. How- IgE receptor triggering of cells from conwith AM a 271-fold augmentation of ever, the presence of endotoxin in some trol and asthmatic subjects, we observed no difference between the increased ratio of monokine production from both AM FROM AM FROM groups of subjects after stimulation. This ASTHMATICS CONTROLS observation was apparently in opposition to the fact that the BM and AM from MEDIUM; ANTI·IGE MEDIUM; ANTI-IGE ; IFN Y ; ANTI·IGE + allergic asthmatics have an increased perIFN Y centage of Pcslcll-positive cells (5, 25). This discrepancy could be due to a defect in the saturation degree of monoFig. 4. Expression of mRNA for TNF by nuclear phagocytes FCERII (a receptor of TNFa _188 AM from asthmatic or control subjects low affinity) from the asthmatics, whereafter IgE-dependent stimulation and its as cells from control subjects were sensimodulation by rhIFN-y. Methylene blue staining of RNA is also shown. AM cultized just before the activation. ture and hybridization are performed as Although the effect of anti-IgE trigdescribed in METHODS. gering is the same on BM from asthmatics as on those from control subjects passively sensitized with IgE-rich medium, AM from allergic asthmatics secrete Methylene Blue spontaneously higher concentrations of Staining monokine and after activation, demonstrating that these cells are activated in

0

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773

TNF AND IL-6 PRODUCTION BY MONONUCLEAR PHAGOCYTES AFTER IgE-DEPENDENT STIMULATION

vivo. Another argument for this priming step is that AM from the asthmatics are poorly reactive to the specific allergen, whereas BM sensitivity is clearly higher, which presumably suggests a desensitization mechanism of AM caused by recent contacts with the related allergen. This hypothesis is supported by the absence of response to IgE-dependent triggering observed with AM from asthmatics after bronchial allergen challenge (15). However, the asthmatics selected in this study were hospitalized and, as far as possible, protected from any massive allergen exposure for at least 24 h before the BAL. This observation as well as the higher spontaneous production of monokines by their AM suggest that this state persists for at least 24 h after allergen contact. This activation kinetic likely implicates the lymphocytes that has been evoked (26), and possibly cytokines (IFN-y or granulocyte monocyte-colony stimulating factor [GM-CSF]) in the AM activation. Indeed, the addition of rhlFN-y to mononuclear phagocyte increases significantly the TNF and IL-6 production triggered by anti-IgE as previously demonstrated with the LPSinduced TNF synthesis (27). However, our results show that rhlFN-y had a weaker effect on IL-6 than on TNF synthesis. To our knowledge, the effect of this lymphokine on the IL-6 production by mononuclear phagocytes has not been studied, so we cannot discuss this result. GM-CSF also amplifies the TNF release in costimulation with LPS, and each alone induces augmentation of TNF mRNA expression (27, 28). These lymphokines can also increase the FceRII expression (29), but our experiments are performed in costimulation, a situation that does not favor this mechanism. So the priming of AM can take place subsequent to allergen contact. Another amplification loop may implicate PAF and TNF, known as able to promote their respective secretion (30, 31). In order to precisely identify the in vivo involvement of monokine in the inflammation accompanying the LAR, mono kine production has been also studied on AM from allergic patients collected 18 h after a bronchial allergen challenge (15). The results demonstrated, in AM supernatants from patients exhibiting a LAR, a significant increase of TNF and IL-6 secretion in comparison with that of patients developing only an early asthmatic reaction (EAR) or no response at all. Moreover, TNF production has been recently demonstrated by sensitized rat lung tissue after IgE-receptor trigger-

ing, although the cellular origin of TNF was not defined (13). Mast cellscould also be another source of TNF as previously described (32). These and our present data suggest that the cross-linking of FceR is one possible but not the only inducer of cytokine release since AM from patients developing a single EAR did not produce these monokines 18 h after the challenge, although AM activation was previously demonstrated by enzyme secretion within a few minutes after local allergen instillation (9). One explanation can be that the bronchial allergen exposure induced a rapid monokine production (but of weak range) in patients exhibiting a single EAR, which can be amplified by another mechanism during the development of LAR. The different cofactors susceptible to potentiate monokine production have been mentioned above. Monokines are likely to participate in several steps of the pathophysiology of allergic asthma. In a primate model, Wegner and coworkers (33) have demonstrated that intercellular adhesion molecule-l (ICAM-l) became strongly expressed after allergen challenge on both endothelial and bronchial epithelial cells. Moreover, perfusion of monoclonal antibodies to ICAM-l resulted in the decrease of both bronchial hyperreactivity and eosinophil infiltration, suggesting thus that expression of ICAM-l on vascular endothelial cells may playa pivotal role in the inflammatory process of allergicasthma. Moreover,TNF increases ICAM-l expression on endothelial cells (34) and supernatants of AM from asthmatic patients developing a LAR have a similar effect (35). Byaddition of a neutralizing antibody, we have demonstrated that among the AM-derived mediators, TNF is the major inducer of ICAM-l expression on endothelial cells. More generally, TNF is able to amplify the generation of PAF by monocytes, endothelial cells, and neutrophils (31, 36) and of leukotrienes by granulocytes (37). TNF also activates macrophages, resulting in increased cytocidal potential and enhances superoxide anion production (38). Furthermore, TNF alone stimulates fibroblast proliferation (39), which is observed in the submucosal area of some asthmatic patients. In vivo, tracheal instillation of TNF induces increased pulmonary inflammatory response and enhances mononuclear phagocyte functions (40). It is difficult at this time to suggest a role for IL-6 in the inflammatory reaction consecutiveto allergen challenge; however, it induces differentiation

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