INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway inflammation in asthma plays an essential role in the pathogenesis of clinical manifestations and has been assessed by the analysis of bronchoalveolar lavage (BAL) and bronchial biopsies (1, 2). This inflammatory process is characterized by epithelial destruction, muscular hypertrophy, thickening of the basement membrane, and inflammatory infiltrate mainly consisting of eosinophils, T lymphocytes, and mast cells (3). During asthma inflammation, many mediators are released and potentially involved in tissue injury. A predominant T helper cell type 2 (Th2) cytokine profile including interleukin-4 (IL-4), IL-13, and IL-5 has been described in biopsies and BAL of asthmatic patients (4). Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor- (TNF-) are also detected in BAL of patients with symptomatic asthma, and an increase of TNF- production by macrophages after late-phase response consecutive to antigen challenge has been demonstrated (5, 6). On the other hand, interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor receptor (sTNFR), and transforming growth factor-1 (TGF-1) have anti-inflammatory properties and contribute to limit the inflammatory process in controlled asthma and in asthmatic patients after allergen exposure (7).

However, little is known of the inflammatory process in patients presenting an acute respiratory failure defined as status asthmaticus (SA). Events susceptible to trigger the development of SA are poorly identified, sometimes related to massive allergen or irritant exposure, infection, discontinuation of treatment, aspirin, or sulfite ingestion (11). Pathological studies from patients who died of fatal asthma showed lung distension, diffuse bronchial obstruction with the presence of mucus, cell destruction, and fibrin described as "plugs." Intensity of the pathological changes was higher in SA than in controlled asthma (12). In some cases of SA, it seems that the sudden onset of asthma may be consecutive to an acute bronchospasm (18, 19), but the inflammatory participation is obvious within these patients, a predominant neutrophil influx (15, 16). Supporting this, Carroll and coworkers (15) showed that in cases of fatal asthma with sudden onset, the numbers of neutrophils and mucus gland area were increased compared with those with asthma of longer duration for which the cells infiltrating the bronchial mucosa and lumen were eosinophils. In patients with acute severe asthma, Fahy and coworkers found a predominance of neutrophils in sputum, associated with an increase in elastase and IL-8 (20).

In patients with SA under optimal medical treatment needing ventilatory support, bronchial lavage (BL) could be helpful in the treatment of refractory SA (21, 22). Analysis of the fluid from eight patients with SA showed that neutrophil was the main cell type detected in BL of patients who required ventilation, independently of a local infection or of the mechanism of failure (23). The aim of this study was to investigate the balance between pro- and anti-inflammatory mediators in SA. In chronic asthma, the role of proinflammatory cytokines such as TNF-, IL-1, and IL-6 has been well demonstrated (5, 6, 24). In other inflammatory processes, mediators such as IL-1Ra, IL-10, sTNFR, and TGF- are secreted in response to proinflammatory mediators and are able to limit the process. In the present study, we have measured concentrations of proinflammatory cytokines (IL-1, IL-6, TNF-) and of anti-inflammatory components (IL-10, TGF-1, IL-1Ra, sTNFRI and II) in BL to determine the net balance of inflammatory activity in BL from patients with SA.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Patients

Characteristics of patients with SA and of the three control groups mechanical ventilation (V), controlled asthma (A), and healthy volunteers (C)are summarized in Table 1. In SA, one was a current smoker and two were ex-smokers whereas in V, two were active smokers. There was no smoker or ex-smoker in the A and C groups. The severity of asthma evaluated by the Aas score (25) did not differ between patients with chronic asthma and patients with SA when studied at the distance of the acute exacerbation episode.

Nine patients with SA were included between January 1995 and May 1996. There was one more patient included, compared with the previously published data on cellularity in BL from SA (23). Patients were admitted to the intensive care unit (ICU) for acute respiratory failure linked to asthma, and required mechanical ventilation (MV). The triggering event for SA was identified in five patients: viral infection was suspected in two patients, psychological conflict in one, and inhalation of nonspecific irritants in two cases. Three patients had sudden onset of SA and six had progressive onset of SA defined as an interval between the first symptom of the acute attack of asthma and the respiratory failure requiring MV of less or more than 3 h. At the time of admission, the treatment consisted of systemic steroids (2 mg/ kg/d of methylprednisolone), ₂-agonist (5 to 20 µg/kg/d), curare, and benzodiazepine according to the American Thoracic Society (ATS) recommendations (21). MV was carried out according to the procedure of controlled hypoventilation for a maximal airway pressure of 50 cm H₂O. Fraction of inspired oxygen (FI_O2) was adapted to obtain a Pa_O2 more than 60 mm Hg. Patients with pneumonia were excluded from the study. Mean duration of time on MV was 17.3 ± 4.8 d.

Four patients without asthma but requiring MV for a central respiratory failure linked to a benzodiazepine intoxication (V) were studied. Patients were admitted to an ICU. No respiratory disease, recent infection (less than 6 wk) or atopy was present.

Eleven patients with mild asthma (A) were included. Asthma was defined according to the ATS criteria (21). Exclusion criteria were infection, systemic or inhaled steroids, or hospitalization during the previous 6 wk. Mean FEV₁ (percentage of predicted value) was 87.2 ± 6.4%. Mean provocative concentration of methacholine causing a 20% reduction in FEV₁ (PC₂₀) was 1.5 ± 0.6 mg/ml.

Eight healthy nonsmoker volunteers (C) were included. None had a history of asthma, atopy, or tobacco use. Lung function was in a normal range. The study was approved by the ethics committee of the University Hospital of Lille (Comité Consultatif pour la Protection des Personnes en Recherche Biomédicale [CCPPRB LILLE:9307]).

Asthma Characteristics

Skin prick tests were performed to common aeroallergens (Dermatophagoides pteronyssinus, D. farinae, cat and dog danders, pollens, mold). Total serum IgE was determined by the Phadebas Paper Radioimmunosorbent Test (Pharmacia Diagnostics, Uppsala, Sweden). Atopy was defined by the presence of two or more positive skin tests. All had a reversible obstructive pattern with an increased forced expiratory volume in one second (FEV₁) of 20% after 200 µg of albuterol and/or bronchial hyperreactivity to methacholine (PC₂₀ < 8 mg/ml). Asthma severity was evaluated by the Aas score, from 1 to 5 (25). It is evaluated according to the clinical history of asthma in the past year. It considers both the symptoms (the number and duration of asthma episodes, total duration of symptoms, and presence or absence of symptom-free interval between attacks) and the requirement for medications. Grade 1 is less than 5 episodes per year with long symptom-free intervals to grade 5 with chronic incapacitating asthma with severe, acute exacerbations in spite of continuous medication following adequate and safe dosage regimens.

Fiberoptic Bronchoscopy and Bronchial Lavage

BL was performed in patients with SA when therapeutic benefits could be expected: in case of atelectasis, or refractory SA despite an optimal medical treatment (21, 22). Eighteen BL were performed in nine patients with SA which means that for six patients, the procedure was repeated several times (not more than four times). The time between the beginning of MV and the fiberoptic bronchoscopy was within 0 to 11 d in SA (median = 3.6 d) and at Day 2 in MV. Indications for bronchoscopy were atelectasis in seven of 18 BL procedures and attempt to remove mucus distal impaction in 11. In nine cases, improvement in clinical and/or normalization of the chest radiography was noted. Fiberoptic bronchoscopy was performed through an adaptor of the endotracheal tube designed to minimize air leak (model 514900; Rüsch AG, Kernen, Germany). FI_O2 was increased to 1 for 15 min before and after the procedure. The tip of the bronchoscope was wedged into different segmental bronchi to remove diffuse mucus impaction in patients without atelectasis or in the relevant bronchus in patients with atelectasis. The lavage was performed by infusion of two or three 15-ml aliquots of sterile 0.9% saline solution at room temperature. Each aliquot was immediatly aspirated and the recovered fractions were pooled. Characteristics concerning the site and the timing of BL are reported in Table 2.

In patients with MV for respiratory depression linked to benzodiazepine intoxication (V), fiberoptic bronchoscopy was performed when typical aspect of atelectasis was observed on the chest radiography. Fiberoptic bronchoscopy was performed as described for the patients with SA.

In asthmatic and healthy patients, fiberoptic bronchoscopy was performed after local anesthesia with lidocaine 2% applied to the upper respiratory tract. BL was performed in a segmental bronchus of the right middle lobe by slow infusion of two 15-ml aliquots of sterile 0.9% saline solution. Each aliquot was immediatly aspirated and the different fractions were pooled. During bronchoscopy, oxygen was available and the patient had an intravenous infusion to provide venous access if needed.

BL Processing and Cell Evaluation

An aliquot of 5 ml of each BL fluid was sent for quantitative and qualitative cultures (the diagnosis of lower respiratory tract infection required > 10⁶ pathogens/ml). Among the 18 BL performed in SA, three were positive: one on Day 9 with Pseudomonas aeruginosa 10⁸ colony-forming units per milliliter (cfu/ml); one with Streptococcus pneumoniae 10⁶ cfu/ml on Day 4; and one with Proteus mirabilis 10⁷ cfu/ml on Day 1. In other patients with SA, V, and A, no infectious agent was identified. The total cell counts were determined on BL using a standard hemacytometer. Cytospins of BL were stained using eosin/methylene blue (RAL, Paris, France) for differential cell counts. At least 400 cells on each slide were read by two investigators blinded to the clinical details of the subjects. Average cell count determinations of the two investigators are reported. Macrophages, neutrophils, lymphocytes, eosinophils, mast cells, and epithelial cells were enumerated and results were given both in percentages and number of cells per milliliter of recovered fluid. The remainder of the lavage samples was centrifuged for 10 min at 400 × g to separate the supernatant, which was used for cytokine assays.

Biochemical Assays: Mediators and Cytokines in BL

Concentrations of IL-10 (Immunotech, Marseille, France), IL-1, IL-6, TNF (Biosource-Medgenix, Fleurus, Belgium), IL-1Ra, sTNFRI, and sTNFRII (R&D, Abingdon, UK) were measured by ELISA. Results were expressed in pg/ml for cytokines and soluble receptors. The active form of TGF-1 was directly evaluated in BL fluids (BLF) by ELISA (Promega, Madison, WI) whereas the total concentration (the latent plus the active forms) was measured after acidification with HCl during 15 min by the same assay. Results were expressed as pg/ ml. All these assays used in this study were unsensitive to the addition of the corresponding soluble receptors, the ligand, or the inhibitor. Moreover, the sensitivity of these assays was not affected by the addition of BLF from the different groups of patients.

Proinflammatory Activity of BL

The human epithelial lung adenocarcinoma A549 cell line (ATCC, Rockville, MD) was used to evaluate the bioactivity of proinflammatory cytokines of BL fluids as defined by their capacity to modulate intercellular adhesion molecule-1 (ICAM-1) expression (26). A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) complemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine (Life Technologies, Eragny, France). ICAM-1 expression was measured on A549 using a cell ELISA technique. Confluent cells were trypsinized and distributed in 96-well plates (FALCON; Becton Dickinson, Franklin Lakes, NJ), at the concentration of 5 × 10⁴ cells/well in 200 µl culture medium. Six hours later, culture medium was removed by inversion of the plates and adherent cells were washed 2 times with RPMI 1640 (Life Technologies). BL fluids were diluted at the final concentration of 33%, 10%, 3.3%, and 1% in RPMI 1640 supplemented with 1% FCS and 2 mM L-glutamine in 100 µl final volume. Dilutions of BLF were tested in triplicates. A standard of recombinant human IL-1 (Genzyme, Cambridge, MA) was also added at different concentrations (0.1, 0.3, 1, 3, and 10 units/ml) and the plates were incubated overnight at 37° C in a humidified atmosphere enriched with 5% CO₂. After incubation, cells were washed two times with phosphate-buffered saline (PBS) and fixed with glutaraldehyde for 10 min. A549 cells were washed with PBS containing 5 mM ethylenediaminetetraacetic acid (EDTA)-0.1% bovine serum albumin (BSA), saturated with the same medium and incubated for 1 h with 100 µl/well of mouse monoclonal antibody to ICAM-1 (Becton Dickinson, France) at the defined dose of 0.4 µg/ml. Cells were washed twice again and incubated for 1 h with 100 µl/well of 1:5,000 (vol/vol) diluted peroxidase-labeled anti-mouse IgG (H + L) goat antibody (Diagnostic Pasteur, France). After four additional washings, 100 µl/ well of substrate buffer containing 0.03% H₂O₂ and 0.4 mg/ml o-phenylenediamine were added for 30 min, after which the reaction was stopped with 100 µl/well of 4 N HCl. Optical density (OD) was read in a multiwell spectrophotometer at 492 nm. Using the software Biolise piloting the ELISA reader we determined the dilution of BLF able to induce an increase of ICAM-1 expression corresponding to 50% of that obtained with an optimal concentration of IL-1. The results were expressed in units/ml as the inverse of the dilution giving 50%. A549 cell expression (OD) of ICAM-1 with and without IL-1 was, respectively, 0.61 ± 0.008 and 0.116 ± 0.024. Inhibition of the inflammatory effect was performed by the preincubation of BLF for 1 h with anti-IL-1 and preimmune rabbit IgG (Endogen, Boston, MA), anti-TNF- (Genzyme) antibodies, both at the dilution of 1/50, before their addition to the A549 cell culture.

Statistical Analysis

Group data are expressed as means ± SEM. Because most of the data were not normally distributed, statistical analysis was performed using the nonparametric Kruskal-Wallis test for differences between the four groups (SA, V, A, C). When this test indicated significant difference, each pairing was examined by means of Mann-Whitney U test. The correlations between cytokines and different cell types (neutrophils, eosinophils) were evaluated with Spearman's rank correlation coefficient test. A probability value of less than 0.05 was taken as statistically significant. Data are expressed as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and the bars the 10th and the 90th percentiles.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cellularity in BL

Data concerning the cellular profile were reported on eight of the nine patients in our previous study (23). Total cell number was significantly higher in SA (3.65 ± 1.5 × 10⁶/ml) compared with the V, A, and C groups (0.4 ± 0.1, 0.37 ± 1.15, and 0.29 ± 0.12 × 10⁶/ml, respectively). The percentage of eosinophils was significantly increased in SA (4.8 ± 1.8%) compared with the V, A, and C groups (0.25 ± 0.2, 1.68 ± 0.4, and 0.2 ± 1%, respectively). The percentage of neutrophils was increased in SA (80.4 ± 4.3%) compared with V, A, and C (44.2 ± 12.2, 6.9 ± 2.7, and 9.4 ± 0.12%, respectively). Regarding the presence or absence of lower respiratory tract infection in SA, total cell count was higher in sterile BL (n = 15) compared with positive bacteriologic cultures (n = 3). Differential cell counts were not different between the two groups. When we differentiated in SA BL performed during the first 48 h of mechanical ventilation (n = 9) and those performed after 48 h (n = 9), total count and neutrophil count were similar. However, the number of eosinophils in SA were higher in the BL performed in the first 48 h after mechanical ventilation than in others (p < 0.05).

Levels of Proinflammatory Cytokines in BL Fluids: IL-1, IL-6, TNF-

IL-1 concentrations were significantly higher in SA than in V, A, and C (Figure 1A). There was no difference between groups A and C. In SA, the number of macrophages and of neutrophils were correlated with IL-1 concentration (r_s = 0.7; p = 0.01 and r_s = 0.7; p = 0.009, respectively).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Proinflammatory cytokine levels: concentrations of IL-1 (A), IL-6 (B), and TNF- (C ) in BLF of control subjects (C), stable asthmatic patients (A), patients under MV (V), and patients with status asthmaticus under MV (SA). Data are expressed as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and the error bars the 10th and the 90th percentiles. Circles represent outliers below 10th and above 90th percentiles. *p < 0.05 compared with V patients; #p < 0.05 compared with A and C.

IL-6 concentrations were significantly higher in SA than in V, A, and C. Levels of IL-6 were also higher in V when compared with A and C. When compared with C, IL-6 concentrations were significantly increased in group A (Figure 1B).

TNF- concentrations were significantly higher in SA compared with V, A, and C. There was no difference in TNF- levels between groups V, A, and C (Figure 1C). In SA, the number of neutrophils was correlated with TNF- level (r_s = 0.5; p = 0.05).

Levels of "Anti-Inflammatory" Mediators in BL Fluids: IL-1Ra, IL-10, Active Fraction, and Total Concentration of TGF-1, sTNFRI and II

Concentrations of IL-1Ra were significantly higher in SA compared with A and C whereas there was no difference between SA and V. IL-1Ra levels were not different between V and A but significantly higher in V than in C. Levels of IL-1Ra were also significantly increased in A when compared with C (Figure 2A).

View larger version (17K): [in this window] [in a new window]   Figure 2.   Anti-inflammatory cytokine levels: concentrations of IL-1Ra (A), IL-10 (B), active form of TGF- (C ), total TGF- (D), sTNFRI (E ), and sTNFRII (F ) in BLF of control subjects (C), stable asthmatic patients (A), patients under MV (V), and patients with status asthmaticus under MV (SA). Data are expressed as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and the error bars the 10th and the 90th percentiles. Circles represent outliers below 10th and above 90th percentiles. *p < 0.05 compared with V patients; #p < 0.05 compared with A and C.

IL-10 concentrations were significantly elevated in SA when compared with A and C, but were not different from levels found in V. Patients with V had greater amounts of IL-10 when compared with A and C. No difference between A and C was detected (Figure 2B).

Concentrations of active TGF- were higher in SA when compared with V, A, and C. The levels were surprisingly lower in V than in A and C. There was no difference between A and C (Figure 2C). Total TGF- values were higher in SA patients compared with groups A and C, but were similar to the concentration found in V. There was no difference between A and C (Figure 2D).

sTNFRI levels were not significantly different between SA and V. No significant difference was detected between A and C whereas the levels of sTNFRI were higher in SA and in V compared with A and C (Figure 2E). Concentrations of sTNFRII were not statistically different between SA and V. They were more elevated in SA compared with A and C and in V compared with A and C (Figure 2F).

The ratio of IL-1Ra/IL-1 was significantly higher in SA than in V (p = 0.008), A (p = 0.0005), and in C (p = 0.002). There was no difference between groups V, A, and C. Mean values of the sTNFRI/TNF- ratio were 11.7 ± 3.1 in SA and 10 ± 3.6 in V; no significant difference was noted between the four groups. With regard to TNFRsII/TNF-, mean values were 10.1 ± 4 in SA and 12.4 ± 4.5 in V; no significant difference was noted between the four groups.

Evaluation of the Proinflammatory Activity of BLF

Proinflammatory activity was significantly higher in SA when compared with V, A, and C (Figure 3). There was no significant difference when compared A, C, and V. Inhibition by neutralizing antibodies revealed that mainly anti-IL-1 antibodies neutralized the proinflammatory activity of BL from SA. Levels of inhibition were lower with anti-TNF- antibodies (Table 3).

View larger version (11K): [in this window] [in a new window]   Figure 3.   Proinflammatory activity: concentrations of proinflammatory activity in BLF from control subjects (C), stable asthmatic patients (A), patients under MV (V), and from status asthmaticus patients under MV (SA). Data are expressed as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and the error bars the 10th and the 90th percentiles. Circles represent outliers below 10th and above 90th percentiles. *p < 0.05 compared with V patients; #p < 0.05 compared with A and C.

Evaluation of Cytokine Levels and of the Proinflammatory Activity in the First BL Performed (n = 9) in the Nine Patients with SA: Comparison with Data Obtained in All the BL (n = 18) Performed in the Nine Patients and with Other Groups (V, A, C)

When we took into account only the first BL of each SA patient, the results of cytokine concentrations and of the proinflammatory activity were not different compared with the statistical analysis performed on the 18 BL of SA patients (p = NS). When we considered only the nine first BL samples, similar differences were observed between SA and the other groups (such as a significant increase of TNF-, IL-1, IL-6, and active TGF- in BL from SA patients) than those obtained when we compared the 18 BL of SA patients with the other groups.

Influence of Duration of MV on Cytokine Levels: Comparison of BL Performed before 48 h (9 BL) and BL Performed after 48 h of MV (9 BL) in SA

The only difference was found in sTNFRI concentrations which were significantly higher in BLF performed in the first 48 h of MV (1,232 ± 660) than in those performed after 48 h (271 ± 77) (p = 0.05). There was no difference for IL-1, IL-6, TNF-, IL-10, IL-1Ra, TGF-, and sTNFRII, the ratio IL-1Ra/ IL-1, sTNFRI/TNF, sTNFRII/TNF, and the proinflammatory activity of BL.

Influence of Infection on Cytokine Levels: Comparison of BL with Positive Bacterial Analysis (n = 3) and in Sterile BL (n = 15) in SA

No significant difference between those two groups of SA patients was detected for the different cytokines evaluated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We previously identified a prominent neutrophilia in bronchial lumen from patients with SA correlated with an increased level of neutrophil elastase demonstrating an important recruitment and activation of neutrophils in BL of these patients. Eosinophil influx to the bronchi associated with a massive increase of eosinophil cationic protein (ECP) level was also observed, indicating eosinophil degranulation (23). The aim of our study was to identify the mediators present in the bronchial compartment, possibly implicated in the inflammatory process of SA. A marked increase in levels of pro- and anti-inflammatory mediators was found in BL from patients with SA compared with the other groups. Using a bioassay measuring the net inflammatory activity of these fluids, we observed that the BLF from patients with SA was proinflammatory, mainly because of the presence of bioactive IL-1 and to a lesser extent of bioactive TNF-. As indicated by a duration of MV greater than 48 h in all patients, our study population represented certainly a subgroup of the patients with the most severe SA. Caution should therefore be used to extrapolate our findings to other less severe asthmatic patient populations such as patients with rapidly progressive attacks (18).

Using ELISA techniques, a marked increase in concentrations of inflammatory cytokines such as TNF, IL-1, IL-6, and the active form of TGF- was found in the bronchial compartment from patients with SA compared with other groups of patients. In contrast, IL-1Ra, IL-10, and total concentration of TGF-1, sTNFRI and II were not different between SA and MV, although higher than in controlled asthma and healthy volunteers. Whereas the ratio sTNFR/TNF- was unchanged in the four groups, the IL-1Ra/IL-1 ratio was significantly increased in SA as compared with the other groups, indicating a specific IL-1/IL-1Ra response in this group of patients.

Several lines of evidence suggest that proinflammatory cytokines play an important role in asthma. TNF- and IL-1 share many biological activities: both induce the expression of adhesion molecules (ICAM-1, vascular cellular adhesion molecule-1 [VCAM-1], and E-selectin) (27) and the secretion of chemokines by epithelial and endothelial cells (28). Both mechanisms are involved in the recruitment and the subsequent activation of leukocytes in the tissue. TNF- and IL-1 are also involved in bronchial hyperresponsiveness (BHR) directly by their effect on smooth muscle cells and indirectly by amplifying bronchial inflammation (29). Bronchial instillation of recombinant TNF- promotes neutrophil recruitment concomitantly in the lung with the appearance of BHR in normal subjects (30). In guinea pigs, pretreatment with IL-1Ra partly inhibits allergen-induced eosinophil influx, increase in histamine BHR, and release of TNF- (31). These cytokines are able to induce the release of additional inflammatory mediators: for example, preincubation of eosinophils with TNF- increases the production of reactive oxygen species and their cytotoxicity toward endothelium (32). IL-1 as well as TNF- also induce the synthesis of cytokines such as IL-1 itself, IL-6, and IL-8 (33). Thus, these data may explain that IL-6 and IL-8 are also significantly increased in BL of SA compared with MV. IL-8 is a potent chemoattractant factor for neutrophils and to some extent, for activated eosinophils. The implication of IL-6 is less evident: it is involved in T-cell and B-cell activation, growth, and differentiation and in the production of acute-phase proteins (34). IL-6 also increases in vivo and in vitro the release of IL-1Ra and sTNFRI (35).

TNF-, IL-1, and IL-6 have been measured at high concentrations in BAL of symptomatic asthma compared with asymptomatic asthma (comparable to our group of controlled asthmatics) (24). Moreover, it has been demonstrated that epithelial cells of symptomatic asthmatics expressed IL-1, IL-6, and IL-8 (36). Although high levels of TNF and IL-1 were observed both in symptomatic asthma and in SA, the intensity of inflammatory reaction seemed to be higher in patients with SA than in symptomatic asthmatics.

Secretion of proinflammatory cytokines in the lung is usually accompanied by an anti-inflammatory response aimed at modulating the inflammatory reaction (37). In our study, we found elevated levels of IL-10, IL-1Ra, sTNFR, and TGF-1 in BL. IL-10 decreases macrophage production of IL-1, IL-6, and TNF- and inhibits the allergen-induced eosinophil recruitment into the airways (9). Although IL-10 concentrations were more elevated in SA compared with the other groups, they remained relatively low. The relevance of low levels of IL-10 detection in BL of SA is unclear. In contrast, IL-1Ra, an antagonist for IL-1 binding to IL-1 receptor type 1, was found in large amount in BL from SA. The implication of IL-1Ra in asthma is not well documented, but its function is probably to dampen IL-1-mediated inflammation in vivo as suggested in models of endotoxic shock, sepsis, graft versus host disease, and rheumatoid arthritis (38). In terms of systemic treatment in humans and animals, it seems that the ratio IL-1Ra/IL-1 needs to be very elevated (about 100,000-fold), to limit the severity of the disease and to represent a therapeutic approach, compared with the 100-fold excess observed naturally (38). We found ratios below 1,000 in SA which did not seem to be high enough to block IL-1 biological activity.

TGF-1 is a multifunctional cytokine involved in the synthesis and the deposition of collagen and other matrix components of the basement membrane (39). It also regulates the synthesis of the protease and protease inhibitors in fibroblasts and epithelial cells (39). In TGF-1-deficient mice, a multifocal inflammatory disease associated with massive lymphocyte and neutrophil influx and with an increased TNF- production was observed after birth. Total TGF-1 was found elevated both in SA and in V, whereas the active fraction is elevated only in patients with SA. This TGF- and IL-1Ra response in SA may reflect a negative feedback reaction consecutive to the acute inflammation and may also participate in airway tissue remodeling.

Because of the multiplicity of pro- and anti-inflammatory mediators present in BL from patients with SA, we next addressed the balance of biological activity of these mediators. For this, we measured the capacity of BL to upregulate ICAM-1 expression in a human alveolar type-II like cell (A549) as an indicator for proinflammatory activity, as previously described in BAL from patients with acute respiratory distress syndrome (ARDS) (26). A strong proinflammatory activity was found in BL from patients with SA and contrasted with low activity measured in BL from stable asthmatics or ventilated control patients. Similarly to BAL from patients with ARDS (26), the proinflammatory activity of BL in patients with SA was mainly due to bioactive IL-1 (70%) and TNF- (30%). However, different pathogenesis, pathological data, and clinical behavior are observed in ARDS when compared with SA. In addition, the BL performed in SA explored a different compartment (bronchi) than BAL performed in ARDS, which is representative mainly of the alveolar space. The site of proinflammatory cytokine production seems to be of critical importance for the pathology of the disease: bronchi in SA and alveoli in ARDS. As indicated earlier, high concentration of IL-1 and TNF- was accompanied by the secretion of natural antagonists such as IL-1Ra and soluble TNF receptors. However, a complete inhibition of the proinflammatory activity was not observed in both SA and ARDS despite the molar excess of inhibitors (37) and the net activity remained proinflammatory. In both syndromes, the cell sources of TNF- and IL-1 as well as the stimuli for the production of these cytokines remain to be determined.

The role of MV in triggering or enhancing lung airway inflammation and cytokine production in SA has been recently proposed by several investigators (40). This is particularly true for injurious ventilatory regimens such as those used for patients with SA. Only a weak influx of neutrophils and a significantly higher secretion of IL-6 and of anti-inflammatory cytokines were observed in ventilated control patients compared with A and C, whereas there was no change in concentrations of IL-1 and TNF-. However, the precise effect of MV on the dramatic inflammatory process observed in bronchi from patients with SA needs to be further studied.

Medical treatment of patients with SA includes high doses of systemic glucocorticoids which inhibit the production of proinflammatory cytokines. Despite this treatment, high concentrations of proinflammatory cytokines were detected in BLF from patients with SA and no significant decrease of their levels was observed after 2 d of treatment compared with BL performed in the first 48 h. These data suggest that our patients with SA were poor responders to steroid therapy or that low levels of steroids reached the bronchi.

In conclusion, the bronchi are the site of an intense production of pro- and anti-inflammatory mediators during the course of SA. The net bioactivity is proinflammatory owing to the presence of bioactive IL-1 and TNF- which are certainly implicated in the massive bronchial influx of neutrophils and eosinophils. Targeting proinflammatory cytokines, particularly in the bronchi, might represent an interesting future strategy for the treatment of SA.