Increased secretion of tumor necrosis factor 70% of the predicted value and stablepulmonary functions for 1 week before allergen challenge and/or BAL. BAL was performedafter corticosteroids and P,-adrenergic drugs had been stopped for at least 1 week. Theophyllin and cromolyn sodium were also stopped at least 1 day before BAL. Patientswith asthmawere divided into four groups: group 0, patients evaluatedat baselineand not submitted to allergen challenge; group 1, patients with asthmaexhibiting no significant responseto allergen inhalation; group 2, subjectswith a single EAR; group 3, patients developing an EAR plus an LAR. The bronchial provocation test was performed when the basal value of FEV, was X0% of the predicted value and after abstaining from bronchodilatators for at least 1 day. Two allergen extracts were used, Dpt or WF (both from StallerghnesLaboratories, Fresnes,France), and were standardized in reactivity index units. Increasing doses of allergens (0.2, 1, 5, and 10 reactivity index units) were successively administeredby inhalation through an electric nebulizer (Mediprom SDC 88, Paris, France).Forcedexpiratory maneuvers were performed 5 and 10 minutes after each inhalation of allergen. The first allergen concentration producing a fall of 20% FEV, was considered as a positive EAR. After development of EAR, FEV, values were followed up to the tenth hour after the beginning of the provocation test. A secondaryfall of FEV, >20% was judged as reflecting an LAR. Fourteenhealthy subjectswere selectedon the following criteria: no history of asthma and absenceof atopy and normal pulmonary function tests. Eleven healthy subjects were not submittedto bronchial allergen challenge. In three cases,control subjectswere exposed to allergen (two with Dpt and one with WF) and submitted 18 to 20 hours later to a BAL. The study was approved by the hospital ethical committee, and written informed consentwas obtainedfrom both patients with asthmaand normal control subjects.

VOLUME NUMBER

88 4

TNF and IL-6 in late asthmatic

TABLE I. Characteristics

No.

Control subjects

of patients

with asthma

Age* iv)

lltopyt

37

0

14

and control

subjects

Positive

cutaneous tests*

reaction

563

prick

Dpt

WF

Cat

Pollen

Serum lgE§

Baseline FEV, (% predicted)

0

0

0

0

0

47 -t 21

98.5 2 2.8

9

6

6

1

4

3

240 -I- 53

88.2 k 5

4

3

2

3

0

1

499 k 240

82.2 rt 5.1

6

4

6

3

2

2

510 * 220

78.3 t 6

5

3

4

3

3

3

201 +- 56

86.4 -c 6

Rhinitist

(20-52) Groups 0

9

1

4

2

6

3

5

34 (20-45) 29 (23-35) 31 (25-46) 29 (25-34)

Group0, patientswith asthmaat baseline;groups1, 2, and 3, patientswith asthmaevaluatedafter provocationtest and distributed accordingto their responseto allergenchallenge. *Limit rangein parentheses. tNumberof subjects. SNumberof subjectswith whealdiameter>5 mmto indicatedallergen. $KIU/L (kilo internationalunit perliter) f geometricmean. BAL BAL wasperformedbetweenthe eighteenthand twentieth hour after allergen exposure, as described previously.4 Briefly, BAL was performed with fiberoptic bronchoscopy by success.Lve instillations of one aliquot of 20 ml followed by five aliquots of 50 ml of physiologic saline in a subsegmental bro’nchus.The first aliquot of 20 ml corresponded to the so-called “bronchial lavage.” The additional five aliquots of 50 ml representedthe BAL itself, only cells recovered fmm this second part of the lavage were cultured and testedfor cytokine production. Although BAL has been proved safe in subjects with mild asthma, it was always performed after careful evaluation of pulmonary function tests. In these conditions, BAL was well tolerated, leading to a bronchospasmin only two casesand was rapidly cured after inhalation of two puffs of a &adrenergic drug. AM isolation

and culture

AMs were isolated as previously described.13Briefly, the lavage fluid was filtered through sterile surgical gauze and centrifuged at 400 g for 10 minutes at 4” C. After three washings, the pellet was resuspendedat a cell concentration of 1.5 X 106/ml in RPM1 1640 containing 5% heat-inactivated fetal calf serum (FCS) and mmol/L of L-glutamine (Gibco, Pontoise, France). Endotoxin contamination of medium was controlled by limulus amoebocytetest (Coatest, Kabevitrum, Austria) and was 90%.

Statistical

analysis

Statistical analysis was performed with Mann-Whitney U test and by Spearman’s linear correlation.

RESULTS Results of bronchial

allergen

challenge

As describedin the MATERIAL AND METHODS section, patients were exposedto the relevant allergen corresponding to their sensitivity: Dpt, N = 6, or WF, N =, 9. Results of pulmonary function testsperformed during the 10 hours after the exposurerevealed (Table II)’ that four patients did not react to the challenge (fall of FEV,