Study Population Forty-eight patients were investigated prior to treatment. Thirty- six had granulomatous lung diseases, including 13 with EAA (related to exposure to birds in 8, molds in 3, and diagnosed by open lung biopsy in 2) and 23 with pulmonary sarcoidosis, all showing shadowing on the chest roentgenogram (2 stage 1; 10 stage 2; 11 stage 3) and all with a biopsy specimen-confirmed diagnosis. The remaining 12 patients all had biopsy evidence of FA, including 3 with CFA, 8 with FA in association with scleroderma, and 1 with FA in association with primary biliary cirrhosis. The details of these study groups are given in Table 1. Seven volunteers without parenchymal lung disease also underwent bronchoalveolar lavage; five were male and two were female, with a mean (±SD) age of 44 ± 10 years (range, 26 to 59 years); three were current cigarette smokers and four had never smoked. All patients and control subjects gave their written consent and the study was approved by the Ethics Committee of the National Heart and Chest Hospitals. Bronchoalveolar Lavage Cells

Alveolar macrophages and other cell types were sampled from the air spaces of the lungs (lateral segment of the right lower lobe) by bronchoalveolar lavage using the standard procedure we have previously reported. Immediately after collection the cells were sedimented by low speed centrifiigation (300 g) at 4°C, then washed twice and resuspended in MEM containing 25 mmol/L of HEPES buffer (Gibco, Paisley, Scotland) at 1 x 107 cells per milliliter.
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Staining of Alveolar Macrophage Surface Markers and Intracellular DNA

Bronchoalveolar lavage cells were double stained for the simul­taneous detection of surface markers and intracellular DNA by flow cytometry using the method we have described14 to improve discrimination of cells in lavage and blood samples for flow cytometric analysis. To investigate surface markers on alveolar macrophages we employed the monoclonal antibodies anti-HLA- DR (monomorphic), an ti-HLA-DQ (Leu-10) (polymorphic), anti- HLA-DP (monomorphic), and anti-transferrin receptor (Becton Dickinson, Cowley, Oxford); 100-(jl1 aliquots of bronchoalveolar lavage cell suspension were stained by adding 20 jjlI of stock monoclonal antibody and incubating for 30 minutes at 4°C. Controls were set up omitting the monoclonal antibody to determine “background” fluorescence emission. Following two washes in MEM, 5 |xl of fluorescein isothiocyanate (FITC) conjugated F(ab)2 fragment of goat anti-mouse IgG, IgM, IgA (DAKO, High Wy­combe, Bucks) was added to the cell pellet made up to 100 ц.1 in MEM (Becton-Dickinson). After incubation for 30 minutes at 4°C, the excess was removed by washing twice in 10 percent degraded gelatin plasma substitute (Haemaccel) in MEM. The pellet was resuspended by vortexing in 1 ml of 5 percent Haemaccel, 45 percent MEM, and 50 percent methyl alcohol and the cells were left to fix for 15 minutes at 4°C followed by two washes in PBS’A’ (Oxoid, Basingstoke Hants). The addition of Haemaccel was found to help prevent clumping of the cells during fixation. Following fixation, the cells were incubated at 37°C for 30 minutes with 0.5 mg/ml of RNAase (Sigma, Poole, Dorset) in PBS’A’ to remove intracellular RNA. They were then resuspended in 1 ml of 0.05 mg/ml of propidium iodide (PI) (Sigma) in PBS’A’ for 15 minutes at 4°C to stain the intracellular DNA, followed by two washes in PBS’A’.

Table 1—Clinical Details of the Groups of Batients Studied

Age, у

Lung Function—Mean

± SD % Predicted (Range)

No. of

Mean ± SD

Sex,

Smoking,

Group*

Patients

(Range)

M/F

N/Ex/S

Deo

KCO

TLC

FVC

FEV,

FEV,/FVC

Granulomatous

lung diseases

EAA

13

45 ±13

5/8

8/4/1

62 ±18

86± 14

83± 19

85 ±20

86± 19

103 ±8

(24-66)

(42-105)

(69-115)

(51-117)

(48-116)

(42-108)

(86-112)

Sarcoidosis

23

42 ±13

16/7

17/4/2

76 ±23

98± 19

89 ±16

92 ±21

91 ±23

100 ±14

(24-66)

(32-113)

(38-127)

(57-111)

(56-122)

(38-127)

(52-122)

Total

36

43± 13

21/15

25/8/3

71 ±22

93± 18

87 ±17

90 ±21

89 ±22

101 ±12

(24-66)

(32-113)

(38-127)

(51-117)

(48-122)

(38-127)

(52-122)

Difluse interstitial

fibrosing lung

diseases

CFA

3

60±6

3/0

1/2/0

47 ±7

81 ±18

73 ±2

71 ±7

77 ±13

114 ±15

(55-66)

(39-53)

(62-97)

(71-75)

(67-79)

(62-86)

(98-127)

FA + scleroderma

8

36± 12

1/7

5/3/0

58± 15

83± 14

81 ±30

81 ±31

84 ±29

106 ±16

(24-56)

(32-78)

(62-108)

(31-119)

(27-115)

(33-129)

(71-121)

FA + PBC

1

51

0/1

0/1/0

66

74

97

125

128

101

Total

12

44± 15

4/8

6/6/0

56± 14

82 ±14

81 ±24

82 ±29

86 ±27

107 ±14

(24-66)

(37-78)

(62-108)

(31-119)

(27-125)

(33-129)

(71-127)

Analysis of Stained Alveolar Macrophages by Flow Cytometry

The stained BAL-cell suspensions were analyzed in a cell sorter (Becton-Dickinson FACS 440) interfaced to a computer system (Consort 40) (Becton-Dickinson, Mountain View, Calif). Analysis was performed using the 488-nm laser line to excite both FITC and PI and the instrument was calibrated daily using glutaraldehyde- fixed turkey erythrocytes. Log FITC fluorescence emission was detected via a 525-nm bandpass filter and linear PI fluorescence emission was detected via a 600-nm long-pass filter. Forward angle and 90° light scatter characteristics were also recorded for each cell, to give an indication of size and granularity, respectively, to aid in distinguishing the different populations (Fig 1). The percentages of positive cells reacting with monoclonal antibody and FITC in the total alveolar macrophage population (gated according to 90° and forward angle light scatter characteristics) were determined by comparison with FITC-only controls without monoclonal antibody. kamagra soft tablets

FIGURE 1. Differentiation of alveolar macrophages (gate 1) from lymphocytes (gate 2) in bronchoalveolar lavage samples by flow cytometric analysis of the light scatter characteristics relating to the size (forward angle light scatter) and granularity (90° light scatter) of each cell in the lavage sample. The figure shows the two-dimensional display of these parameters in a patient with extrinsic allergic alveolitis.