Measurement of Intensity of Expression of Surface Markers

The logarithmic green (525 nm) fluorescence data (Fig 2) were mathematically transformed (using software by D.J.P) to calculate the linear mean, modal, and median intensity value for the total alveolar macrophage population. The measure of the fluorescence intensity of the specific FITC fluorescence relating to bound monoclonal antibody (which is proportional to the absolute amount of antigen present on the cells) was obtained by subtracting the linear median intensity value for the macrophages in the control sample stained only with FITC antimouse F(ab)2 from that of the monoclonal-stained sample. This calculation prevents inaccuracies in the results since it includes correction for the variation in cell autofluorescence between patients. It was not the aim of this study to explore the density of antigen per unit area of the alveolar macrophage cell surface, which is not possible without accurate measurement of cell size, but only to explore the relative median fluorescent intensities that reflect differences in the relative amounts of monoclonal antibody bound to the total alveolar macrophage population in different patients.

Enumeration of BAL Lymphocytes and Their Subpopulations

Total and differential counts of lymphocytes and other cells in the lavage samples of the patients (Table 2) were made using an Improved Neubauer Counting Chamber and May-Griinwald-Giemsa-stained cytocentrifuge preparations, respectively, as described in detail previously. The results for the seven control subjects were all within the normal range for our laboratory, as indicated in Table 2.
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Measurement of lymphocyte subsets by flow cytometry was undertaken by staining (method above) aliquots of cells from each lavage with the following monoclonal antibodies: anti-Leu-14(B cell; CD22), anti-Leu-4 (pan-T cell; CD3), anti-Leu-3a+3b (helper/ inducer T cell; CD4), and anti-Leu-2a (suppressor/cytotoxic T cell; CD8) (Becton Dickinson). The percentage of each subpopulation in the total lymphocyte gate (selected according to 90° and forward angle light scatter characteristics; see Fig 1) was determined following the methodology described above for alveolar macro­phages.

FIGURE 2. Intensity of green fluorescence emission from alveolar macrophages stained only with FITC-anti mouse F(ab)2 (control peak 1, open area) compared with the emission (peak 2, hatched area) from the same population stained with a monoclonal antibody to HLA-DR as well as FITC-antimouse F(ab)2. The difference in the median fluorescence emission for peak 2 compared with peak 1 (after linear transformation of the logarithmic values) was used as a measure of the relative fluorescent intensity reflecting the amount of monoclonal antibody bound to HLA-DR antigens expressed on the mac-rophage population.

Measurement of lymphocyte activation was undertaken by stain­ing aliquots of cells with a monoclonal antibody to detect expression of surface receptors for the mediator interleukin 2 (Becton Dickin­son) and by measuring the percentages of lymphocytes in the S + G2 + M stage of the cell cycle from flow cytometric analysis of intracellular DNA content indicated by PI fluorescence emission (see methods of staining and analysis described above for macro­phages).

Table 2—Bronchoalveolar Lavage Cell Counts


No. of Patients

Total No. of Cells/mix 10» ±SD (Range) (*1.20)t

Mean ± SD Differential Percentage Counts (Range)

Macro (*80)t









lung diseases



0.516 ±0.539

46 ±23

45 ±20


1.9± 1.9

0.45 ±0.74









0.253 ±0.256

61 ±19

29 ±21

9± 13

0.87 ±1.5

0.19 ±0.49









0.348 ±0.397

56 ±21

35 ±22

8± 11

1.3± 1.7

0.28 ±0.59







Diffuse interstitial

fibrosing lung




0.309 ±0.095

81 ±3




0.03 ±0.06







FA + scleroderma


0.513 ±0.604

76 ±18


12 ±16


















0.462 ±0.493

78 ±15


11 ±14


0.008 ±0.029







Flow Cytometric Measurement of HLA-D Region Antigens and Transferrin Receptors on Blood Monocytes

Peripheral blood mononuclear cells (monocytes and lymphocytes) were obtained from defibrinated human blood by sedimentation on lymphocyte separation medium (Flow Labs, UK). The collected cells were washed twice in MEM/HEPES at 4°C and resuspended at 1 x 107 cells per milliliter. To determine the percentages of blood monocytes expressing HLA-DR, DQ, and DP antigens and trans­ferrin receptors, a double-immunofluorescence staining method was used to overcome the problem that blood monocytes cannot be completely differentiated from lymphocytes by their 90° and forward angle light scatter characteristics. First, 100-ul aliquots of the peripheral blood mononuclear cell suspension (I x 107 cells per milliliter in MEM/HEPES) were incubated with 20 p.1 of mouse monoclonal anti-HLA-DR, DQ, or DP or anti-transferrin receptor (Becton Dickinson) for 30 minutes at 4°C, washed with MEM/ HEPES, then incubated with 5 ц.1 of FITC-conjugated F(ab)2 goat antimouse IgG, IgM, and IgA (DAKO) made up to 100 (il in MEM/ HEPES for 30 minutes at 4°C to stain the first cell-bound monoclonal antibody. Second, the cells were washed and incubated with a 1 in 50 dilution of normal mouse serum for 10 minutes to block any nonspecific uptake of the second layer monoclonal antibody. Excess mouse serum was removed and the second layer monoclonal antibody, phycoerythrin (PE)-conjugated Leu-M3 (Becton Dickin­son), was added (20 to 100 ul of the cell suspension) for 30 minutes at 4°C to specifically stain monocytes. The cells were finally washed in MEM/HEPES, fixed in 2 percent paraformaldehyde in PBSA (Oxoid) for 30 minutes, washed, then left at 4°C in PBSA prior to analysis in the flow cytometer. For analysis, the 488-nm laser line was used to excite both FITC and PE and the monocyte population was selectively gated on the basis of red fluorescence emission from the PE-Leu-M3-stained cells detected using a 575-nm bandpass filter. The percentage of monocytes (M3+ cells) simultaneously binding FITC-conjugated anti-HLA-DR, DQ, DP, or transferrin receptors was determined by simultaneous detection of green fluorescence emission from the same cells using a 525-nm bandpass filter.
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Statistical Analysis

For the analysis of quantitative data, groups were compared using the Mann-Whitney U test for nonparametric data and correlations were made using the Spearman Rank correlation coefficient.