Journal of Immunological Methods, 129 (1990) 127-133 Elsevier
127
JIM05553
A cytofluorometric method to quantify membrane antigens on individual alveolar macrophages W. Pankow 1, K. N e u m a n n 2, j. giischoff 2, j. H e y m a n n s i and P. Von Wichert 1 ] Department of Internal Medicine, and ~ Department of Pathology, University Hospital, BaldingerstraBe, D-3550 Marbur~ F.ILG.
(Received 26 June 1989, revised received 3 November 1989, accepted 3 January 1990)
An immunofluorescence method for assaying membrane-bound antigens on individual alveolar macrophages (AM) collected by bronchoalveolar lavage (BAL) is described. Cells were labelled with FITC-conjugated anti-HLA-DR antibodies in a single (direct) step. Quantification of the fluorescence was performed by computer-assisted cytophotometry. Alveolar macrophages, especially when obtained from cigarette smokers, exhibited an autofluorescence which interfered with the measurement of specific fluorescence. The specific quorescence was calculated by determination of total fluorescence in the wave-length optimal for FITE and the non-specific fluorescence in a different wavelength during a second •measurement. Specificity and reproducibility testing confirmed the reliability of the method. Key words: Cytophotometry; Immtmofluorescence; Alveolar macrophage; HI.A-DR antigen; Monoclonal antibody
Introduction There has recently been a substantial increase in interest in the quantitation of surface antigens on alveolar macrophages (AM). It has been shown that AM taken from patients with sarcoidosis have a higher density of membrane HLA class II antigens when compared with the AM found in healthy controls (Cambell et al., 1986; Spurzem et al., 1989). It was suggested that this enhanced class II expression by AM in sarcoidosis might be responsible for the previously observed increased induction of T lymphocyte proliferation (Venet et al., 1985).
Correspondence to: W. Pankow, Department of Internal Medicine, University Hospital, BaldingerstralSe, D-3550 Marburg, F.R.G. Abbreviations: AM, alveolar macrophage; BAL, bronchoalveolar lavage; FITC, fluoresceinisothiocyanate; HLA, human leukocyte antigen.
The quantification of cellular constituents on AM using flow cytometry is complicated by two major factors. First, matching cell size and fluorescence is difficult due to the wide variation in cell size which is found among AM. The second complication involves the intense autofluorescence emitted by AM which interferes with the specific fluorescence of antibody-fluorophore complexes. AM has an autofluorescence spectrum with excitation maxima at 370 and 490 nm and an emission maximum at 541 nm with a shoulder at 580 nm (Edelson et al., 1985). These fluorescence characteristics overlap significantly with the fluorochromes FITC and rhodamine. In view of these problems Rossman and Douglas (1988) concluded that 'other techniques ... will have to be applied to identify alveolar macrophages that bind to monoclonal antibodies'. We have developed a microscopic fluorometric method which measures cell size and fluorescence on individual ceils simultaneously. At the same
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
128 time autofluorescence is recorded at a different wavelength and this makes it possible to calculate the specific fluorescence.
Materials and methods
Cell preparation Human alveolar macrophages were collected by bronchoalveolar lavage (BAL) during routine bronchoscopic examination of smokers and nonsmokers after informed consent had been obtained. Bronchoscopy was performed because of minor hemoptysis or the presence of a peripheral tumor in the contralateral lung. After local anesthesia with 1% lidocaine a flexible Olympus bronchoscope was wedged in one segment of the right middle lobe. 200 ml of a 0.9% sterile saline solution were instilled in four 50 ml aliquots and then recollected by gentle aspiration. Recollected fluid always exceeded 50% of the instilled volume. The collected fluid was filtered through several layers of gauze. Cell viability was between 80 and 907o as assessed by the trypan blue exclusion test. Before the first centrifugation step differential cell counts were performed on hematoxylin-eosin stained Millipore filter preparations as previously described (Saltini et al., 1984). In all cases the differential cell count was normal: macrophages > 857o, lymphocytes < 157o, granulocytes < 3%. After centrifugation and resuspension of the cell pellet in Hanks' balanced saline solution, three different types of cell preparation were obtained by the following procedures: -sedimentation and adherence on Lab-Tek tissue culture slides (Miles, Naperville, U.S.A.) during incubation at 37 °C for 3 h; -sedimentation and adherence on poly-Llysine-coated glass slides (Bross et al., 1978); -cytocentrifugation (500 rpm/10 rain). Cells were covered with cover glasses (0.17 mm) using Quantafluor Mounting Medium (KaUestad, Austin, U.S.A.). Glass slides with fluorescencestained macrophages were stored in the dark at 4 ° C until use. Non-stained cells on glass slides were fixed in acetone and frozen at - 4 0 °C until use.
lmmunocytochemical staining Before sedimentation on slides or cytocentrifuge preparation viable macrophages were suspended in bovine serum to block non-specific binding sites and then stained with a 1/10 dilution of FITC-conjugated monoclonal antibodies for 60 rain on ice. Two antibodies recognizing HLA-DR membrane antigens were used: antiHLA-DR-FITC (IgG2a) from Becton Dickinson (Mountain View, U.S.A.) and OKDR-FITC (IgG2a) from Ortho (Neckargemtind, F.R.G.). Quantitative measurements were performed with the anti-HLA-DR antibody and the OKDR antibody served as a positive control. OKT3-FITC (Ortho), a monoclonal antibody of the IgG2a subclass, but with no relevant specifity, was used as a negative control.
Cytophotometry Determination of fluorescence intensity t,n the cell surface was performed with a Zeiss microscope fluorometer with epi°illumination and transillumination phase contrast. The microscope was equipped with a microscope photometer 03 containing a variable measuring diaphragm and a photometer electronic multiplier (MPC 64) with a motorized quick-scanning table (Carl Zeiss, Oberkochen, F.R.G.). A mercury short arc lamp (Osram) with a HBO 100W Power Supply (Zeiss) served as the light source. Two filter combinations (Zeiss) were used: -blue light excitation filter BP 450-490 (maximum 490 nm; optimal for FITC) and barrier filter LP 515; -green light excitation filter BP 510-560 and barrier filter LP 590. The photometer system was interfaced with a computerized cytomorphometry system (IBAS 1, Kontron, Eching, F.R.G.) equipped with an intensity measuring program for fluorescence (Cyflan, Zeiss). After prewarming the mercury lamp for a minimum of 30 rain, the system was calibrated with a uranyl glass using the computerized procedure of the Cyflan program. Macrophages were identified using phase contrust illumination. For each cell two diameters were measured (using the photometer diaphragm)
129 and all parameters were stored in a personal computer. Since non-specific autofluorescence of AM interferes with the specific fluorescence of FITC, special attention was given to this problem. FITC has only a narrow excitation and emission range, whereas autofluorescence is excited over a wide light spectrum. Therefore, provided the autofluorescence of different cells at various wavelengths maintained a constant ratio it was possible to calculate the autofluorescence in the following manner. In the first step the fluorescence ratio for 50 unstained cells was calculated by measuring the excitation with blue light and then with green light (factor = f ) . Then in the the second step excitation with blue light and green light of FITClabelled cells was measured. Since FITC was excited only by blue light this measurement represented the specific fluorescence plus the autofluorescence: the fluorescence measured with green light excitation represented only autofluorescence. Therefore from the two measurements for each cell, it was possible to calculate the specific fluorescence intensity (sFI): sFI = blue fluorescence-(green fluorescencex f ) For the determination of antigen density on the cell surface, specific fluorescence was divided by the projected surface area of each macrophage: sFI//~m2. Cells expressing the HLA-DR antigen were dis.tinguished from those lacking this antigen by setting the threshold s F I / # m 2 at: (green fluorescence× f ) + 2 x standard deviation ~m2
Flow cytometry Multiparameter flow cytometric analysis was performed using a FACS analyser (Becton Dickinson, Mountain View, U.S.A.). In each probe 10,000 cells were analyzed for cell volume, right angle light scatter and green fluorescence. Analysis of list mode data and the generation of histograms
were performed using the software program Consort 30 (Becton Dickinson).
Statistical analyses Statistical analyses were performed with a Statgraphics software package (Statistical Graphics Corporation, Rockville, MD, U.S.A.). Interobserver reliability was assessed by Pearson's product-moment correlation coefficient. The comparison of various distribution profiles from separate measurements were evaluated with the Kolmogarov-Smirnov test.
Results
Specificity of monoclonal antibody binding AM in a heterogeneous cell population were identified by cell size and morphological criteria using phase contrast microscopy. HLA-DR antigens detected with the two monoclonal antibodies specified above were expressed on 100% of the AM of non-smoking and 50-60% of cigarette smoking patients. BAL lymphocytes, of which the majority are known to be T lymphocytes, did not show specific fluorescence. Non-specific binding was excluded by control antibody staining with OKT3.
Monoclonal antibody saturation Before undertaking quantitative studies the monoclonal antibodies were titrated: antigen binding sites were saturated with a 1/20 dilution of each of the monoclonal antibodies used (data not shown).
Assessment of non-specificfluorescence The factor ( f ) determined by fluorescence measurements of unstained cells with blue and green light excitation proved to be constant (standard deviation -10%). f was found to be slightly higher in non-smokers (2.6 + 0.28; mean of eight experiments) than in smokers AM (3.3 + 0.28; mean of seven experiments).
Reproducibility Several critical points which might influence the reproducibility of the results were considered:
130
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Fig. l. Fluorescence reading of one cell for 1.2 s: fading for a period of 12 ms is below 0.15%. 0
a
-Constancy of photometer measurements; -Fading; -Stability of fluorescence staining; -Inter-observer variance and retest reliability; -Cell preparation.
0,5
1
1,5
2
2,5
50~ 40-
Constancy of photometer measurements
3O--
In order to obtain reproducible results it was necessary to allow sufficient time for the light source to warm up: 30 rain proved to be adequate. After the calibration of fluorescence intensity, photometer readings were mainly influenced by the environmental temperature (Jongsma et al., 1971). Our measurements were performed at a constant temperature of 2 0 " C , which was kept stable by air-conditioning. During a 4 h period ten uranyl-glass measurements were performed with standardized adjustments of the microscope and
20. 100
~ 0
0,5
1
_~ 1,5
, 2
2,5
b
Fig. 3. a: Population histogram of 100 cells collected by sedimentation and adherence on glass slides showing the relative distribution of HLA-DR antigen density on the cell surface. b: Cytocentrifugepreparation of the same sample: the docrcasc in fluorescenceintensity is due to an increase in the projcctcd cell surface.
photometer system. Under these conditions, fluctuations in fluorescence intensity were < 2%.
Fading
°
1234
?
IO
13
16
22
Z8
Days
Fig. 2. Fluorescence intensity for nine individual A M during a 4 wcck period. Cells were labelled with FITC-conjugatcd antiH L A - D R antibody. A linear decrease of fluorcscence intensity is dcmonstratod. Separate measurements were made by a second observer on day 2 (o): inter-obscrver reliability 0.996.
Fading is defined as a decrease in fluorescence during excitation, regardless of its exact cause. The degree of fading depends on the intensity of the excitation fight and exposure time (Jongsma et al., 1971). All of our measurements were performed rapidly with an exposure time of 12 ms. Fig. 1 shows 100 fluorescence readings of the ¢oame cell: fading for a single reading was < 0.15%.
Stability of fluorescence staining T o assess the stability of the fluorescence staining nine cells stained with the anti-HLA-DR-FITC
131
stain were repeatedly measured during a 4 week period (Fig. 2). There was a 2.3~ per day linear decrease of fluorescence intensity.
lnterobserver variance and retest reliability Fluorescence intensity readings of the same nine cells mentioned above were performed by a second observer and similar results were obtained (Fig. 2): interobserver reliability was 0.996. Reproducibility was also confirmed by two separate
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Fig. 5. a: flow cytometer analysis of non-smoker AM: the fluorescence curve of unstained cells (left) shows no-overlapping with the curve for HLA-DR antigen labelled cells (fight) demonstrating minimal non-specific fluorescence, b: strong autofluorescence of smoker AM results in overlapping of the
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127
JIM05553
A cytofluorometric method to quantify membrane antigens on individual alveolar macrophages W. Pankow 1, K. N e u m a n n 2, j. giischoff 2, j. H e y m a n n s i and P. Von Wichert 1 ] Department of Internal Medicine, and ~ Department of Pathology, University Hospital, BaldingerstraBe, D-3550 Marbur~ F.ILG.
(Received 26 June 1989, revised received 3 November 1989, accepted 3 January 1990)
An immunofluorescence method for assaying membrane-bound antigens on individual alveolar macrophages (AM) collected by bronchoalveolar lavage (BAL) is described. Cells were labelled with FITC-conjugated anti-HLA-DR antibodies in a single (direct) step. Quantification of the fluorescence was performed by computer-assisted cytophotometry. Alveolar macrophages, especially when obtained from cigarette smokers, exhibited an autofluorescence which interfered with the measurement of specific fluorescence. The specific quorescence was calculated by determination of total fluorescence in the wave-length optimal for FITE and the non-specific fluorescence in a different wavelength during a second •measurement. Specificity and reproducibility testing confirmed the reliability of the method. Key words: Cytophotometry; Immtmofluorescence; Alveolar macrophage; HI.A-DR antigen; Monoclonal antibody
Introduction There has recently been a substantial increase in interest in the quantitation of surface antigens on alveolar macrophages (AM). It has been shown that AM taken from patients with sarcoidosis have a higher density of membrane HLA class II antigens when compared with the AM found in healthy controls (Cambell et al., 1986; Spurzem et al., 1989). It was suggested that this enhanced class II expression by AM in sarcoidosis might be responsible for the previously observed increased induction of T lymphocyte proliferation (Venet et al., 1985).
Correspondence to: W. Pankow, Department of Internal Medicine, University Hospital, BaldingerstralSe, D-3550 Marburg, F.R.G. Abbreviations: AM, alveolar macrophage; BAL, bronchoalveolar lavage; FITC, fluoresceinisothiocyanate; HLA, human leukocyte antigen.
The quantification of cellular constituents on AM using flow cytometry is complicated by two major factors. First, matching cell size and fluorescence is difficult due to the wide variation in cell size which is found among AM. The second complication involves the intense autofluorescence emitted by AM which interferes with the specific fluorescence of antibody-fluorophore complexes. AM has an autofluorescence spectrum with excitation maxima at 370 and 490 nm and an emission maximum at 541 nm with a shoulder at 580 nm (Edelson et al., 1985). These fluorescence characteristics overlap significantly with the fluorochromes FITC and rhodamine. In view of these problems Rossman and Douglas (1988) concluded that 'other techniques ... will have to be applied to identify alveolar macrophages that bind to monoclonal antibodies'. We have developed a microscopic fluorometric method which measures cell size and fluorescence on individual ceils simultaneously. At the same
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
128 time autofluorescence is recorded at a different wavelength and this makes it possible to calculate the specific fluorescence.
Materials and methods
Cell preparation Human alveolar macrophages were collected by bronchoalveolar lavage (BAL) during routine bronchoscopic examination of smokers and nonsmokers after informed consent had been obtained. Bronchoscopy was performed because of minor hemoptysis or the presence of a peripheral tumor in the contralateral lung. After local anesthesia with 1% lidocaine a flexible Olympus bronchoscope was wedged in one segment of the right middle lobe. 200 ml of a 0.9% sterile saline solution were instilled in four 50 ml aliquots and then recollected by gentle aspiration. Recollected fluid always exceeded 50% of the instilled volume. The collected fluid was filtered through several layers of gauze. Cell viability was between 80 and 907o as assessed by the trypan blue exclusion test. Before the first centrifugation step differential cell counts were performed on hematoxylin-eosin stained Millipore filter preparations as previously described (Saltini et al., 1984). In all cases the differential cell count was normal: macrophages > 857o, lymphocytes < 157o, granulocytes < 3%. After centrifugation and resuspension of the cell pellet in Hanks' balanced saline solution, three different types of cell preparation were obtained by the following procedures: -sedimentation and adherence on Lab-Tek tissue culture slides (Miles, Naperville, U.S.A.) during incubation at 37 °C for 3 h; -sedimentation and adherence on poly-Llysine-coated glass slides (Bross et al., 1978); -cytocentrifugation (500 rpm/10 rain). Cells were covered with cover glasses (0.17 mm) using Quantafluor Mounting Medium (KaUestad, Austin, U.S.A.). Glass slides with fluorescencestained macrophages were stored in the dark at 4 ° C until use. Non-stained cells on glass slides were fixed in acetone and frozen at - 4 0 °C until use.
lmmunocytochemical staining Before sedimentation on slides or cytocentrifuge preparation viable macrophages were suspended in bovine serum to block non-specific binding sites and then stained with a 1/10 dilution of FITC-conjugated monoclonal antibodies for 60 rain on ice. Two antibodies recognizing HLA-DR membrane antigens were used: antiHLA-DR-FITC (IgG2a) from Becton Dickinson (Mountain View, U.S.A.) and OKDR-FITC (IgG2a) from Ortho (Neckargemtind, F.R.G.). Quantitative measurements were performed with the anti-HLA-DR antibody and the OKDR antibody served as a positive control. OKT3-FITC (Ortho), a monoclonal antibody of the IgG2a subclass, but with no relevant specifity, was used as a negative control.
Cytophotometry Determination of fluorescence intensity t,n the cell surface was performed with a Zeiss microscope fluorometer with epi°illumination and transillumination phase contrast. The microscope was equipped with a microscope photometer 03 containing a variable measuring diaphragm and a photometer electronic multiplier (MPC 64) with a motorized quick-scanning table (Carl Zeiss, Oberkochen, F.R.G.). A mercury short arc lamp (Osram) with a HBO 100W Power Supply (Zeiss) served as the light source. Two filter combinations (Zeiss) were used: -blue light excitation filter BP 450-490 (maximum 490 nm; optimal for FITC) and barrier filter LP 515; -green light excitation filter BP 510-560 and barrier filter LP 590. The photometer system was interfaced with a computerized cytomorphometry system (IBAS 1, Kontron, Eching, F.R.G.) equipped with an intensity measuring program for fluorescence (Cyflan, Zeiss). After prewarming the mercury lamp for a minimum of 30 rain, the system was calibrated with a uranyl glass using the computerized procedure of the Cyflan program. Macrophages were identified using phase contrust illumination. For each cell two diameters were measured (using the photometer diaphragm)
129 and all parameters were stored in a personal computer. Since non-specific autofluorescence of AM interferes with the specific fluorescence of FITC, special attention was given to this problem. FITC has only a narrow excitation and emission range, whereas autofluorescence is excited over a wide light spectrum. Therefore, provided the autofluorescence of different cells at various wavelengths maintained a constant ratio it was possible to calculate the autofluorescence in the following manner. In the first step the fluorescence ratio for 50 unstained cells was calculated by measuring the excitation with blue light and then with green light (factor = f ) . Then in the the second step excitation with blue light and green light of FITClabelled cells was measured. Since FITC was excited only by blue light this measurement represented the specific fluorescence plus the autofluorescence: the fluorescence measured with green light excitation represented only autofluorescence. Therefore from the two measurements for each cell, it was possible to calculate the specific fluorescence intensity (sFI): sFI = blue fluorescence-(green fluorescencex f ) For the determination of antigen density on the cell surface, specific fluorescence was divided by the projected surface area of each macrophage: sFI//~m2. Cells expressing the HLA-DR antigen were dis.tinguished from those lacking this antigen by setting the threshold s F I / # m 2 at: (green fluorescence× f ) + 2 x standard deviation ~m2
Flow cytometry Multiparameter flow cytometric analysis was performed using a FACS analyser (Becton Dickinson, Mountain View, U.S.A.). In each probe 10,000 cells were analyzed for cell volume, right angle light scatter and green fluorescence. Analysis of list mode data and the generation of histograms
were performed using the software program Consort 30 (Becton Dickinson).
Statistical analyses Statistical analyses were performed with a Statgraphics software package (Statistical Graphics Corporation, Rockville, MD, U.S.A.). Interobserver reliability was assessed by Pearson's product-moment correlation coefficient. The comparison of various distribution profiles from separate measurements were evaluated with the Kolmogarov-Smirnov test.
Results
Specificity of monoclonal antibody binding AM in a heterogeneous cell population were identified by cell size and morphological criteria using phase contrast microscopy. HLA-DR antigens detected with the two monoclonal antibodies specified above were expressed on 100% of the AM of non-smoking and 50-60% of cigarette smoking patients. BAL lymphocytes, of which the majority are known to be T lymphocytes, did not show specific fluorescence. Non-specific binding was excluded by control antibody staining with OKT3.
Monoclonal antibody saturation Before undertaking quantitative studies the monoclonal antibodies were titrated: antigen binding sites were saturated with a 1/20 dilution of each of the monoclonal antibodies used (data not shown).
Assessment of non-specificfluorescence The factor ( f ) determined by fluorescence measurements of unstained cells with blue and green light excitation proved to be constant (standard deviation -10%). f was found to be slightly higher in non-smokers (2.6 + 0.28; mean of eight experiments) than in smokers AM (3.3 + 0.28; mean of seven experiments).
Reproducibility Several critical points which might influence the reproducibility of the results were considered:
130
.~ vvo
40-
v~-
~ es.
2O
ols
~;o
~;s
io f,)"
10
Fig. l. Fluorescence reading of one cell for 1.2 s: fading for a period of 12 ms is below 0.15%. 0
a
-Constancy of photometer measurements; -Fading; -Stability of fluorescence staining; -Inter-observer variance and retest reliability; -Cell preparation.
0,5
1
1,5
2
2,5
50~ 40-
Constancy of photometer measurements
3O--
In order to obtain reproducible results it was necessary to allow sufficient time for the light source to warm up: 30 rain proved to be adequate. After the calibration of fluorescence intensity, photometer readings were mainly influenced by the environmental temperature (Jongsma et al., 1971). Our measurements were performed at a constant temperature of 2 0 " C , which was kept stable by air-conditioning. During a 4 h period ten uranyl-glass measurements were performed with standardized adjustments of the microscope and
20. 100
~ 0
0,5
1
_~ 1,5
, 2
2,5
b
Fig. 3. a: Population histogram of 100 cells collected by sedimentation and adherence on glass slides showing the relative distribution of HLA-DR antigen density on the cell surface. b: Cytocentrifugepreparation of the same sample: the docrcasc in fluorescenceintensity is due to an increase in the projcctcd cell surface.
photometer system. Under these conditions, fluctuations in fluorescence intensity were < 2%.
Fading
°
1234
?
IO
13
16
22
Z8
Days
Fig. 2. Fluorescence intensity for nine individual A M during a 4 wcck period. Cells were labelled with FITC-conjugatcd antiH L A - D R antibody. A linear decrease of fluorcscence intensity is dcmonstratod. Separate measurements were made by a second observer on day 2 (o): inter-obscrver reliability 0.996.
Fading is defined as a decrease in fluorescence during excitation, regardless of its exact cause. The degree of fading depends on the intensity of the excitation fight and exposure time (Jongsma et al., 1971). All of our measurements were performed rapidly with an exposure time of 12 ms. Fig. 1 shows 100 fluorescence readings of the ¢oame cell: fading for a single reading was < 0.15%.
Stability of fluorescence staining T o assess the stability of the fluorescence staining nine cells stained with the anti-HLA-DR-FITC
131
stain were repeatedly measured during a 4 week period (Fig. 2). There was a 2.3~ per day linear decrease of fluorescence intensity.
lnterobserver variance and retest reliability Fluorescence intensity readings of the same nine cells mentioned above were performed by a second observer and similar results were obtained (Fig. 2): interobserver reliability was 0.996. Reproducibility was also confirmed by two separate
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Fluorescence Intensity
Fig. 5. a: flow cytometer analysis of non-smoker AM: the fluorescence curve of unstained cells (left) shows no-overlapping with the curve for HLA-DR antigen labelled cells (fight) demonstrating minimal non-specific fluorescence, b: strong autofluorescence of smoker AM results in overlapping of the
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