Glucocorticoid Resistance in Chronic Asthma Peripheral Blood T Lymphocyte Activation and Comparison of the T Lymphocyte Inhibitory Effects of Glucocorticoids and Cyclosporin A1-3
C. J. CORRIGAN, P. H. BROWN, N. C. BARNES, J.-J. TSAI, A. J. FREW, and A. B. KAY
Introduction We have described a group of nonsmoking asthmatic subjects with documented reversibility of airways obstruction who showed no clinical response to a 2-wk course of systemic glucocorticoid therapy (1). Compared to age- and sexmatched glucocorticoid-sensitive asthmatic patients, the resistant patients showed no physiologically significant differences in their clearance of orally administered glucocorticoid or in the characteristics of their T lymphocyte and monocyte glucocorticoid receptors. The most striking difference between the two groups was observed in a standard test of T lymphocyte function. Phytohemagglutinin (PHA)-induced proliferation of T lymphocytes from the sensitive, but not the resistant, asthmatic patients was significantly inhibited by dexamethasone at a concentration of 10-7 mol/L. Furthermore, there was a significant correlation between the degree of peripheral blood T lymphocyte inhibition by dexamethasone and the changes in FEV 1 after 1 wk of treatment with oral prednisolone. It was hypothesized that this may reflect the fact that one therapeutic effect of glucocorticoids in asthma is directly to inhibit the proliferation of activated T lymphocytes, which are concerned with the maintenance of asthmatic bronchial mucosal inflammation. We recently established that patients hospitalized with acute severe asthma (status asthmaticus) have elevated percentages of activated (Class II histocompatibility antigen, HLA-DR; interleukin-2 receptor IL-2R; and very late activation antigen 1, VLA -1 positive) peripheral blood T lymphocytes, whereas these are not detectable in normal subjects (2). At the time of hospital admission, when their mean peak expiratory flow (PEF) was less than 20% of the predicted value and before the administration of parenteral glucocorticoids, these patients also 1026
SUMMARY A total of 37 chronic severe asthmatic patients with documented reversible airways obstruction were classified as glucocorticoid sensitive or resistant according to changes In the FEV1 following a course of oral prednisolone. The phenotype and expression of activation molecules on peripheral blood T lymphocytes from these patients just before the course of prednisolone were studied using flow cytometry. The resistant patients had significantly elevated percentages of T lymphocytes expressing the activation molecules IL-2Rand HLA-DR compared to the sensitive patients. There were no differences between the patient groups In the percentages of peripheral blood T lymphocytes expressing the phenotypic markers CD4and CDS.Peripheral blood mononuclear cells (PBMC) from 29 patients were cultured In vitro with the T lymphocyte mitogen PHA In the presence or absence of dexamethasone or cyclosporln A. Dexamethasone (10-7 mollL) significantly Inhibited the proliferation of T lymphocytes from the sensitive but not the resistant asthmatic subjects. In contrast, cyclosporln A (500 ng/ml) Inhibited proliferation of T lymphocytes from both the sensitive and the resistant asthmatic subjects, although the effect was less mark8d In the latter group. Inhibition of elaboration of Interleukln-2 and Interferon-y by mitogen-stimulated T lymphocytes from sensitive and resistant asthmatic patients was also studied. Dexamethasone (10-7 mollL) significantly Inhibited the production of Interleukln-2 and Interferon-y by proliferating T lymphocytes Isolated from the glucocorticoid-sensitive but not the resistant chronic asthmatic patients. Cyclosporln A (500 ng/ml) Inhibited the elaboration of both Iymphoklnes by T lymphocytes derived from both patient groups. These observations demonstrate that ongoing T lymphocyte activation can be detected In the peripheral blood of chronic asthmatic patients who are receiving glucocortlcolds but not demonstrating clhJlcallmprovement. They also provide further evidence that clinical glucocorticoid sensitivity In asthma correlates with the degree of Inhibition of T lymphocyte function by glucocortlcolds In vitro. This suggests that activated T lymphocytes may be one target for glucocorticoid therapy In this disease, with the Implication that alternative antl-T lymphocyte drugs, such as cyclosporln A, may be of therapeutic value In glucocorticoid-resistant asthmatic patients. AM REV RESPIR DIS 1991; 144:1026-1032
had increased serum concentrations of soluble IL-2 receptors and interferon-v, which were reduced following therapy to a degree that correlated with clinical responsiveness (3). For these reasons we have extended our studies of T lymphocyte function in glucocorticoid-resistant asthma to include comparisons of the phenotype and activation status of peripheral blood T lymphocytes from the sensitive and resistant groups of patients. We also measured the effects of dexamethasone on IL-2 and interferon-y release by PHA-stimulated T lymphocytes in vitro to determine whether proliferation and cytokine release can be pharmacologically dissociated in these groups of patients. Cyclosporin A, a potent antiT lymphocyte drug used to prevent allograft rejection, also inhibits a number of T lymphocyte functions (4, 5). We
compared dexamethasone with cyclosporin A in its capacity to inhibit PHAinduced thymidine uptake and release of soluble mediators in vitro by T lymphocytes from both glucocorticoid-resistant and sensitive patients.
(Receivedin originalform August 13, 1990and in revised form April 4, 1991) 1 From the Department of Allergy and Clinical Immunology, the National Heart and Lung Institute, the Brompton Hospital, and the London Chest Hospital, London, England, and the Northern General Hospital, Edinburgh, Scotland. 2 Supported by grants from the Asthma Research Council (UK), the WellcomeTrust, and Glaxo, Ltd. 3 Correspondence and requests for reprints should be addressed to A. B. Kay, Professor and Director, Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.
GWCOCORTICOIDS AND CYCLOSPORIN A IN CHRONIC ASTHMA
Methods
Patient Selection This has been described in detail elsewhere (1). Briefly, 37 nonsmoking adult asthmatic patients with stable moderate to severe disease were recruited from the outpatient departments of the London Chest Hospital, London and the Northern General Hospital, Edinburgh. All were maintained on regular inhaled glucocorticoid therapy for control of their disease; seven patients were, in addition, taking up to 10 mg oral prednisolone daily. Strict criteria were invoked for the diagnosis of asthma and to exclude patients with irreversible obstructive airways disease. Patients were classified as glucocorticoid sensitive or resistant according to their FEV. response to a course of oral prednisolone (20 mg daily for 1wk followed by 40 mg daily for a second week). Patients who responded to glucocorticoid after the first week of therapy were designated as sensitive, Week 1 and those who responded after 2 wk as sensitive, Week 2. A positive response was defined as an increase in baseline FEV. (expressed as a percentage of the predicted value) of 300/0 or greater. Informed consent was obtained from all patients before their entry into the study, which was approved by the ethical committees of the National Heart and Chest and Northern General Hospitals. Peripheral Blood Mononuclear Cell (PBMCj Isolation Heparinized peripheral venous blood was obtained from all asthmatic patients just before the clinical trial of oral prednisolone and 8 h after a dose of inhaled or oral glucocorticoid. Blood was mixed with an equal volume of RPMI-1640 medium (GIBCO, Paisley, Scotland). Aliquots (30 ml) were layered onto 20ml aliquots of Ficoll-Hypaque'" (Pharmacia, Uppsala, Sweden) in 50-ml sterile conical tubes (Falcons; Becton Dickinson, Cowley, England). After centrifuging at 1,200 x g for 20 min at 20° C, PBMC were removed from the plasma/Ficoll interface using gentle suction, washed twice, and resuspended at 4 x 106/ml for culture. All in vitro studies were performed without knowledge of clinical data. PBMC Culture Stock solutions of drugs were prepared as follows. Dexamethasone (Sigma, Poole, England) was dissolved in ethanol at a concentration of 200 umol/L and 20-J,11 aliquots stored at - 20° C. In use, aliquots wereevaporated to dryness and resuspended in 2 ml RPMI-1640. After filtering sterile through a 0.22-J,1m pore size filter, appropriate serial dilutions of the solution were made in a total of 1.5 ml RPMI-I640. Cyclosporin A (100 mg/ml) in oily suspension (a gift from Sandoz, Feltham, England) was diluted in RPM I1640to a concentration of 20 ug/rnl. The solution was filtered sterile and aliquots stored at - 20° C. In use, the stock solution wasdiluted to appropriate concentrations in sterile
RPMI-I640. Identical placebo diluent (Sandoz) was diluted in a similar fashion. Phytohemagglutinin (Sigma) was dissolved at 100 ug/rnl in RPMI-1640 and filtered sterile. In use, PHA was added to cultured cells at a final concentration of 5 ug/rnl. In proliferation inhibition experiments, 1-mlaliquots of cell suspension with or without PHA were added to 1-mlaliquots of drug solution or medium control in sterile disposable culture tubes (Cell-Cults; Sterilin, Hounslow, England) with thorough mixing. Triplicate 150-J,11 aliquots of the mixtures were transferred to sterile round-bottomed 96-well culture plates (Sterilin) for measurement of cellular proliferation. Culture tubes and plates were incubated at 37° C in a humidified 5% CO 2 atmosphere for periods of 24, 48, and 72 h. At these time points, culture supernatants were recovered from the tube cultures and stored at - 80° C pending mediator measurements. Cellular proliferation was assessed by uptake of tritiated methylthymidine. Sterile tritiated thymidine solution (Amersham PLC, Amersham, England) (0.66 J,1Cilwell in a volume of 10 J,11) was added to cell culture plates for the last 6 h of the quoted incubation periods. Cells were then harvested onto glass fiber paper using a cell harvester apparatus (11aeon, Tonbridge, England) and incorporated radio label counted using a beta-spectrometer. Results were expressed as the mean counts per minute of triplicate cultures.
Immunofluorescent Staining and Flow Cytometry Staining was performed with the following murine IgG monoclonal antibodies: anti-CD3 conjugated to phycoerythrin (PE) (Becton Dickinson, Cowley, England); anti-HLA-DR (Becton Dickinson); anti-IL-2R (the "anti-lac" antibody; a gift from Dr. T. Waldmann). These unconjugated antibodies were stained with a second layer Ftab'), fragment of rabbit antimouse immunoglobulin conjugated to fluorescein isothiocyanate (RAM-FITC) (Dako, High Wycombe, England) used diluted 1:8. PBMC freshly isolated from blood were resuspended at 107 cells per ml in phosphatebuffered saline (PBS) containing 0.1% wt/vol sodium azide and 0.5% wt/vol bovine serum albumin (PAB medium). Aliquots (50 J,11) of the mononuclear cell suspension were used for each staining procedure. All steps were performed at 4°C. To measure IL-2R and HLA-DR expression on T lymphocytes, cell aliquots were first incubated with 10J,11 of the relevant antibody for 15min. Control aliquots were incubated with an irrelevant murine monoclonal antibody of identical isotype. After washing once with PAB medium the cells were incubated with 25 J,11 RAM-FITC for 15 min and then washed again. An excess of murine pooled IgG (0.3 mg/rnl, 10 J,11) was then added to block any unoccupied sites on the RAM-FITC antibody. After 10 min of incubation, 10J,11 ofanti-CD3-PE was added without washing and incubation continued for a
1027
further 15 min. The cells were then washed twice, fixed by suspension in 0.5 ml PBS containing 0.1% wt/vol sodium azide and 1% wt/vol paraformaldehyde, and stored in the dark at 4° C pending flow cytometric analysis. For measurement of the percentages of CD4 and CD8 T lymphocytes in peripheral blood, cell aliquots were incubated once for 15min with a mixture of anti-CD4-FITC and anti-CD8-PE (Simultests; Becton Dickinson). Control aliquots were incubated with a mixture of irrelevant, isotype-identical antibodies coupled to the same fluorochromes. After washing once the cells were then fixed and stored as before. Flow cytometry was performed on a Becton Dickinson FACS analyzer interfaced with a Hewlett-Packard C30 computer and calibrated by standard techniques. A total of 15,000 events were counted for each sample and gated on volume/90° light scatter plots to excludecontaminating erythrocytes and debris. For measurement of expression of HLADR and IL-2R on CD3 T lymphocytes, further gates were set on red fluorescence (PE) histograms to isolate CD3+cells. Green fluorescence (FITC) histograms of these same cells were then analyzed to detect surface expression of IL-2R and HLA-DR. Specifically stained cell populations werecounted as positive only if their fluorescence histograms differed from those of the negative control populations at the 99.9% confidence level (Kolmogorov-Smirnov two-sample statistics) (6). In such cases, the percentages of positively stained cells were obtained by subtracting the fluorescence histogram of the control cells from those of the specifically stained cells. The percentages of CD4 and CD8 cells in gated lymphocytes were measured by direct subtraction of red and green fluorescence histograms of cells stained with the mixture of irrelevant antibodies conjugated to FITC and PE from those stained with a mixutre of anti-CD8-PE and anti-CD4-FITC.
Measurement of Interleukin-Z and Interferon-s Concentrations in Cell Culture Supernatants IL-2 concentrations in culture supernatants were measured using a competitive radioimmunoassay (Amersham PLC). The IL-2 standard employed was human recombinant IL-2 with an amino-terminal methionine residue in addition to the native 133 amino acid residues of specific activity 3.0 x 106 units/ mg. Sensitivity of the assay, defined as the amount of IL-2 required to reduce zero binding by 2 SD, was 0.3 ng/ml. The coefficients of interassay variation using standards of nominal concentration 1.25and 10ng/ml were 21.6 and 19.0%, respectively. The intraassay coefficient of variation wasestimated as 5.7%. All the samples from anyone patient were always analyzed in the same assay so that within-patient comparisons were subject only to intraassay variability. Interferon-y was measured in culture supernatants using an ELISA technique sup-
1028 60
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CORRIGAN, BROWN, BARNES. TSAI, FREW, AND KAY
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C. J. CORRIGAN, P. H. BROWN, N. C. BARNES, J.-J. TSAI, A. J. FREW, and A. B. KAY
Introduction We have described a group of nonsmoking asthmatic subjects with documented reversibility of airways obstruction who showed no clinical response to a 2-wk course of systemic glucocorticoid therapy (1). Compared to age- and sexmatched glucocorticoid-sensitive asthmatic patients, the resistant patients showed no physiologically significant differences in their clearance of orally administered glucocorticoid or in the characteristics of their T lymphocyte and monocyte glucocorticoid receptors. The most striking difference between the two groups was observed in a standard test of T lymphocyte function. Phytohemagglutinin (PHA)-induced proliferation of T lymphocytes from the sensitive, but not the resistant, asthmatic patients was significantly inhibited by dexamethasone at a concentration of 10-7 mol/L. Furthermore, there was a significant correlation between the degree of peripheral blood T lymphocyte inhibition by dexamethasone and the changes in FEV 1 after 1 wk of treatment with oral prednisolone. It was hypothesized that this may reflect the fact that one therapeutic effect of glucocorticoids in asthma is directly to inhibit the proliferation of activated T lymphocytes, which are concerned with the maintenance of asthmatic bronchial mucosal inflammation. We recently established that patients hospitalized with acute severe asthma (status asthmaticus) have elevated percentages of activated (Class II histocompatibility antigen, HLA-DR; interleukin-2 receptor IL-2R; and very late activation antigen 1, VLA -1 positive) peripheral blood T lymphocytes, whereas these are not detectable in normal subjects (2). At the time of hospital admission, when their mean peak expiratory flow (PEF) was less than 20% of the predicted value and before the administration of parenteral glucocorticoids, these patients also 1026
SUMMARY A total of 37 chronic severe asthmatic patients with documented reversible airways obstruction were classified as glucocorticoid sensitive or resistant according to changes In the FEV1 following a course of oral prednisolone. The phenotype and expression of activation molecules on peripheral blood T lymphocytes from these patients just before the course of prednisolone were studied using flow cytometry. The resistant patients had significantly elevated percentages of T lymphocytes expressing the activation molecules IL-2Rand HLA-DR compared to the sensitive patients. There were no differences between the patient groups In the percentages of peripheral blood T lymphocytes expressing the phenotypic markers CD4and CDS.Peripheral blood mononuclear cells (PBMC) from 29 patients were cultured In vitro with the T lymphocyte mitogen PHA In the presence or absence of dexamethasone or cyclosporln A. Dexamethasone (10-7 mollL) significantly Inhibited the proliferation of T lymphocytes from the sensitive but not the resistant asthmatic subjects. In contrast, cyclosporln A (500 ng/ml) Inhibited proliferation of T lymphocytes from both the sensitive and the resistant asthmatic subjects, although the effect was less mark8d In the latter group. Inhibition of elaboration of Interleukln-2 and Interferon-y by mitogen-stimulated T lymphocytes from sensitive and resistant asthmatic patients was also studied. Dexamethasone (10-7 mollL) significantly Inhibited the production of Interleukln-2 and Interferon-y by proliferating T lymphocytes Isolated from the glucocorticoid-sensitive but not the resistant chronic asthmatic patients. Cyclosporln A (500 ng/ml) Inhibited the elaboration of both Iymphoklnes by T lymphocytes derived from both patient groups. These observations demonstrate that ongoing T lymphocyte activation can be detected In the peripheral blood of chronic asthmatic patients who are receiving glucocortlcolds but not demonstrating clhJlcallmprovement. They also provide further evidence that clinical glucocorticoid sensitivity In asthma correlates with the degree of Inhibition of T lymphocyte function by glucocortlcolds In vitro. This suggests that activated T lymphocytes may be one target for glucocorticoid therapy In this disease, with the Implication that alternative antl-T lymphocyte drugs, such as cyclosporln A, may be of therapeutic value In glucocorticoid-resistant asthmatic patients. AM REV RESPIR DIS 1991; 144:1026-1032
had increased serum concentrations of soluble IL-2 receptors and interferon-v, which were reduced following therapy to a degree that correlated with clinical responsiveness (3). For these reasons we have extended our studies of T lymphocyte function in glucocorticoid-resistant asthma to include comparisons of the phenotype and activation status of peripheral blood T lymphocytes from the sensitive and resistant groups of patients. We also measured the effects of dexamethasone on IL-2 and interferon-y release by PHA-stimulated T lymphocytes in vitro to determine whether proliferation and cytokine release can be pharmacologically dissociated in these groups of patients. Cyclosporin A, a potent antiT lymphocyte drug used to prevent allograft rejection, also inhibits a number of T lymphocyte functions (4, 5). We
compared dexamethasone with cyclosporin A in its capacity to inhibit PHAinduced thymidine uptake and release of soluble mediators in vitro by T lymphocytes from both glucocorticoid-resistant and sensitive patients.
(Receivedin originalform August 13, 1990and in revised form April 4, 1991) 1 From the Department of Allergy and Clinical Immunology, the National Heart and Lung Institute, the Brompton Hospital, and the London Chest Hospital, London, England, and the Northern General Hospital, Edinburgh, Scotland. 2 Supported by grants from the Asthma Research Council (UK), the WellcomeTrust, and Glaxo, Ltd. 3 Correspondence and requests for reprints should be addressed to A. B. Kay, Professor and Director, Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.
GWCOCORTICOIDS AND CYCLOSPORIN A IN CHRONIC ASTHMA
Methods
Patient Selection This has been described in detail elsewhere (1). Briefly, 37 nonsmoking adult asthmatic patients with stable moderate to severe disease were recruited from the outpatient departments of the London Chest Hospital, London and the Northern General Hospital, Edinburgh. All were maintained on regular inhaled glucocorticoid therapy for control of their disease; seven patients were, in addition, taking up to 10 mg oral prednisolone daily. Strict criteria were invoked for the diagnosis of asthma and to exclude patients with irreversible obstructive airways disease. Patients were classified as glucocorticoid sensitive or resistant according to their FEV. response to a course of oral prednisolone (20 mg daily for 1wk followed by 40 mg daily for a second week). Patients who responded to glucocorticoid after the first week of therapy were designated as sensitive, Week 1 and those who responded after 2 wk as sensitive, Week 2. A positive response was defined as an increase in baseline FEV. (expressed as a percentage of the predicted value) of 300/0 or greater. Informed consent was obtained from all patients before their entry into the study, which was approved by the ethical committees of the National Heart and Chest and Northern General Hospitals. Peripheral Blood Mononuclear Cell (PBMCj Isolation Heparinized peripheral venous blood was obtained from all asthmatic patients just before the clinical trial of oral prednisolone and 8 h after a dose of inhaled or oral glucocorticoid. Blood was mixed with an equal volume of RPMI-1640 medium (GIBCO, Paisley, Scotland). Aliquots (30 ml) were layered onto 20ml aliquots of Ficoll-Hypaque'" (Pharmacia, Uppsala, Sweden) in 50-ml sterile conical tubes (Falcons; Becton Dickinson, Cowley, England). After centrifuging at 1,200 x g for 20 min at 20° C, PBMC were removed from the plasma/Ficoll interface using gentle suction, washed twice, and resuspended at 4 x 106/ml for culture. All in vitro studies were performed without knowledge of clinical data. PBMC Culture Stock solutions of drugs were prepared as follows. Dexamethasone (Sigma, Poole, England) was dissolved in ethanol at a concentration of 200 umol/L and 20-J,11 aliquots stored at - 20° C. In use, aliquots wereevaporated to dryness and resuspended in 2 ml RPMI-1640. After filtering sterile through a 0.22-J,1m pore size filter, appropriate serial dilutions of the solution were made in a total of 1.5 ml RPMI-I640. Cyclosporin A (100 mg/ml) in oily suspension (a gift from Sandoz, Feltham, England) was diluted in RPM I1640to a concentration of 20 ug/rnl. The solution was filtered sterile and aliquots stored at - 20° C. In use, the stock solution wasdiluted to appropriate concentrations in sterile
RPMI-I640. Identical placebo diluent (Sandoz) was diluted in a similar fashion. Phytohemagglutinin (Sigma) was dissolved at 100 ug/rnl in RPMI-1640 and filtered sterile. In use, PHA was added to cultured cells at a final concentration of 5 ug/rnl. In proliferation inhibition experiments, 1-mlaliquots of cell suspension with or without PHA were added to 1-mlaliquots of drug solution or medium control in sterile disposable culture tubes (Cell-Cults; Sterilin, Hounslow, England) with thorough mixing. Triplicate 150-J,11 aliquots of the mixtures were transferred to sterile round-bottomed 96-well culture plates (Sterilin) for measurement of cellular proliferation. Culture tubes and plates were incubated at 37° C in a humidified 5% CO 2 atmosphere for periods of 24, 48, and 72 h. At these time points, culture supernatants were recovered from the tube cultures and stored at - 80° C pending mediator measurements. Cellular proliferation was assessed by uptake of tritiated methylthymidine. Sterile tritiated thymidine solution (Amersham PLC, Amersham, England) (0.66 J,1Cilwell in a volume of 10 J,11) was added to cell culture plates for the last 6 h of the quoted incubation periods. Cells were then harvested onto glass fiber paper using a cell harvester apparatus (11aeon, Tonbridge, England) and incorporated radio label counted using a beta-spectrometer. Results were expressed as the mean counts per minute of triplicate cultures.
Immunofluorescent Staining and Flow Cytometry Staining was performed with the following murine IgG monoclonal antibodies: anti-CD3 conjugated to phycoerythrin (PE) (Becton Dickinson, Cowley, England); anti-HLA-DR (Becton Dickinson); anti-IL-2R (the "anti-lac" antibody; a gift from Dr. T. Waldmann). These unconjugated antibodies were stained with a second layer Ftab'), fragment of rabbit antimouse immunoglobulin conjugated to fluorescein isothiocyanate (RAM-FITC) (Dako, High Wycombe, England) used diluted 1:8. PBMC freshly isolated from blood were resuspended at 107 cells per ml in phosphatebuffered saline (PBS) containing 0.1% wt/vol sodium azide and 0.5% wt/vol bovine serum albumin (PAB medium). Aliquots (50 J,11) of the mononuclear cell suspension were used for each staining procedure. All steps were performed at 4°C. To measure IL-2R and HLA-DR expression on T lymphocytes, cell aliquots were first incubated with 10J,11 of the relevant antibody for 15min. Control aliquots were incubated with an irrelevant murine monoclonal antibody of identical isotype. After washing once with PAB medium the cells were incubated with 25 J,11 RAM-FITC for 15 min and then washed again. An excess of murine pooled IgG (0.3 mg/rnl, 10 J,11) was then added to block any unoccupied sites on the RAM-FITC antibody. After 10 min of incubation, 10J,11 ofanti-CD3-PE was added without washing and incubation continued for a
1027
further 15 min. The cells were then washed twice, fixed by suspension in 0.5 ml PBS containing 0.1% wt/vol sodium azide and 1% wt/vol paraformaldehyde, and stored in the dark at 4° C pending flow cytometric analysis. For measurement of the percentages of CD4 and CD8 T lymphocytes in peripheral blood, cell aliquots were incubated once for 15min with a mixture of anti-CD4-FITC and anti-CD8-PE (Simultests; Becton Dickinson). Control aliquots were incubated with a mixture of irrelevant, isotype-identical antibodies coupled to the same fluorochromes. After washing once the cells were then fixed and stored as before. Flow cytometry was performed on a Becton Dickinson FACS analyzer interfaced with a Hewlett-Packard C30 computer and calibrated by standard techniques. A total of 15,000 events were counted for each sample and gated on volume/90° light scatter plots to excludecontaminating erythrocytes and debris. For measurement of expression of HLADR and IL-2R on CD3 T lymphocytes, further gates were set on red fluorescence (PE) histograms to isolate CD3+cells. Green fluorescence (FITC) histograms of these same cells were then analyzed to detect surface expression of IL-2R and HLA-DR. Specifically stained cell populations werecounted as positive only if their fluorescence histograms differed from those of the negative control populations at the 99.9% confidence level (Kolmogorov-Smirnov two-sample statistics) (6). In such cases, the percentages of positively stained cells were obtained by subtracting the fluorescence histogram of the control cells from those of the specifically stained cells. The percentages of CD4 and CD8 cells in gated lymphocytes were measured by direct subtraction of red and green fluorescence histograms of cells stained with the mixture of irrelevant antibodies conjugated to FITC and PE from those stained with a mixutre of anti-CD8-PE and anti-CD4-FITC.
Measurement of Interleukin-Z and Interferon-s Concentrations in Cell Culture Supernatants IL-2 concentrations in culture supernatants were measured using a competitive radioimmunoassay (Amersham PLC). The IL-2 standard employed was human recombinant IL-2 with an amino-terminal methionine residue in addition to the native 133 amino acid residues of specific activity 3.0 x 106 units/ mg. Sensitivity of the assay, defined as the amount of IL-2 required to reduce zero binding by 2 SD, was 0.3 ng/ml. The coefficients of interassay variation using standards of nominal concentration 1.25and 10ng/ml were 21.6 and 19.0%, respectively. The intraassay coefficient of variation wasestimated as 5.7%. All the samples from anyone patient were always analyzed in the same assay so that within-patient comparisons were subject only to intraassay variability. Interferon-y was measured in culture supernatants using an ELISA technique sup-
1028 60
50
40
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CORRIGAN, BROWN, BARNES. TSAI, FREW, AND KAY
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