The Effect of T-Cell Depletion on Enhanced Basophil Histamine Release after In Vitro Incubation with Live Influenza A Vrrus M. A. Huftel, c. A. Swensen, w. R. Borcherding, E. C. Dick, R. Hong, H. Kita, G. J. Gleich, and W. W. Busse
Departments of Medicine, Pediatrics, and Preventive Medicine, University of Wisconsin Medical School, Madison, Wisconsin, and Department of Immunology, Mayo Clinic, Rochester, Minnesota
A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C 4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P < 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-y. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.
Viral upper respiratory infections exacerbate asthma in some patients (1-5). Several mechanisms have been proposed to explain these wheezing episodes including the production of virus-specific IgE antibodies (6-8), diminished {j-adrenergic function (9-12), airway epithelial damage (13, 14), enhanced cholinergic-dependent bronchospasm (15), the development of late asthmatic reactions (16), and changes in mediator release (17, 18). We have reported that patients undergoing allergen bronchoprovocation during a rhinovirus upper respiratory infection are more likely to develop late asthmatic reactions (16). In skin, nasal mucosa, and lower airway models, the profile of cellular influx and mediator release during late responses to allergen suggests that basophils may play a prominent role in this reaction (19-22). Thus, factors that promote basophil (Received in original form March 12, 1992 and in revised form April 24, 1992) Address correspondence to: William W. Busse, M.D., H6/367, Clinical Science Center, 600 Highland Ave., Madison, WI 53792. • Abbreviations: antigen E, AgE; defined calf serum, DCS; 50% egg infectious doses/mI, EID so; granulocyte/macrophage colony-stimulating factor, GM-CSF; Hanks' balanced salt solution, HBSS; histamine release, HR; interferon, IFN; interleukin, IL; leukotriene C4 , L1C4 ; peripheral blood mononuclear cell(s), PBMC; Pipes buffer with Ca2+ and Mg2 + , PCM. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp, 434-440,1992
migration to the lung or enhance its mediator-release activity could influence either the likelihood and/or intensity of a pulmonary late-phase reaction. In earlier experiments, we found that peripheral blood mononuclear cell (PBMC) suspensions (which contain basophils) have increased antigen-stimulated histamine release (HR) when evaluated during rhinovirus infections; further, the rhinovirus infection appeared to promote the development of late asthmatic reactions (16). Moreover, in vitro incubation of PBMC with respiratory viruses results in enhanced leukocyte HR (17, 18). Consequently, incubation of leukocytes with respiratory viruses has similarities to events occurring in vivo and may thus provide an in vitro model to further study the cellular interactions that lead to enhanced mediator release during viral infections. The objectives of this study were principally twofold: first, to determine the effect of virus incubation on the generation and release of leukotriene C4 (LTC4) , a nonpreformed cell mediator, and second, to observe the effect of T-cell depletion before virus incubation on basophil HR.
Materials and Methods Subjects Twelve subjects (eight men and four women), ranging in age from 22 to 54 yr (mean, 33 yr), were selected for study. Each
Huftel, Swenson, Borcherding et al.: Effect of T-Cell Depletion on Basophil Histamine Release
subject had symptoms consistent with seasonal allergic rhinitis and had a positive prick skin test to mixed giant and short ragweed (1:20 wt/vol; Greer Laboratories, Lenoir, NC). Further, all subjects had leukocytes that released histamine when incubated with ragweed antigen E (AgE) in vitro. Finally, each subject was studied outside of the well-defined Wisconsin ragweed season (August through September). None required daily medication nor were taking antihistamines at the time of the study. Reagents The Pipes buffer (Sigma Chemical Co., St. Louis, MO), which was used for basophil HR experiments, contained 1 mM of Ca2+ and Mg2+ (PCM). Hanks' balanced salt solution (HBSS), without phenol red, was obtained from GIBCO (Grand Island, NY). Saline (0.85%) buffered with Tris was made by adding 8.5 g NaCI and 2.06 g Tris-preset crystals (Sigma) to 1 liter double-distilled deionized water. Leukocyte Separation Procedure PBMC with basophils were isolated as previously described (17). Briefly, peripheral blood from ragweed-allergic subjects was anticoagulated with EDTA (0.5 ml of 2.7% EDTA/10 ml of blood) and diluted 1:1 with Tris-buffered saline before separation by density gradient centrifugation on a Ficoll-Hypaque cushion. The mononuclear cell band, containing lymphocytes, monocytes, and basophils, was removed and washed twice with HBSS. Platelets had been removed by low-speed centrifugation. The remaining PBMC were resuspended in RPMI 1640 at a concentration of 3 x 1Q6 cells/ml for subsequent incubations with influenza A virus. Influenza A The influenza A used for in vitro incubation was a live H3N2 recombinant of A/Eng/42172 and A/PR8/34. Its infectivity was 106,75 50 % egg infectious doses/ml (EID5o) ; the hemagglutinin titer was 1:256. All virus-leukocyte incubations were performed in RPMI 1640 (GmCO), supplemented with 4 mM L-glutamine, 100 D/ml penicillin, and 100 /-tg/ml streptomycin. The following experimental design was used for incubation of influenza A or appropriate control (allantoic fluid) with leukocytes to determine the effect on basophil HR. Influenza A or allantoic fluid was added to the leukocyte suspension at a concentration equivalent to EID 50 per cell. All incubations were conducted at 37 0 C in 5 % CO 2 atmosphere. After a 2-h incubation, the leukocytes were collected, resuspended in fresh RPMI with 10% autologous serum, and incubated for an additional 22 h (37 0 C). After this incubation, the cells were collected, washed, and resuspended in PCM before measuring basophil histamine release to ragweed AgE. Leukocyte HR Leukocyte suspensions containing basophils were added to 12 x 75 mm plastic tubes (Sarstedt, Princeton, NJ) in 0.5-ml replicate samples. After temperature equilibration (37 0 C for 10 min), varying concentrations (1 x 10- 4 to 1 X 10- 1 /-tg/ml) ragweed AgE (Research Resource Branch of the National Institutes of Health, Bethesda, MD) were added. The suspensions were incubated in a shaking water bath for 20
435
min (37 0 C). The reaction was terminated by immersion of the leukocyte suspension into an ice-water bath for 10 min followed by centrifugation (1.5 min in a Serofuge; Clay Adams Division, Becton-Dickinson, Parsippany, NJ). The supernatants were then recovered for histamine analysis. Total leukocyte histamine content was determined by the addition of 3 % perchloric acid to the cell suspension. Histamine analysis was performed by an automated fluorometric method that uses the organic extraction of histamine and its linkage to o-phthal-aldehyde (23, 24). This method is sensitive to 1 ng/ml. The percent HR was calculated by the following formula: %HR = 100 X (experimental supernatant HR - spontaneous HR)/(total HR - spontaneous HR). LTC4 Determination Assay Leukocyte suspensions (5 X 106 cells/ml) were added to 12 X 75 mm plastic tubes in 0.5-ml triplicate samples. Equilibration, ragweed AgE challenge, and incubation were performed as described above. The reaction was terminated by the addition of 0.5 ml LTC4 assay buffer (0.9% NaCl, 0.1% gelatin, 0.01 M EDTA, 0.1% sodium azide in 10 mM phosphate buffer) and placement of the leukocyte suspensions in an ice-water bath for 10 min. One of each of the triplicate samples was used for LTC4 determination by a radioimmunoassay method (LTC4 PH] RIA Kit; E. I. Du Pont de Remours and Co., Boston, MA). This assay is sensitive to 0.2 ng/ml. The remaining duplicate samples were used for histamine analysis. T-Cell Depletion A T-cell-depleted suspension of leukocytes was achieved by a negative selection technique utilizing a monoclonal antibody directed against the pan T-cell marker, CD3 (produced by CRL 8001; American Type Culture Collection, Rockville, MD). PBMC were isolated as described above and divided into two aliquots, one of which underwent T-cell depletion before incubation with influenza A virus or control media. After the second wash and centrifugation, the cell pellet, which was to be depleted of T cells, was resuspended in tissue culture media containing mouse IgG2 anti-CD3 monoclonal antibody and incubated for 30 min at 4 0 C. After incubation, the cells were pelleted and resuspended in RPMI 1640 containing 10% fetal calf serum. Goat anti-mouse IgGcoated magnetic beads (Dynabeads; Dynal Inc., Great Neck, NY) were then added to the monoclonal antibodycoated T cells at a concentration of 10 beads to one lymphocyte and rotated (Rotc-Torque; Cole-Parmer Instrument Co., Chicago, IL) at 4 0 C for 1 h. The suspension was then placed in a magnetic field, and the T-cell-depleted supernatant was recovered. Flow cytometry was performed using anti-CD3 to quantitate the number of T cells remaining in the depleted mixture. The T-cell-depleted and nontreated leukocyte suspensions were then incubated with either influenza A virus or control media as described above. After incubation, ragweed AgE was added to stimulate HR as described earlier. Detection of Influenza A-generated Cytokine A number of cytokines have been shown to promote eosinophil survival in vitro (25). This function was used to detect
436
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
the presence of cytokines in supernatants from an influenza A virus- or placebo-treated leukocyte in culture. Eosinophil Purification Eosinophils were purified from peripheral blood as previously described (26). Briefly, heparinized (10 U/ml) venous blood was obtained from normal volunteers or patients with mild asthma or hay fever and sedimented with 6 % dextran in 0.9% NaCI (Gentran 70; Travenol Laboratories, Deerfield, IL) at 5:1 (vol/vol) ratio for 60 min at 37 0 C. The buffy coat was collected and washed twice in Pipes buffer (25 mM Pipes, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose) (Sigma). The cells were suspended in 2.4 ml of PercolI (Sigma), density 1.070 g/ml, with 5% heat-inactivated defined calf serum (DCS) (Hyclone Laboratories, Logan, UT) and overlayed on a discontinuous Percoll gradient consisting of the following densities (g/ml): 1.080, 1.085, 1.090, 1.100, and 1.120. The osmolarity of PercoIl ranged from 290 to 315 mOsm/kg and the pH was 7.4. Cells were centrifuged through the gradient at 1,500 x g in a JA-20fixed angle rotor on a Beckman J2-21 centrifuge at 4 0 C for 45 min. Fractions were collected and eosinophil numbers were determined utilizing Randolph's stain. Eosinophil-rich fractions were pooled, washed twice in the Pipes buffer with 1% DCS, and used immediately. The eosinophil preparations were> 80% pure and> 98 % viable, as determined using Randolph's stain and by trypan blue dye exclusion, respectively. The few contaminating cells were neutrophils. There was no contamination of lymphocytes or monocytes. Eosinophil Survival Assay Purified eosinophils were washed by Hybri-Care medium containing gentamicin, 50 p.g/ml, and 0.1% human serum albumin, and resuspended in this medium supplemented with 10% DCS at 2.5 x l()'i cells/ml, A 100-p.l aliquot of the cell suspension was incubated with 100 p.l of medium containing the supernatant from influenza A virus- or placebotreated leukocyte (25 % final concentration) for 4 days at 37 0 C and 5 % CO2 in 96-well, flat-bottom tissue culture plates. The supernatants were pretreated with anti-IFN-I', anti-IL-3, anti-IL-5, or anti-GM-CSF antibody for 1 h at 4 0 C. All the antibodies were rat monoclonal antibodies that
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are specific for their respective cytokine with no crossreactivity with other cytokines (27). After 4 days of incubation, 120 p.l of supernate was removed from each well and 10 p.l of fluorescein diacetate (28) (0.2 mg/ml in phosphatebuffered saline) was added to the cell suspension. After a 30min incubation at 4 0 C, 10 Jotl of propidium iodide (29) (2 mg/ml in Hybri-Care medium) was added. After an additional 5 min at 4 0 C, the number of viable cells (green fluorescence) and dead cells (red fluorescence) were counted using a hemocytometer and an epifluorescent microscope. Data were calculated using the number of the viable cells divided by the total number of cells. Inhibition of cell survival after exposure of supernatant fluids to anticytokine monoclonal antibodies was determined. There was no difference in total number of cells before and after culture. All experiments were carried out in duplicate. Statistical Analysis A two-tailed paired Student's t test (Abstat Release 4.13; Anderson-Bell, Parker, CO) was used to compare basophil HR and LTC4 release between the virus-treated and control-treated groups at specific concentrations of ragweed AgE. A P value < 0.05 was considered significant.
Results Effect of Influenza A Virus on Ragweed AgE-induced HR Control- and virus-treated leukocytes were incubated with ragweed AgE (10-4, 3 x 10-4, 10- 3 , 3 X 10- 3 , 10- 2 , 3 X 10- 2 , and 10- 1 p.g/ml); histamine released into the supernatant was measured by an automated fluorometric method. In all experiments, cell viability was maintained at > 90 %, as indicated by the exclusion of trypan blue dye. After incubation with influenza A virus, spontaneous HR was greater (P = 0.003) in the virus-treated (12.3 ± 1.1%)versus control (6.7 ± 0.9%). This value was subtracted in the calculation of antigen-induced HR. Ragweed AgE-stimulated HR was enhanced at all antigen concentrations, significantly so at the lower concentrations of antigen (l x 10- 4 to 3 X 10- 3 p.glml) (Figure 1). Maximal HR was not significantly enhanced; however, the concentration of antigen required for
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Figure 1. The effect of an in vitro influenza A virus incubation on leukocyte histamine release to ragweed antigen E (AgE). Values are mean ± SEM, n = 9.
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Departments of Medicine, Pediatrics, and Preventive Medicine, University of Wisconsin Medical School, Madison, Wisconsin, and Department of Immunology, Mayo Clinic, Rochester, Minnesota
A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C 4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P < 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-y. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.
Viral upper respiratory infections exacerbate asthma in some patients (1-5). Several mechanisms have been proposed to explain these wheezing episodes including the production of virus-specific IgE antibodies (6-8), diminished {j-adrenergic function (9-12), airway epithelial damage (13, 14), enhanced cholinergic-dependent bronchospasm (15), the development of late asthmatic reactions (16), and changes in mediator release (17, 18). We have reported that patients undergoing allergen bronchoprovocation during a rhinovirus upper respiratory infection are more likely to develop late asthmatic reactions (16). In skin, nasal mucosa, and lower airway models, the profile of cellular influx and mediator release during late responses to allergen suggests that basophils may play a prominent role in this reaction (19-22). Thus, factors that promote basophil (Received in original form March 12, 1992 and in revised form April 24, 1992) Address correspondence to: William W. Busse, M.D., H6/367, Clinical Science Center, 600 Highland Ave., Madison, WI 53792. • Abbreviations: antigen E, AgE; defined calf serum, DCS; 50% egg infectious doses/mI, EID so; granulocyte/macrophage colony-stimulating factor, GM-CSF; Hanks' balanced salt solution, HBSS; histamine release, HR; interferon, IFN; interleukin, IL; leukotriene C4 , L1C4 ; peripheral blood mononuclear cell(s), PBMC; Pipes buffer with Ca2+ and Mg2 + , PCM. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp, 434-440,1992
migration to the lung or enhance its mediator-release activity could influence either the likelihood and/or intensity of a pulmonary late-phase reaction. In earlier experiments, we found that peripheral blood mononuclear cell (PBMC) suspensions (which contain basophils) have increased antigen-stimulated histamine release (HR) when evaluated during rhinovirus infections; further, the rhinovirus infection appeared to promote the development of late asthmatic reactions (16). Moreover, in vitro incubation of PBMC with respiratory viruses results in enhanced leukocyte HR (17, 18). Consequently, incubation of leukocytes with respiratory viruses has similarities to events occurring in vivo and may thus provide an in vitro model to further study the cellular interactions that lead to enhanced mediator release during viral infections. The objectives of this study were principally twofold: first, to determine the effect of virus incubation on the generation and release of leukotriene C4 (LTC4) , a nonpreformed cell mediator, and second, to observe the effect of T-cell depletion before virus incubation on basophil HR.
Materials and Methods Subjects Twelve subjects (eight men and four women), ranging in age from 22 to 54 yr (mean, 33 yr), were selected for study. Each
Huftel, Swenson, Borcherding et al.: Effect of T-Cell Depletion on Basophil Histamine Release
subject had symptoms consistent with seasonal allergic rhinitis and had a positive prick skin test to mixed giant and short ragweed (1:20 wt/vol; Greer Laboratories, Lenoir, NC). Further, all subjects had leukocytes that released histamine when incubated with ragweed antigen E (AgE) in vitro. Finally, each subject was studied outside of the well-defined Wisconsin ragweed season (August through September). None required daily medication nor were taking antihistamines at the time of the study. Reagents The Pipes buffer (Sigma Chemical Co., St. Louis, MO), which was used for basophil HR experiments, contained 1 mM of Ca2+ and Mg2+ (PCM). Hanks' balanced salt solution (HBSS), without phenol red, was obtained from GIBCO (Grand Island, NY). Saline (0.85%) buffered with Tris was made by adding 8.5 g NaCI and 2.06 g Tris-preset crystals (Sigma) to 1 liter double-distilled deionized water. Leukocyte Separation Procedure PBMC with basophils were isolated as previously described (17). Briefly, peripheral blood from ragweed-allergic subjects was anticoagulated with EDTA (0.5 ml of 2.7% EDTA/10 ml of blood) and diluted 1:1 with Tris-buffered saline before separation by density gradient centrifugation on a Ficoll-Hypaque cushion. The mononuclear cell band, containing lymphocytes, monocytes, and basophils, was removed and washed twice with HBSS. Platelets had been removed by low-speed centrifugation. The remaining PBMC were resuspended in RPMI 1640 at a concentration of 3 x 1Q6 cells/ml for subsequent incubations with influenza A virus. Influenza A The influenza A used for in vitro incubation was a live H3N2 recombinant of A/Eng/42172 and A/PR8/34. Its infectivity was 106,75 50 % egg infectious doses/ml (EID5o) ; the hemagglutinin titer was 1:256. All virus-leukocyte incubations were performed in RPMI 1640 (GmCO), supplemented with 4 mM L-glutamine, 100 D/ml penicillin, and 100 /-tg/ml streptomycin. The following experimental design was used for incubation of influenza A or appropriate control (allantoic fluid) with leukocytes to determine the effect on basophil HR. Influenza A or allantoic fluid was added to the leukocyte suspension at a concentration equivalent to EID 50 per cell. All incubations were conducted at 37 0 C in 5 % CO 2 atmosphere. After a 2-h incubation, the leukocytes were collected, resuspended in fresh RPMI with 10% autologous serum, and incubated for an additional 22 h (37 0 C). After this incubation, the cells were collected, washed, and resuspended in PCM before measuring basophil histamine release to ragweed AgE. Leukocyte HR Leukocyte suspensions containing basophils were added to 12 x 75 mm plastic tubes (Sarstedt, Princeton, NJ) in 0.5-ml replicate samples. After temperature equilibration (37 0 C for 10 min), varying concentrations (1 x 10- 4 to 1 X 10- 1 /-tg/ml) ragweed AgE (Research Resource Branch of the National Institutes of Health, Bethesda, MD) were added. The suspensions were incubated in a shaking water bath for 20
435
min (37 0 C). The reaction was terminated by immersion of the leukocyte suspension into an ice-water bath for 10 min followed by centrifugation (1.5 min in a Serofuge; Clay Adams Division, Becton-Dickinson, Parsippany, NJ). The supernatants were then recovered for histamine analysis. Total leukocyte histamine content was determined by the addition of 3 % perchloric acid to the cell suspension. Histamine analysis was performed by an automated fluorometric method that uses the organic extraction of histamine and its linkage to o-phthal-aldehyde (23, 24). This method is sensitive to 1 ng/ml. The percent HR was calculated by the following formula: %HR = 100 X (experimental supernatant HR - spontaneous HR)/(total HR - spontaneous HR). LTC4 Determination Assay Leukocyte suspensions (5 X 106 cells/ml) were added to 12 X 75 mm plastic tubes in 0.5-ml triplicate samples. Equilibration, ragweed AgE challenge, and incubation were performed as described above. The reaction was terminated by the addition of 0.5 ml LTC4 assay buffer (0.9% NaCl, 0.1% gelatin, 0.01 M EDTA, 0.1% sodium azide in 10 mM phosphate buffer) and placement of the leukocyte suspensions in an ice-water bath for 10 min. One of each of the triplicate samples was used for LTC4 determination by a radioimmunoassay method (LTC4 PH] RIA Kit; E. I. Du Pont de Remours and Co., Boston, MA). This assay is sensitive to 0.2 ng/ml. The remaining duplicate samples were used for histamine analysis. T-Cell Depletion A T-cell-depleted suspension of leukocytes was achieved by a negative selection technique utilizing a monoclonal antibody directed against the pan T-cell marker, CD3 (produced by CRL 8001; American Type Culture Collection, Rockville, MD). PBMC were isolated as described above and divided into two aliquots, one of which underwent T-cell depletion before incubation with influenza A virus or control media. After the second wash and centrifugation, the cell pellet, which was to be depleted of T cells, was resuspended in tissue culture media containing mouse IgG2 anti-CD3 monoclonal antibody and incubated for 30 min at 4 0 C. After incubation, the cells were pelleted and resuspended in RPMI 1640 containing 10% fetal calf serum. Goat anti-mouse IgGcoated magnetic beads (Dynabeads; Dynal Inc., Great Neck, NY) were then added to the monoclonal antibodycoated T cells at a concentration of 10 beads to one lymphocyte and rotated (Rotc-Torque; Cole-Parmer Instrument Co., Chicago, IL) at 4 0 C for 1 h. The suspension was then placed in a magnetic field, and the T-cell-depleted supernatant was recovered. Flow cytometry was performed using anti-CD3 to quantitate the number of T cells remaining in the depleted mixture. The T-cell-depleted and nontreated leukocyte suspensions were then incubated with either influenza A virus or control media as described above. After incubation, ragweed AgE was added to stimulate HR as described earlier. Detection of Influenza A-generated Cytokine A number of cytokines have been shown to promote eosinophil survival in vitro (25). This function was used to detect
436
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
the presence of cytokines in supernatants from an influenza A virus- or placebo-treated leukocyte in culture. Eosinophil Purification Eosinophils were purified from peripheral blood as previously described (26). Briefly, heparinized (10 U/ml) venous blood was obtained from normal volunteers or patients with mild asthma or hay fever and sedimented with 6 % dextran in 0.9% NaCI (Gentran 70; Travenol Laboratories, Deerfield, IL) at 5:1 (vol/vol) ratio for 60 min at 37 0 C. The buffy coat was collected and washed twice in Pipes buffer (25 mM Pipes, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose) (Sigma). The cells were suspended in 2.4 ml of PercolI (Sigma), density 1.070 g/ml, with 5% heat-inactivated defined calf serum (DCS) (Hyclone Laboratories, Logan, UT) and overlayed on a discontinuous Percoll gradient consisting of the following densities (g/ml): 1.080, 1.085, 1.090, 1.100, and 1.120. The osmolarity of PercoIl ranged from 290 to 315 mOsm/kg and the pH was 7.4. Cells were centrifuged through the gradient at 1,500 x g in a JA-20fixed angle rotor on a Beckman J2-21 centrifuge at 4 0 C for 45 min. Fractions were collected and eosinophil numbers were determined utilizing Randolph's stain. Eosinophil-rich fractions were pooled, washed twice in the Pipes buffer with 1% DCS, and used immediately. The eosinophil preparations were> 80% pure and> 98 % viable, as determined using Randolph's stain and by trypan blue dye exclusion, respectively. The few contaminating cells were neutrophils. There was no contamination of lymphocytes or monocytes. Eosinophil Survival Assay Purified eosinophils were washed by Hybri-Care medium containing gentamicin, 50 p.g/ml, and 0.1% human serum albumin, and resuspended in this medium supplemented with 10% DCS at 2.5 x l()'i cells/ml, A 100-p.l aliquot of the cell suspension was incubated with 100 p.l of medium containing the supernatant from influenza A virus- or placebotreated leukocyte (25 % final concentration) for 4 days at 37 0 C and 5 % CO2 in 96-well, flat-bottom tissue culture plates. The supernatants were pretreated with anti-IFN-I', anti-IL-3, anti-IL-5, or anti-GM-CSF antibody for 1 h at 4 0 C. All the antibodies were rat monoclonal antibodies that
-e-
- 0 - Control Q)
are specific for their respective cytokine with no crossreactivity with other cytokines (27). After 4 days of incubation, 120 p.l of supernate was removed from each well and 10 p.l of fluorescein diacetate (28) (0.2 mg/ml in phosphatebuffered saline) was added to the cell suspension. After a 30min incubation at 4 0 C, 10 Jotl of propidium iodide (29) (2 mg/ml in Hybri-Care medium) was added. After an additional 5 min at 4 0 C, the number of viable cells (green fluorescence) and dead cells (red fluorescence) were counted using a hemocytometer and an epifluorescent microscope. Data were calculated using the number of the viable cells divided by the total number of cells. Inhibition of cell survival after exposure of supernatant fluids to anticytokine monoclonal antibodies was determined. There was no difference in total number of cells before and after culture. All experiments were carried out in duplicate. Statistical Analysis A two-tailed paired Student's t test (Abstat Release 4.13; Anderson-Bell, Parker, CO) was used to compare basophil HR and LTC4 release between the virus-treated and control-treated groups at specific concentrations of ragweed AgE. A P value < 0.05 was considered significant.
Results Effect of Influenza A Virus on Ragweed AgE-induced HR Control- and virus-treated leukocytes were incubated with ragweed AgE (10-4, 3 x 10-4, 10- 3 , 3 X 10- 3 , 10- 2 , 3 X 10- 2 , and 10- 1 p.g/ml); histamine released into the supernatant was measured by an automated fluorometric method. In all experiments, cell viability was maintained at > 90 %, as indicated by the exclusion of trypan blue dye. After incubation with influenza A virus, spontaneous HR was greater (P = 0.003) in the virus-treated (12.3 ± 1.1%)versus control (6.7 ± 0.9%). This value was subtracted in the calculation of antigen-induced HR. Ragweed AgE-stimulated HR was enhanced at all antigen concentrations, significantly so at the lower concentrations of antigen (l x 10- 4 to 3 X 10- 3 p.glml) (Figure 1). Maximal HR was not significantly enhanced; however, the concentration of antigen required for
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Paired t-test
*
P < 0.005
+ p < 0.05
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Figure 1. The effect of an in vitro influenza A virus incubation on leukocyte histamine release to ragweed antigen E (AgE). Values are mean ± SEM, n = 9.
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