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821 Reduced interferon-gamma (IFN-γ) production in response to respiratory viruses in atopic asthmatic adults
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 1, PART 2
vated HSVEC was more efficient at inducing CXCR3 intemalization than HSVEC supematants. I-TAC (1000 rig/ml) induced a 94% decrease in CXCR3 MF, while the same concentration of IP- IO and Mig only induced a 43% and 33% decrease, respectively. Our data suggest that these chemokines are more effective at inducing CXCR3 internalization when bound to endothelial cells than when in solution. Immunofhtorescence confocal staining confirmed that CXCR3 underwent internalization into transfertin receptor containing vacuoles following I-TAC treatment. T cells pretreatement with the inhibitors genestein, wortmannin, staurosporin and pertussis toxin, all known to inhibit chemokine induced chemotaxis or calcium flux responses had no effect on I-TAC-induced internalization. Furthermore, treatment of HSVEC with heparin or with a combination of heparitinase I, III, and chondroitinase did not significantly affect I-TAC-induced CXCR3 internalization. In contrast, CXCR3 internalization by activated HSVEC supematants could be inhibited by more than 90% using anti-I-TAC neutralizing antibodies, indicating that I-TAC is the principal factor responsible for the rapid and profound CXCR3 sequestration during T-cell interaction with IFNg-activated endothelium. In conclusion, our study shows that while IFNg-activated endothelial cells express the 3 known ligands for CXCR3, I-TAC is principally responsible for inducing CXCR3 internalization. These studies also reveal that these 3 IFNginducible chemokines differentially signal CXCR3, leading to differences in surface expression of CXCR3. 820
Estimation of the Frequency of Infectable Cells in Human Bronchial Epithelium by Rbinovirus (RV) AC Mosser, LJ Burchell, R Brockman-Schneider; WW Busse, JE Gem Departments of Medicine and Pediatrics, University of Wisconsin Medical School, Madison, WI Human RV infections are characteristic upper airway infections, yet they provoke clinically significant lower airway symptoms for asthma patients. Therefore, it is of interest to determine if lower airway epithelial cells are infectable by RV. Previous work has shown that during an RV-induced cold, there is no gross loss of cells from the upper airway, and infection appears to be confined to a few cells. Although viral RNA has been detected in BAL samples from individuals infected with RV16, the frequency of infection has not been determined. Therefore, to quantitate the infectivity of RV in lower airway cells, we infected primary and low-passage bronchial epithelial (BE) cells, and analyzed these cells using two techniques; infectious center assay and immunohistochemistry (IHC). For the infectious center assays, low passage BE cells or HeLa cells (positive control) were infected with varying amounts of RV16. After 6 hr (HeLa cells) or 24 hr (BE cells), the cells were trypsinized and plated over a confluent monolayer of HeLa cells, the indicator cells; if the infected cells release virus, then a plaque should develop at the site. Analysis of plaque formation showed that all of the HeLa cells could be infected, as predicted. In contrast, even when large multiplicities of infecting virus were used, only about 3% of the BE cells were infected (Figure 1). a
Infedability
of Cells !o RVl6
S279
For IHC, low passage BE cell monolayers grown on microscope slides were inoculated with RV16, fixed and stained with a monoclonal antibody (R16-7) specific for this virus. Using this technique, approximately 1% of the BE cells stained positive for RV 16. When considered together, these results suggest that, even when large amounts of input virus were used, RV infected only a small subset of BE cells, and this pattern was remarkably similar to previous reports of RV growth in upper airway cells. Further definition of these RV-susceptible cells is likely to yield important insights into the pathogenesis of RV infection and virus-induced asthma. 821
Reduced Interferon-Gamma (IFN-$ Production in Response to Respiratory Viruses in Atopic Asthmatic Adults M Moss, K Anklam, L Rosenthal, L Mikus, S Elletman. L Zing, M Kosorok P Schult, K Roberg, K Carlson-Dakes, K Adler; J Gem, R Lemanske University of Wisconsin Hospital and Clinics, Madison, WI, In a rodent model (F344 versus Brown Norway rats) of virusinduced airway dysfunction, decreased levels of IFN-y appear to contribute to the development of a chronic asthma-like phenotype (AJRCCM 160:705, 1999). As a result of these observations, we are conducting a four-year prospective study [Childhood Origins of Asthma (COAST) project] to investigate the relationships among a genetic predisposition for atopy (cytokine response patterns), early respiratory viral infections in children, and the development of childhood asthma. To evaluate familial patterns of cytokine responses, as well as differences between parents with and without asthma, peripheral blood samples from parents of the children in the study were obtained for cytokine analyses. Since enrollment requires that at least one parent have allergies (2 1 positive aeroallergen skin test) and/or asthma, we evaluated whether a difference in stimulated peripheral blood mononuclear cell (PBMC) production of IFN-ybetween allergic adults with and without asthma could be demonstrated. PBMCs were incubated with either phytohemagglutinin (PHA), interleukin-12 (IL-12). Staphylococcus aureus cells (SAC), respiratory syncytial virus (RSV), tetanus toxoid (TT). ovomucoid (OVO), rhinovirus (RV). phorbal myristate acetateiionomycin (PMA), or medium alone. Supematant fluids were collected at I day and 5 days, and IFN-y was quantified using a sandwich ELISA (Pharmingen). Of 32 allergic parents evaluated thus far, the following results have been noted (Table 1).
IFN- PRODUCTION
(PG/ML) CONTROL 1 DAY
ASTHMA (N=l7) NO ASTHMA (N=lS) P-VALUE*
12 d NS
PHA
PMA
IL-12
SAC
371 484 NS
477 500 NS
22 57 d.01
145 257 cQ.05
CONTROL 5 DAY RSV 12 45 NS
RV
332 255 52.5 430 ccl.05 cu.05
* NS = not significant (p-value >0.05). Mitogen (PHA and PMA)-induced IFN-y was similar in individuals with or without asthma. However, in comparison to nonasthmatic allergic individuals, IFN-y production by PBMC obtained from allergic adults with asthma was significantly decreased in response to IL-12, SAC, and two viruses known to be associated with asthma exacerbations, RSV and RV. These data indicate IFN-ydysregulation in response to specific cytokine and/or antigen stimulation may be an important factor associated with the asthmatic phenotype. Moreover it is possible that diminished IFNy secretion during viral infections may contribute to the pathogenesis of viral-induced asthma exacerbations. Supported by NIH grant I-IL98005
J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 1, PART 2
vated HSVEC was more efficient at inducing CXCR3 intemalization than HSVEC supematants. I-TAC (1000 rig/ml) induced a 94% decrease in CXCR3 MF, while the same concentration of IP- IO and Mig only induced a 43% and 33% decrease, respectively. Our data suggest that these chemokines are more effective at inducing CXCR3 internalization when bound to endothelial cells than when in solution. Immunofhtorescence confocal staining confirmed that CXCR3 underwent internalization into transfertin receptor containing vacuoles following I-TAC treatment. T cells pretreatement with the inhibitors genestein, wortmannin, staurosporin and pertussis toxin, all known to inhibit chemokine induced chemotaxis or calcium flux responses had no effect on I-TAC-induced internalization. Furthermore, treatment of HSVEC with heparin or with a combination of heparitinase I, III, and chondroitinase did not significantly affect I-TAC-induced CXCR3 internalization. In contrast, CXCR3 internalization by activated HSVEC supematants could be inhibited by more than 90% using anti-I-TAC neutralizing antibodies, indicating that I-TAC is the principal factor responsible for the rapid and profound CXCR3 sequestration during T-cell interaction with IFNg-activated endothelium. In conclusion, our study shows that while IFNg-activated endothelial cells express the 3 known ligands for CXCR3, I-TAC is principally responsible for inducing CXCR3 internalization. These studies also reveal that these 3 IFNginducible chemokines differentially signal CXCR3, leading to differences in surface expression of CXCR3. 820
Estimation of the Frequency of Infectable Cells in Human Bronchial Epithelium by Rbinovirus (RV) AC Mosser, LJ Burchell, R Brockman-Schneider; WW Busse, JE Gem Departments of Medicine and Pediatrics, University of Wisconsin Medical School, Madison, WI Human RV infections are characteristic upper airway infections, yet they provoke clinically significant lower airway symptoms for asthma patients. Therefore, it is of interest to determine if lower airway epithelial cells are infectable by RV. Previous work has shown that during an RV-induced cold, there is no gross loss of cells from the upper airway, and infection appears to be confined to a few cells. Although viral RNA has been detected in BAL samples from individuals infected with RV16, the frequency of infection has not been determined. Therefore, to quantitate the infectivity of RV in lower airway cells, we infected primary and low-passage bronchial epithelial (BE) cells, and analyzed these cells using two techniques; infectious center assay and immunohistochemistry (IHC). For the infectious center assays, low passage BE cells or HeLa cells (positive control) were infected with varying amounts of RV16. After 6 hr (HeLa cells) or 24 hr (BE cells), the cells were trypsinized and plated over a confluent monolayer of HeLa cells, the indicator cells; if the infected cells release virus, then a plaque should develop at the site. Analysis of plaque formation showed that all of the HeLa cells could be infected, as predicted. In contrast, even when large multiplicities of infecting virus were used, only about 3% of the BE cells were infected (Figure 1). a
Infedability
of Cells !o RVl6
S279
For IHC, low passage BE cell monolayers grown on microscope slides were inoculated with RV16, fixed and stained with a monoclonal antibody (R16-7) specific for this virus. Using this technique, approximately 1% of the BE cells stained positive for RV 16. When considered together, these results suggest that, even when large amounts of input virus were used, RV infected only a small subset of BE cells, and this pattern was remarkably similar to previous reports of RV growth in upper airway cells. Further definition of these RV-susceptible cells is likely to yield important insights into the pathogenesis of RV infection and virus-induced asthma. 821
Reduced Interferon-Gamma (IFN-$ Production in Response to Respiratory Viruses in Atopic Asthmatic Adults M Moss, K Anklam, L Rosenthal, L Mikus, S Elletman. L Zing, M Kosorok P Schult, K Roberg, K Carlson-Dakes, K Adler; J Gem, R Lemanske University of Wisconsin Hospital and Clinics, Madison, WI, In a rodent model (F344 versus Brown Norway rats) of virusinduced airway dysfunction, decreased levels of IFN-y appear to contribute to the development of a chronic asthma-like phenotype (AJRCCM 160:705, 1999). As a result of these observations, we are conducting a four-year prospective study [Childhood Origins of Asthma (COAST) project] to investigate the relationships among a genetic predisposition for atopy (cytokine response patterns), early respiratory viral infections in children, and the development of childhood asthma. To evaluate familial patterns of cytokine responses, as well as differences between parents with and without asthma, peripheral blood samples from parents of the children in the study were obtained for cytokine analyses. Since enrollment requires that at least one parent have allergies (2 1 positive aeroallergen skin test) and/or asthma, we evaluated whether a difference in stimulated peripheral blood mononuclear cell (PBMC) production of IFN-ybetween allergic adults with and without asthma could be demonstrated. PBMCs were incubated with either phytohemagglutinin (PHA), interleukin-12 (IL-12). Staphylococcus aureus cells (SAC), respiratory syncytial virus (RSV), tetanus toxoid (TT). ovomucoid (OVO), rhinovirus (RV). phorbal myristate acetateiionomycin (PMA), or medium alone. Supematant fluids were collected at I day and 5 days, and IFN-y was quantified using a sandwich ELISA (Pharmingen). Of 32 allergic parents evaluated thus far, the following results have been noted (Table 1).
IFN- PRODUCTION
(PG/ML) CONTROL 1 DAY
ASTHMA (N=l7) NO ASTHMA (N=lS) P-VALUE*
12 d NS
PHA
PMA
IL-12
SAC
371 484 NS
477 500 NS
22 57 d.01
145 257 cQ.05
CONTROL 5 DAY RSV 12 45 NS
RV
332 255 52.5 430 ccl.05 cu.05
* NS = not significant (p-value >0.05). Mitogen (PHA and PMA)-induced IFN-y was similar in individuals with or without asthma. However, in comparison to nonasthmatic allergic individuals, IFN-y production by PBMC obtained from allergic adults with asthma was significantly decreased in response to IL-12, SAC, and two viruses known to be associated with asthma exacerbations, RSV and RV. These data indicate IFN-ydysregulation in response to specific cytokine and/or antigen stimulation may be an important factor associated with the asthmatic phenotype. Moreover it is possible that diminished IFNy secretion during viral infections may contribute to the pathogenesis of viral-induced asthma exacerbations. Supported by NIH grant I-IL98005