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Hyaluronan Dependent Leukocyte Adhesion Is Enhanced by Viral Infection, but Not by Aeroallergen Exposure in Human Lung Fibroblasts
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A2857 - Hyaluronan Dependent Leukocyte Adhesion Is Enhanced by Viral Infection, but Not by Aeroallergen Exposure in Human Lung Fibroblasts
Author Block: S. R. Reeves1, I. Kang2, K. A. Barrow1, M. P. White1, T. N. Wight2, J. S. Debley1; 1Center for Immunity and Immunotherapies, Seattle Children's, Seattle, WA, United States, 2Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States.
Rationale: Previous studies have demonstrated that exposure of human lung fibroblasts (HLFs) to viral mimetics such as polyinosine-polycytidylic acid (poly I:C) enhance the ability of the extracellular matrix (ECM) to bind leukocytes. Enhanced binding to ECM following treatment of HLFs with poly I:C was dependent on hyaluronan (HA) and increased HA-associated proteoglycans. Both viruses and aeroallergens are known to enhance airway inflammation in vivo; however, their role in promoting HA-dependent leukocyte adhesion is currently unknown.
Aim: To determine if viral infection or exposure to aeroallergens enhances HA-dependent binding of leukocytes to ECM generated by healthy HLFs.
Methods: HLFs were purchased from a commercial vendor and used for all experiments (n=3/group). HLFs were treated with the following conditions for 96 h prior to conducting leukocyte binding studies; respiratory syncytial virus (RSV; MOI=1) infection, human rhinovirus-16 (HRV16; MOI=0.1) infection, exposure to cockroach antigen (CRA; 100 μg/mL), or house dust mite antigen (HDM; 100 μg/mL). A hyaluronidase-treated condition was included for each group. Following treatment, leukocyte binding assays were conducted using fluorescently-labeled U937 (monocyte, calcein-AM), HL-60 (eosinophil, calcein-red/orange), and LUVA (mast cell, calcein-blue) cell lines mixed 1:1:1 at a concentration of 1x106/mL. Following wash steps, cell counts were obtained using quantitative fluorescent microscopy.
Results: Total HA-dependent leukocyte adhesion was greater in the RSV-infected HLF group relative to controls (80.4±1.87% vs. 37.4±11.6% decrease following hyaluronidase treatment; P=0.02). A non-significant trend was also noted for HRV16-infected HLFs compared to controls (69.8±2.43% vs. 37.4±11.6% decrease; P=0.052). No significant differences were noted in total leukocyte adhesion in CRA- or HDM-treated HLFs compared to controls. U937 cell subsets displayed greater HA-dependent binding compared to controls for HLFs infected with RSV (31.0±8.46% vs. 75.5±2.94% decrease; P=0.008) and HRV16 (31.0±8.46% vs. 67.6±5.13% decrease; P=0.02). For HL-60 cells, RSV-infected HLFs displayed greater HA-dependent adhesion compared to controls (83.5±1.77% vs. 45.8±9.11% decrease; P=0.02).
Conclusions: Direct viral infection of HLFs with RSV or HRV16 may contribute to inflammatory changes via a HA-dependent mechanism. HA-dependent adhesion to ECM generated by HLFs following direct stimulation with CRA or HDM was not observed. These data suggest that viral infection of HLFs may contribute to the establishment of a HA-enriched ECM that is more permissive for leukocytes. Direct exposure of HLFs to CRA and HDM did not lead to enhanced HA-dependent binding of leukocytes, suggesting that inflammatory changes via aeroallergen exposure may work through a separate mechanism.
Author Block: S. R. Reeves1, I. Kang2, K. A. Barrow1, M. P. White1, T. N. Wight2, J. S. Debley1; 1Center for Immunity and Immunotherapies, Seattle Children's, Seattle, WA, United States, 2Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States.
Rationale: Previous studies have demonstrated that exposure of human lung fibroblasts (HLFs) to viral mimetics such as polyinosine-polycytidylic acid (poly I:C) enhance the ability of the extracellular matrix (ECM) to bind leukocytes. Enhanced binding to ECM following treatment of HLFs with poly I:C was dependent on hyaluronan (HA) and increased HA-associated proteoglycans. Both viruses and aeroallergens are known to enhance airway inflammation in vivo; however, their role in promoting HA-dependent leukocyte adhesion is currently unknown.
Aim: To determine if viral infection or exposure to aeroallergens enhances HA-dependent binding of leukocytes to ECM generated by healthy HLFs.
Methods: HLFs were purchased from a commercial vendor and used for all experiments (n=3/group). HLFs were treated with the following conditions for 96 h prior to conducting leukocyte binding studies; respiratory syncytial virus (RSV; MOI=1) infection, human rhinovirus-16 (HRV16; MOI=0.1) infection, exposure to cockroach antigen (CRA; 100 μg/mL), or house dust mite antigen (HDM; 100 μg/mL). A hyaluronidase-treated condition was included for each group. Following treatment, leukocyte binding assays were conducted using fluorescently-labeled U937 (monocyte, calcein-AM), HL-60 (eosinophil, calcein-red/orange), and LUVA (mast cell, calcein-blue) cell lines mixed 1:1:1 at a concentration of 1x106/mL. Following wash steps, cell counts were obtained using quantitative fluorescent microscopy.
Results: Total HA-dependent leukocyte adhesion was greater in the RSV-infected HLF group relative to controls (80.4±1.87% vs. 37.4±11.6% decrease following hyaluronidase treatment; P=0.02). A non-significant trend was also noted for HRV16-infected HLFs compared to controls (69.8±2.43% vs. 37.4±11.6% decrease; P=0.052). No significant differences were noted in total leukocyte adhesion in CRA- or HDM-treated HLFs compared to controls. U937 cell subsets displayed greater HA-dependent binding compared to controls for HLFs infected with RSV (31.0±8.46% vs. 75.5±2.94% decrease; P=0.008) and HRV16 (31.0±8.46% vs. 67.6±5.13% decrease; P=0.02). For HL-60 cells, RSV-infected HLFs displayed greater HA-dependent adhesion compared to controls (83.5±1.77% vs. 45.8±9.11% decrease; P=0.02).
Conclusions: Direct viral infection of HLFs with RSV or HRV16 may contribute to inflammatory changes via a HA-dependent mechanism. HA-dependent adhesion to ECM generated by HLFs following direct stimulation with CRA or HDM was not observed. These data suggest that viral infection of HLFs may contribute to the establishment of a HA-enriched ECM that is more permissive for leukocytes. Direct exposure of HLFs to CRA and HDM did not lead to enhanced HA-dependent binding of leukocytes, suggesting that inflammatory changes via aeroallergen exposure may work through a separate mechanism.
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