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Role of T cells in a gp91phox knockout murine model of acute allergic asthma.

Banerjee ER, Henderson WR - Allergy Asthma Clin Immunol (2013)

Bottom Line: Molecular regulation of inflammation, especially, the role of effector cells in NADPH oxidase-mediated redox reactions for producing O2- (superoxide anion) is a critical step.Materials and Methods and Treatment: To clarify the role of NADPH oxidase in the pathophysiology of T cell-initiatedmacrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox-/- SKO (single knockout) and a gp91phox-/- MMP-12-/- DKO (double knockout) mouse and analysed trafficking and functionality of various cell types, the T cell function and T cell-macrophage interaction being given special emphasis.Composite asthma symptoms expressed in a more aggravated manner in both the KO (SKO and DKO) mice compared to WT indicating that some redundancy may exist in the response pathways of gp91phox and MMP-12.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Allergy and Infectious Diseases, Center for Allergy and Inflammation, University of Washington, Room 254, 850 Republican Street, Seattle, WA 98109, USA. enarb1@gmail.com.

ABSTRACT

Objective: Molecular regulation of inflammation, especially, the role of effector cells in NADPH oxidase-mediated redox reactions for producing O2- (superoxide anion) is a critical step. This study explores the roles of macrophages and neutrophils and their cross-talk with extra-cellular matrix components in the light of the role essayed by T cells. Materials and Methods and Treatment: To clarify the role of NADPH oxidase in the pathophysiology of T cell-initiatedmacrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox-/- SKO (single knockout) and a gp91phox-/- MMP-12-/- DKO (double knockout) mouse and analysed trafficking and functionality of various cell types, the T cell function and T cell-macrophage interaction being given special emphasis.

Results: Composite asthma symptoms expressed in a more aggravated manner in both the KO (SKO and DKO) mice compared to WT indicating that some redundancy may exist in the response pathways of gp91phox and MMP-12. On the one hand, upregulation in macrophage functions such as proliferation, mixed lymphocyte reaction, and MCP-1 directed chemotaxis, may indicate that a regulatory cross-talk is switched on between T cell and macrophage and on the other, downregulation of respiratory burst response hints at a dichotomy in their signaling pathways. Increased B7.1 but reduced B7.2 and MHC class II expression on KO alveolar macrophages may suggest that a switching on-off mechanism is operative where alteration of co-stimulatory molecule expression selectively activating T cell is a critical step.

Inference: T cell mediated functions such as Th2 cytokine secretion, and T cell proliferation in response to OVA were upregulated synchronous with the overall robustness of the asthma phenotype.

Conclusions: As far as cell-cell interaction is concerned, the data is indicative of the existence of a plethora of networks where molecular switches may exist that selectively induce activation and deactivation of regulatory pathways that ultimately manifest in the overall response. gp91phox and MMP-12 either redundantly or synergistically but not additively, provide a regulatory checkpoint for restricting T cell cross-talk with macrophages and keep excessive tissue damage and ECM degradation during acute allergic inflammation under control.

No MeSH data available.


Related in: MedlinePlus

Inhibition of oxidative burst response by KO alveolar leukocytes. Alveolar leukocytes (0.5 × 106 cells) were stained with F4/80-Cy-Chrome and Gr1-APC for 30 min on ice, washed in PBS, warmed up at 370C for 5 min and loaded with 5mM dihydrorhodamine 123 (Molecular Probes, Eugene, OR). After 10 min at 370C, cells were split in two equal aliquots, and PMA (Sigma, St. Louis, MO) was added to one aliquot at final concentration of 1mM. After 10 min incubation cells were washed in ice-cold PBS and immediately subjected to FACS analysis. Cells were gated on neutrophils (Gr1hi), or monocyte/macrophages (F4/80+) and percentage of cells positive for dihydrorhodamine 123 fluorescence with or without PMA treatment was determined for each gate. Results shown are mean of 3 independent experiments ± SEM. (n=5/group). * denotes p value<0.05 compared to WT without PMA treatment and # denotes p value<0.05 compared to WT post-PMA treatment. While WT cells respond to PMA before as well as after OVA challenge, cells from both KO mice before as well as after OVA, failed to respond appreciably. DHR was measured at Fluorescent channel 1 in using a BD Facscaliber and DHR+ cells (CD45+gated and Gr-1+ gated or F4/80+ gated) were analyzed using CellQuestpro software.
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Figure 5: Inhibition of oxidative burst response by KO alveolar leukocytes. Alveolar leukocytes (0.5 × 106 cells) were stained with F4/80-Cy-Chrome and Gr1-APC for 30 min on ice, washed in PBS, warmed up at 370C for 5 min and loaded with 5mM dihydrorhodamine 123 (Molecular Probes, Eugene, OR). After 10 min at 370C, cells were split in two equal aliquots, and PMA (Sigma, St. Louis, MO) was added to one aliquot at final concentration of 1mM. After 10 min incubation cells were washed in ice-cold PBS and immediately subjected to FACS analysis. Cells were gated on neutrophils (Gr1hi), or monocyte/macrophages (F4/80+) and percentage of cells positive for dihydrorhodamine 123 fluorescence with or without PMA treatment was determined for each gate. Results shown are mean of 3 independent experiments ± SEM. (n=5/group). * denotes p value<0.05 compared to WT without PMA treatment and # denotes p value<0.05 compared to WT post-PMA treatment. While WT cells respond to PMA before as well as after OVA challenge, cells from both KO mice before as well as after OVA, failed to respond appreciably. DHR was measured at Fluorescent channel 1 in using a BD Facscaliber and DHR+ cells (CD45+gated and Gr-1+ gated or F4/80+ gated) were analyzed using CellQuestpro software.

Mentions: Macrophages and neutrophils are the key downstream cells contributing to the inflammation in asthma. Their functions were measured by oxidative burst response to PMA and chemotaxis to MCP-1. Figure 5 shows drastic downregulation of DHR+ cells by FACS gated on both Gr-1+ or F4/80+ populations showing either myeloid population to be incapable of showing respiratory burst response by generating reactive oxygen species by responding to PMA. Figure 6 however, surprisingly shows upregulation calcein fluorophore (proportional to cells showing directed migration or specific chemotaxis) in both gp91phox−/− and DKO alveolar macrophages post-OVA to MCP-1.


Role of T cells in a gp91phox knockout murine model of acute allergic asthma.

Banerjee ER, Henderson WR - Allergy Asthma Clin Immunol (2013)

Inhibition of oxidative burst response by KO alveolar leukocytes. Alveolar leukocytes (0.5 × 106 cells) were stained with F4/80-Cy-Chrome and Gr1-APC for 30 min on ice, washed in PBS, warmed up at 370C for 5 min and loaded with 5mM dihydrorhodamine 123 (Molecular Probes, Eugene, OR). After 10 min at 370C, cells were split in two equal aliquots, and PMA (Sigma, St. Louis, MO) was added to one aliquot at final concentration of 1mM. After 10 min incubation cells were washed in ice-cold PBS and immediately subjected to FACS analysis. Cells were gated on neutrophils (Gr1hi), or monocyte/macrophages (F4/80+) and percentage of cells positive for dihydrorhodamine 123 fluorescence with or without PMA treatment was determined for each gate. Results shown are mean of 3 independent experiments ± SEM. (n=5/group). * denotes p value<0.05 compared to WT without PMA treatment and # denotes p value<0.05 compared to WT post-PMA treatment. While WT cells respond to PMA before as well as after OVA challenge, cells from both KO mice before as well as after OVA, failed to respond appreciably. DHR was measured at Fluorescent channel 1 in using a BD Facscaliber and DHR+ cells (CD45+gated and Gr-1+ gated or F4/80+ gated) were analyzed using CellQuestpro software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643823&req=5

Figure 5: Inhibition of oxidative burst response by KO alveolar leukocytes. Alveolar leukocytes (0.5 × 106 cells) were stained with F4/80-Cy-Chrome and Gr1-APC for 30 min on ice, washed in PBS, warmed up at 370C for 5 min and loaded with 5mM dihydrorhodamine 123 (Molecular Probes, Eugene, OR). After 10 min at 370C, cells were split in two equal aliquots, and PMA (Sigma, St. Louis, MO) was added to one aliquot at final concentration of 1mM. After 10 min incubation cells were washed in ice-cold PBS and immediately subjected to FACS analysis. Cells were gated on neutrophils (Gr1hi), or monocyte/macrophages (F4/80+) and percentage of cells positive for dihydrorhodamine 123 fluorescence with or without PMA treatment was determined for each gate. Results shown are mean of 3 independent experiments ± SEM. (n=5/group). * denotes p value<0.05 compared to WT without PMA treatment and # denotes p value<0.05 compared to WT post-PMA treatment. While WT cells respond to PMA before as well as after OVA challenge, cells from both KO mice before as well as after OVA, failed to respond appreciably. DHR was measured at Fluorescent channel 1 in using a BD Facscaliber and DHR+ cells (CD45+gated and Gr-1+ gated or F4/80+ gated) were analyzed using CellQuestpro software.
Mentions: Macrophages and neutrophils are the key downstream cells contributing to the inflammation in asthma. Their functions were measured by oxidative burst response to PMA and chemotaxis to MCP-1. Figure 5 shows drastic downregulation of DHR+ cells by FACS gated on both Gr-1+ or F4/80+ populations showing either myeloid population to be incapable of showing respiratory burst response by generating reactive oxygen species by responding to PMA. Figure 6 however, surprisingly shows upregulation calcein fluorophore (proportional to cells showing directed migration or specific chemotaxis) in both gp91phox−/− and DKO alveolar macrophages post-OVA to MCP-1.

Bottom Line: Molecular regulation of inflammation, especially, the role of effector cells in NADPH oxidase-mediated redox reactions for producing O2- (superoxide anion) is a critical step.Materials and Methods and Treatment: To clarify the role of NADPH oxidase in the pathophysiology of T cell-initiatedmacrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox-/- SKO (single knockout) and a gp91phox-/- MMP-12-/- DKO (double knockout) mouse and analysed trafficking and functionality of various cell types, the T cell function and T cell-macrophage interaction being given special emphasis.Composite asthma symptoms expressed in a more aggravated manner in both the KO (SKO and DKO) mice compared to WT indicating that some redundancy may exist in the response pathways of gp91phox and MMP-12.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Allergy and Infectious Diseases, Center for Allergy and Inflammation, University of Washington, Room 254, 850 Republican Street, Seattle, WA 98109, USA. enarb1@gmail.com.

ABSTRACT

Objective: Molecular regulation of inflammation, especially, the role of effector cells in NADPH oxidase-mediated redox reactions for producing O2- (superoxide anion) is a critical step. This study explores the roles of macrophages and neutrophils and their cross-talk with extra-cellular matrix components in the light of the role essayed by T cells. Materials and Methods and Treatment: To clarify the role of NADPH oxidase in the pathophysiology of T cell-initiatedmacrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox-/- SKO (single knockout) and a gp91phox-/- MMP-12-/- DKO (double knockout) mouse and analysed trafficking and functionality of various cell types, the T cell function and T cell-macrophage interaction being given special emphasis.

Results: Composite asthma symptoms expressed in a more aggravated manner in both the KO (SKO and DKO) mice compared to WT indicating that some redundancy may exist in the response pathways of gp91phox and MMP-12. On the one hand, upregulation in macrophage functions such as proliferation, mixed lymphocyte reaction, and MCP-1 directed chemotaxis, may indicate that a regulatory cross-talk is switched on between T cell and macrophage and on the other, downregulation of respiratory burst response hints at a dichotomy in their signaling pathways. Increased B7.1 but reduced B7.2 and MHC class II expression on KO alveolar macrophages may suggest that a switching on-off mechanism is operative where alteration of co-stimulatory molecule expression selectively activating T cell is a critical step.

Inference: T cell mediated functions such as Th2 cytokine secretion, and T cell proliferation in response to OVA were upregulated synchronous with the overall robustness of the asthma phenotype.

Conclusions: As far as cell-cell interaction is concerned, the data is indicative of the existence of a plethora of networks where molecular switches may exist that selectively induce activation and deactivation of regulatory pathways that ultimately manifest in the overall response. gp91phox and MMP-12 either redundantly or synergistically but not additively, provide a regulatory checkpoint for restricting T cell cross-talk with macrophages and keep excessive tissue damage and ECM degradation during acute allergic inflammation under control.

No MeSH data available.


Related in: MedlinePlus