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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 MCP-1-driven chemotaxis of alveolar macrophages in post-OVA KO mice. 15mM MCP-1 was put in 29μl volume in the lower well and 10 × 106 alveolar macrophages (from 4 mice/experimental group), also in 29μl volume in the upper wells of a 96 well Neuroprobe CTX plates (Chemicon) in high glucose medium for 2 h followed by detachment by mechanical scraping and resuspension in Phenol red-free high glucose DMEM (Gibco) with 5% FBS with 0.5μg/ml Calcein-AM (1:2000 dilution) and incubation for 20 min at 370C. Migrated cells were quantified by fluorescence (excitation at 488 nm, emission at 520 nm) using a Victor 3V (Perkin Elmer laboratories) using a Wallac1420 software. 2.5-fold and 1.26-fold increase in OD (proportinate to number of fluorescing cells in the upper well equivalent to the number of cells migrated) was found in post-OVA gp91phox−/− and DKO mice respectively. * denotes p value<0.05 compared to values in OVA-treated wildtype group.
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Figure 6: Inhibition of MCP-1-driven chemotaxis of alveolar macrophages in post-OVA KO mice. 15mM MCP-1 was put in 29μl volume in the lower well and 10 × 106 alveolar macrophages (from 4 mice/experimental group), also in 29μl volume in the upper wells of a 96 well Neuroprobe CTX plates (Chemicon) in high glucose medium for 2 h followed by detachment by mechanical scraping and resuspension in Phenol red-free high glucose DMEM (Gibco) with 5% FBS with 0.5μg/ml Calcein-AM (1:2000 dilution) and incubation for 20 min at 370C. Migrated cells were quantified by fluorescence (excitation at 488 nm, emission at 520 nm) using a Victor 3V (Perkin Elmer laboratories) using a Wallac1420 software. 2.5-fold and 1.26-fold increase in OD (proportinate to number of fluorescing cells in the upper well equivalent to the number of cells migrated) was found in post-OVA gp91phox−/− and DKO mice respectively. * denotes p value<0.05 compared to values in OVA-treated wildtype group.

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 MCP-1-driven chemotaxis of alveolar macrophages in post-OVA KO mice. 15mM MCP-1 was put in 29μl volume in the lower well and 10 × 106 alveolar macrophages (from 4 mice/experimental group), also in 29μl volume in the upper wells of a 96 well Neuroprobe CTX plates (Chemicon) in high glucose medium for 2 h followed by detachment by mechanical scraping and resuspension in Phenol red-free high glucose DMEM (Gibco) with 5% FBS with 0.5μg/ml Calcein-AM (1:2000 dilution) and incubation for 20 min at 370C. Migrated cells were quantified by fluorescence (excitation at 488 nm, emission at 520 nm) using a Victor 3V (Perkin Elmer laboratories) using a Wallac1420 software. 2.5-fold and 1.26-fold increase in OD (proportinate to number of fluorescing cells in the upper well equivalent to the number of cells migrated) was found in post-OVA gp91phox−/− and DKO mice respectively. * denotes p value<0.05 compared to values in OVA-treated wildtype group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Inhibition of MCP-1-driven chemotaxis of alveolar macrophages in post-OVA KO mice. 15mM MCP-1 was put in 29μl volume in the lower well and 10 × 106 alveolar macrophages (from 4 mice/experimental group), also in 29μl volume in the upper wells of a 96 well Neuroprobe CTX plates (Chemicon) in high glucose medium for 2 h followed by detachment by mechanical scraping and resuspension in Phenol red-free high glucose DMEM (Gibco) with 5% FBS with 0.5μg/ml Calcein-AM (1:2000 dilution) and incubation for 20 min at 370C. Migrated cells were quantified by fluorescence (excitation at 488 nm, emission at 520 nm) using a Victor 3V (Perkin Elmer laboratories) using a Wallac1420 software. 2.5-fold and 1.26-fold increase in OD (proportinate to number of fluorescing cells in the upper well equivalent to the number of cells migrated) was found in post-OVA gp91phox−/− and DKO mice respectively. * denotes p value<0.05 compared to values in OVA-treated wildtype group.
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