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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

T cells response in WT vs. KO mice. A. Splenocytes from control (saline treated) and OVA treated mice were made into single cell suspensions in DMEM+10% heat-inactivated FCS. 0.1 million cells were plated per well without and with increasing concentrations of anti-CD3 antibody and a constant concentration of anti-CD28 antibody (1μg/ml) and cultured for 3 days. B. 1μM PMA and 10ng/ml ionomycin was used to stimulate splenocytes from the above experimental mice and proliferation measured after 3 days. To measure proliferation, MTT assay called CellTiter96 (Promega) was used. OD 546 nm is directly proportional to the number of cells in culture. Abbreviations used are: WT=wildtype, NOX=gp91phox−/−, DKO=gp91phox-MMP-12 double knockout, WA=WT+alum, WO=WT+OVA, NOXA=gp91phox−/−+alum, NOXO=gp91phox−/−+OVA, DKOA= gp91phox-MMP-12 double knockout+alum, DKOO= gp91phox-MMP-12 double knockout+OVA. Data presented are average of 3 independent experiments ± SEM. (n=5/group).
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Figure 4: T cells response in WT vs. KO mice. A. Splenocytes from control (saline treated) and OVA treated mice were made into single cell suspensions in DMEM+10% heat-inactivated FCS. 0.1 million cells were plated per well without and with increasing concentrations of anti-CD3 antibody and a constant concentration of anti-CD28 antibody (1μg/ml) and cultured for 3 days. B. 1μM PMA and 10ng/ml ionomycin was used to stimulate splenocytes from the above experimental mice and proliferation measured after 3 days. To measure proliferation, MTT assay called CellTiter96 (Promega) was used. OD 546 nm is directly proportional to the number of cells in culture. Abbreviations used are: WT=wildtype, NOX=gp91phox−/−, DKO=gp91phox-MMP-12 double knockout, WA=WT+alum, WO=WT+OVA, NOXA=gp91phox−/−+alum, NOXO=gp91phox−/−+OVA, DKOA= gp91phox-MMP-12 double knockout+alum, DKOO= gp91phox-MMP-12 double knockout+OVA. Data presented are average of 3 independent experiments ± SEM. (n=5/group).

Mentions: Proliferation of MACS-purified (>86-92%) CD4+ and CD8+ splenocytes by MTT incorporation assay and OD measurement at 545nm of anti CD3/CD28 (0.01-1ug/ml)induced proliferation of CD4+ shows a 8.4-folds increase in post-OVA WT compared to a 7.4-fold increase in both KO mice. In CD8+ while post-OVA WT increased by 6.4-fold, the KO mice showed 7.9-fold increase compared to the corresponding saline treated mice. With PMA/ionomycin (10ng/ml), CD4+ (post-OVA WT was 2.3-fold more than that in NOX−/−) while CD8+ was 1.5-fold more in post-OVA WT than in either KO mice. Overall, whereas proliferation of both T cell subsets to anti CD3/CD28 is comparable, response to PMA/ionomycin is somewhat compromised in KO post-OVA. (Figure 4 Panels A,B).


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

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

T cells response in WT vs. KO mice. A. Splenocytes from control (saline treated) and OVA treated mice were made into single cell suspensions in DMEM+10% heat-inactivated FCS. 0.1 million cells were plated per well without and with increasing concentrations of anti-CD3 antibody and a constant concentration of anti-CD28 antibody (1μg/ml) and cultured for 3 days. B. 1μM PMA and 10ng/ml ionomycin was used to stimulate splenocytes from the above experimental mice and proliferation measured after 3 days. To measure proliferation, MTT assay called CellTiter96 (Promega) was used. OD 546 nm is directly proportional to the number of cells in culture. Abbreviations used are: WT=wildtype, NOX=gp91phox−/−, DKO=gp91phox-MMP-12 double knockout, WA=WT+alum, WO=WT+OVA, NOXA=gp91phox−/−+alum, NOXO=gp91phox−/−+OVA, DKOA= gp91phox-MMP-12 double knockout+alum, DKOO= gp91phox-MMP-12 double knockout+OVA. Data presented are average of 3 independent experiments ± SEM. (n=5/group).
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Figure 4: T cells response in WT vs. KO mice. A. Splenocytes from control (saline treated) and OVA treated mice were made into single cell suspensions in DMEM+10% heat-inactivated FCS. 0.1 million cells were plated per well without and with increasing concentrations of anti-CD3 antibody and a constant concentration of anti-CD28 antibody (1μg/ml) and cultured for 3 days. B. 1μM PMA and 10ng/ml ionomycin was used to stimulate splenocytes from the above experimental mice and proliferation measured after 3 days. To measure proliferation, MTT assay called CellTiter96 (Promega) was used. OD 546 nm is directly proportional to the number of cells in culture. Abbreviations used are: WT=wildtype, NOX=gp91phox−/−, DKO=gp91phox-MMP-12 double knockout, WA=WT+alum, WO=WT+OVA, NOXA=gp91phox−/−+alum, NOXO=gp91phox−/−+OVA, DKOA= gp91phox-MMP-12 double knockout+alum, DKOO= gp91phox-MMP-12 double knockout+OVA. Data presented are average of 3 independent experiments ± SEM. (n=5/group).
Mentions: Proliferation of MACS-purified (>86-92%) CD4+ and CD8+ splenocytes by MTT incorporation assay and OD measurement at 545nm of anti CD3/CD28 (0.01-1ug/ml)induced proliferation of CD4+ shows a 8.4-folds increase in post-OVA WT compared to a 7.4-fold increase in both KO mice. In CD8+ while post-OVA WT increased by 6.4-fold, the KO mice showed 7.9-fold increase compared to the corresponding saline treated mice. With PMA/ionomycin (10ng/ml), CD4+ (post-OVA WT was 2.3-fold more than that in NOX−/−) while CD8+ was 1.5-fold more in post-OVA WT than in either KO mice. Overall, whereas proliferation of both T cell subsets to anti CD3/CD28 is comparable, response to PMA/ionomycin is somewhat compromised in KO post-OVA. (Figure 4 Panels A,B).

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