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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

Altered Mixed Lymphocyte Reaction in post-OVA KO mice. 0.1 × 105 CD4+ T cells isolated by magnetic activated cell separation (MACS) by positive selection from spleen of the mice were co-cultured with γ-irradiated 0.1x103 adhering alveolar macrophages from BALf of the same experimental animal. Both cell types were from the experimental animals themselves viz. C57Bl/6 WT and KO mice. This is Syngeneic MLR where the APCs (alveolar macrophages) were γ-irradiated (3300rads). Allogeneic MLR reaction involves co-culturing 0.1x105 CD4+ T cells from the spleen of BALB/c mice with 0.1x103 γ-irradiated APCs from BALf of the experimental mice. Both control (saline-treated) and OVA-treated of each group were tested. Each culture was incubated with 1μg/ml OVA.The responders here are the T cells and the stimulators are the APCs, viz. alveolar macrophages which are γ-irradiated to inhibit their own proliferation. Since acute allergic asthma is a Th2 mediated phenomenon, interaction between T cells and macrophages will elucidate functional cross-talk between the two cell types when responders are autologous as well as when they are from a different species. 2-fold increase in post-OVA NOX vs. post-OVA WT and 2.16-fold increase in post OVA-DKO vs. post-OVA shows that T cell:APC interaction is actually more efficient in the absence of the gp91phox and MMP-12 as well as gp91phox.
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Figure 7: Altered Mixed Lymphocyte Reaction in post-OVA KO mice. 0.1 × 105 CD4+ T cells isolated by magnetic activated cell separation (MACS) by positive selection from spleen of the mice were co-cultured with γ-irradiated 0.1x103 adhering alveolar macrophages from BALf of the same experimental animal. Both cell types were from the experimental animals themselves viz. C57Bl/6 WT and KO mice. This is Syngeneic MLR where the APCs (alveolar macrophages) were γ-irradiated (3300rads). Allogeneic MLR reaction involves co-culturing 0.1x105 CD4+ T cells from the spleen of BALB/c mice with 0.1x103 γ-irradiated APCs from BALf of the experimental mice. Both control (saline-treated) and OVA-treated of each group were tested. Each culture was incubated with 1μg/ml OVA.The responders here are the T cells and the stimulators are the APCs, viz. alveolar macrophages which are γ-irradiated to inhibit their own proliferation. Since acute allergic asthma is a Th2 mediated phenomenon, interaction between T cells and macrophages will elucidate functional cross-talk between the two cell types when responders are autologous as well as when they are from a different species. 2-fold increase in post-OVA NOX vs. post-OVA WT and 2.16-fold increase in post OVA-DKO vs. post-OVA shows that T cell:APC interaction is actually more efficient in the absence of the gp91phox and MMP-12 as well as gp91phox.

Mentions: Based on the aforementioned responses of T cells and macrophages it seems apparent that both cells are able to function well in response to OVA on their own at least as far as the asthma phenotype is concerned. They migrate in increased numbers from blood and resident as well as recruited cells are found in impressive inflammatory exudates around the airways. So the next question was whether there is efficient cross-talk between the T cells upstream and the macrophages downstream. To this end we did a mixed lymphocyte reaction using first the CD4+ T cells as the responders and the γ-irradiated alveolar macrophages as the stimulators from the experimental mice themselves and then used CD4+ T cells from splenocytes of BALB/c mice. Increase in proliferation measured by MTT assay (OD 570nm) (Figure 7) shows increased T cell: APC interaction both when autologous APCs (macrophages from adherent cell population in BALf of the same animal) were used and then APCs from experimental animals were used as stimulators to CD4+ T cells from spleen of BALB/c mice which were the responders.


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

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

Altered Mixed Lymphocyte Reaction in post-OVA KO mice. 0.1 × 105 CD4+ T cells isolated by magnetic activated cell separation (MACS) by positive selection from spleen of the mice were co-cultured with γ-irradiated 0.1x103 adhering alveolar macrophages from BALf of the same experimental animal. Both cell types were from the experimental animals themselves viz. C57Bl/6 WT and KO mice. This is Syngeneic MLR where the APCs (alveolar macrophages) were γ-irradiated (3300rads). Allogeneic MLR reaction involves co-culturing 0.1x105 CD4+ T cells from the spleen of BALB/c mice with 0.1x103 γ-irradiated APCs from BALf of the experimental mice. Both control (saline-treated) and OVA-treated of each group were tested. Each culture was incubated with 1μg/ml OVA.The responders here are the T cells and the stimulators are the APCs, viz. alveolar macrophages which are γ-irradiated to inhibit their own proliferation. Since acute allergic asthma is a Th2 mediated phenomenon, interaction between T cells and macrophages will elucidate functional cross-talk between the two cell types when responders are autologous as well as when they are from a different species. 2-fold increase in post-OVA NOX vs. post-OVA WT and 2.16-fold increase in post OVA-DKO vs. post-OVA shows that T cell:APC interaction is actually more efficient in the absence of the gp91phox and MMP-12 as well as gp91phox.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Altered Mixed Lymphocyte Reaction in post-OVA KO mice. 0.1 × 105 CD4+ T cells isolated by magnetic activated cell separation (MACS) by positive selection from spleen of the mice were co-cultured with γ-irradiated 0.1x103 adhering alveolar macrophages from BALf of the same experimental animal. Both cell types were from the experimental animals themselves viz. C57Bl/6 WT and KO mice. This is Syngeneic MLR where the APCs (alveolar macrophages) were γ-irradiated (3300rads). Allogeneic MLR reaction involves co-culturing 0.1x105 CD4+ T cells from the spleen of BALB/c mice with 0.1x103 γ-irradiated APCs from BALf of the experimental mice. Both control (saline-treated) and OVA-treated of each group were tested. Each culture was incubated with 1μg/ml OVA.The responders here are the T cells and the stimulators are the APCs, viz. alveolar macrophages which are γ-irradiated to inhibit their own proliferation. Since acute allergic asthma is a Th2 mediated phenomenon, interaction between T cells and macrophages will elucidate functional cross-talk between the two cell types when responders are autologous as well as when they are from a different species. 2-fold increase in post-OVA NOX vs. post-OVA WT and 2.16-fold increase in post OVA-DKO vs. post-OVA shows that T cell:APC interaction is actually more efficient in the absence of the gp91phox and MMP-12 as well as gp91phox.
Mentions: Based on the aforementioned responses of T cells and macrophages it seems apparent that both cells are able to function well in response to OVA on their own at least as far as the asthma phenotype is concerned. They migrate in increased numbers from blood and resident as well as recruited cells are found in impressive inflammatory exudates around the airways. So the next question was whether there is efficient cross-talk between the T cells upstream and the macrophages downstream. To this end we did a mixed lymphocyte reaction using first the CD4+ T cells as the responders and the γ-irradiated alveolar macrophages as the stimulators from the experimental mice themselves and then used CD4+ T cells from splenocytes of BALB/c mice. Increase in proliferation measured by MTT assay (OD 570nm) (Figure 7) shows increased T cell: APC interaction both when autologous APCs (macrophages from adherent cell population in BALf of the same animal) were used and then APCs from experimental animals were used as stimulators to CD4+ T cells from spleen of BALB/c mice which were the responders.

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