Plasma glutamate-modulated interaction of A2AR and mGluR5 on BMDCs aggravates traumatic brain injury-induced acute lung injury

Abstract

The bone marrow-derived cell (BMDC)-associated inflammatory response plays a key role in the development of acute lung injury (ALI). Activation of adenosine A2A receptor (A2AR) is generally considered to be antiinflammatory, inhibiting BMDC activities to protect against ALI. However, in the present study, we found that in a mouse model of neurogenic ALI induced by severe traumatic brain injury (TBI), BMDC A2AR exerted a proinflammatory effect, aggravating lung damage. This is in contrast to the antiinflammatory effect observed in the mouse oleic acid-induced ALI model (a nonneurogenic ALI model.) Moreover, the A2AR agonist CGS21680 aggravated, whereas the antagonist ZM241385 attenuated, the severe TBI-induced lung inflammatory damage in mice. Further investigation of white blood cells isolated from patients or mouse TBI models and of cultured human or mouse neutrophils demonstrated that elevated plasma glutamate after severe TBI induced interaction between A2AR and the metabotropic glutamate receptor 5 (mGluR5) to increase phospholipase C-protein kinase C signaling, which mediated the proinflammatory effect of A2AR. These results are in striking contrast to the well-known antiinflammatory and protective role of A2AR in nonneurogenic ALI and indicate different therapeutic strategies should be used for nonneurogenic and neurogenic ALI treatment when targeting A2AR.

Figure 1.

BMDC A_2AR differently regulates the lung injury parameters in the severe TBI-induced ALI model and oleic acid–induced ALI model. At 24 h after injury, lung injury parameters were assessed. WT: WT mice; KO: global A_2AR KO mice; WT→WT: WT recipient mice with BMDCs from WT mice; KO→KO: KO recipient mice with BMDCs from KO mice; KO→WT: WT recipient mice with BMDCs from KO mice (selective inactivation of A_2AR in BMDCs of WT mice); WT→KO: KO recipient mice with BMDCs from WT mice (selective reconstitution of A_2AR in BMDCs of A_2AR KO mice). (A) The lung water content was assayed by the wet–dry method. (B) Blood gas analyses of arterial blood. (C) H&E staining for lung sections. Bars, 100 µm. (A and B) Data are expressed as mean ± SEM. *, P < 0.01, compared with the WT group; #, P < 0.01 compared with the KO group; NS, no significant difference between the two groups. For every parameter measured, n = 8–10 in each group, and each experiment was repeated three times.

Figure 2.

BMDC A_2AR inversely modulates the neutrophil infiltration and inflammatory cytokine expression in the severe TBI- and oleic acid–induced ALI models. (A) Alterations of neutrophil infiltration and A_2AR expression on neutrophils in lung tissues. Neutrophil counting and statistical analysis were performed by Image-Pro plus version 4.5 and presented as an index of CD-177–positive cells. Bars, 100 µm. (B) Alterations of protein expression of TNF and IL-1β in lung tissues. Values are expressed as mean ± SEM of three independent experiments performed in triplicate. *, P < 0.01, compared with the WT group; #, P < 0.01 compared with the KO group; NS, no significant difference between the two groups; n = 8–10 for all groups. Each experiment was repeated three times.

Figure 3.

Plasma glutamate levels in severe TBI-induced ALI and nonneurogenic ALI over time. Plasma glutamate levels in mouse models and patients were detected at 6, 12, and 24 h after injury by HPLC. (A) Changes of plasma glutamate levels in mouse models (TBI- vs. oleic acid–induced ALI model). (B) Changes of plasma glutamate levels in patients (TBI- vs. lung trauma–induced ALI patients). Values are expressed as mean ± SEM, and each experiment was repeated three times. *, P < 0.01 between the two groups; NS, no significant difference between the two groups. 10 patients with TBI-induced ALI and 8 patients with lung trauma–induced ALI were investigated. The number of mice for the assay at each time point was 8–10 in each group.

Figure 4.

A_2AR–mGluR5 interaction is detected in WBCs of severe TBI-induced ALI mouse model or severe TBI-induced ALI patients. (A) Co-IP of A_2AR and mGluR2/3 or mGluR5 in WBCs of mouse models using anti-A_2AR antibody as the immunoprecipitating antibody. (B) Co-IP of A_2AR and mGluR2/3 or mGluR5 in WBCs of patients using anti-A_2AR antibody as the immunoprecipitating antibody. (C and D) Interaction of A_2AR and mGluR5 in WBCs of mouse models (C) or of patients (D) was confirmed by co-IP using anti-mGluR5 antibody as the immunoprecipitating antibody. Each experiment was repeated three times. IB, immunoblot.

Figure 5.

Plasma glutamate level, lung injury parameters, and WBC A_2AR–mGluR5 interaction in moderate TBI mouse model. Moderate TBI was induced in 40 mice via cortical impact with the weight-dropping method. Only two of these mice were diagnosed with ALI at 24 h after TBI. (A) Changes of plasma glutamate levels at 6, 12, and 24 h after moderate TBI. (B) Lung water content. (C) Blood gas analyses of arterial blood. (A–C) Values are expressed as mean ± SEM. (D) H&E staining for lung sections. Bars, 100 µm. (E) Co-IP of A_2AR and mGluR5 in WBCs using anti-A_2AR antibody as the immunoprecipitating antibody. (F) Co-IP of A_2AR and mGluR5 in WBCs using anti-mGluR5 antibody as the immunoprecipitating antibody. IB, immunoblot. *, P < 0.01 when compared with the baseline. Each experiment was repeated three times.

Figure 6.

Effect of plasma glutamate levels on A_2AR–mGluR5 interaction and associated inflammatory cytokine expressions. (A and B) Co-IP of A_2AR and mGluR5 at varying glutamate concentrations in mouse neutrophils using anti-A_2AR antibody or anti-mGluR5 as the immunoprecipitating antibody. IB, immunoblot. (C) Western blot for mGluR5 expression in mouse neutrophils to detect the efficiency of mGluR5 RNAi. (D) Real-time PCR for IL-1β relative mRNA levels in mouse neutrophils. CGS21680: an A_2AR agonist; A_2AR KO: A_2AR gene KO; mGluR5 RNAi: RNA interference of mGluR5 expression. (E and F) Co-IP of A_2AR and mGluR5 at varying glutamate concentrations in human neutrophils using anti-A_2AR antibody or anti-mGluR5 as the immunoprecipitating antibody. (G) Real-time PCR for IL-1β relative mRNA levels in human neutrophils. To confirm the effect of A_2AR–mGluR5 interaction on IL-1β expression, the LPS-stimulated human neutrophils were treated with the selective A_2AR agonist CGS21680, A_2AR antagonist ZM241385, and mGluR5 antagonist MPEP. Data for real-time PCR are expressed as mean ± SEM. Each experiment was repeated three times. *, P < 0.01 between the two groups; NS, no significant difference between the two groups.

Figure 7.

Signaling of the A_2AR–mGluR5 interaction–associated proinflammatory effect. (A) Real-time PCR for IL-1β relative mRNA levels in mouse neutrophils. CGS21680: an A_2AR agonist; H-89: a PKA inhibitor; GF109203X: a PKC inhibitor. (B) Western blot for AC and PLC expression changes in mouse neutrophils. (C) Real-time PCR for IL-1β relative mRNA levels in human neutrophils. (D) Western blot for AC and PLC expression changes in human neutrophils. Data for real-time PCR are reported as mean ± SEM of three independent experiments with three replications in each experiment. *, P < 0.01 between the two groups; NS, no significant difference between the two groups.

Figure 8.

A_2AR agonist aggravates, whereas antagonist attenuates lung damage and neutrophil infiltration in the mouse severe TBI-induced ALI model. Effects of A_2AR agonist CGS21680 and antagonist ZM241385 on the treatment of severe TBI-induced ALI. (A) Lung water content. (B) Blood gas analyses of arterial blood. (C) H&E staining for lung sections. (D) Immunofluorescence for neutrophil infiltration with anti–CD-177 antibody; nucleoli were stained by DAPI in WT or A_2AR KO mice. Neutrophil counting and statistical analysis were performed by Image-Pro plus version 4.5 and presented as an index of CD-177–positive cells. (A, B, and D) Values are expressed as mean ± SEM. *, P < 0.01 when compared with the vehicle treatment; NS, no significant difference between the two groups. Each experiment was repeated three times. Bars, 100 µm.