Adenosine A2A receptor activation on CD4+ T lymphocytes and neutrophils attenuates lung ischemia-reperfusion injury

Abstract

Objective: Adenosine A(2A) receptor activation potently attenuates lung ischemia-reperfusion injury. This study tests the hypothesis that adenosine A(2A) receptor activation attenuates ischemia-reperfusion injury by inhibiting CD4+ T cell activation and subsequent neutrophil infiltration.

Methods: An in vivo model of lung ischemia-reperfusion injury was used. C57BL/6 mice were assigned to either sham group (left thoracotomy) or 7 study groups that underwent ischemia-reperfusion (1 hour of left hilar occlusion plus 2 hours of reperfusion). ATL313, a selective adenosine A(2A) receptor agonist, was administered 5 minutes before reperfusion with or without antibody depletion of neutrophils or CD4+ T cells. After reperfusion, the following was measured: pulmonary function using an isolated, buffer-perfused lung system, T cell infiltration by immunohistochemistry, myeloperoxidase and proinflammatory cytokine/chemokine levels in bronchoalveolar lavage fluid, lung wet/dry weight, and microvascular permeability.

Results: ATL313 significantly improved pulmonary function and reduced edema and microvascular permeability after ischemia-reperfusion compared with control. Immunohistochemistry and myeloperoxidase content demonstrated significantly reduced infiltration of neutrophils and CD4+ T cells after ischemia-reperfusion in ATL313-treated mice. Although CD4+ T cell-depleted and neutrophil-depleted mice displayed significantly reduced lung injury, no additional protection occurred when ATL313 was administered to these mice. Expression of tumor necrosis factor-alpha, interleukin 17, KC, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and RANTES were significantly reduced in neutrophil- and CD4+ T cell-depleted mice and reduced further by ATL313 only in neutrophil-depleted mice.

Conclusions: These results demonstrate that CD4+ T cells play a key role in mediating lung inflammation after ischemia-reperfusion. ATL313 likely exerts its protective effect largely through activation of adenosine A(2A) receptors on CD4+ T cells and neutrophils.

FIGURE 1

Effect of ATL313 on mouse hemodynamics and complete blood cell counts. A, Heart rate remained stable in vehicle (saline)-treated mice (n=4/group). Heart rate was mildly elevated during the first 25 min after intravenous injection of 3μg/kg ATL313 compared to vehicle-treated mice. B, Mean arterial pressure remained stable in vehicle-treated mice (n=4/group). Small but insignificant changes in mean arterial pressure occurred after injection of ATL313 during the first 5 minutes. The time of injection of vehicle and ATL313 is indicated by an arrow. C, Complete blood cell counts after reperfusion are presented as the percentage of corresponding pre-operative baseline counts. Intra-group comparison showed a 3.5-fold increase in circulating neutrophils in both IR and IR+ATL313 groups and a >50% reduction in circulating lymphocytes after IR. Comparisons between the three groups revealed no significant differences in total WBC counts. Circulating neutrophils were significantly elevated in the IR and IR+ATL313 groups. Circulating lymphocytes were significantly reduced in the IR group (*p<0.05 vs. sham; ^#p<0.05 vs. other groups; n=5/group).

FIGURE 2

Pulmonary function. A, Airway resistance and pulmonary artery pressure were significantly increased while lung compliance was significantly reduced after IR compared to Sham. Pulmonary function was significantly improved after IR by ATL313 treatment (IR+ATL313) (*p<0.05 vs. Sham, ^#p<0.05 vs. IR, n=10/group). B, Pulmonary function in antibody-treated mice after IR. Compared to IgG-treated mice, airway resistance and pulmonary artery pressure were significantly decreased, and lung compliance was significantly increased in neutrophil-depleted (NE dep) and CD4+ T cell-depleted (CD4 dep) mice. No additional protection occurred after ATL313 treatment (ATL) (^§p<0.05 vs. IgG, n=9/group).

FIGURE 3

Pulmonary infiltration of T cells. The average number of CD4+ T cells per high power field (HPF) was assessed via immunohistochemical staining of peripheral lung sections. The quantitative analysis is shown in the graph, and examples of immunohistochemical staining are shown for each group above the corresponding bars. CD4+ T cell numbers were significantly increased after IR compared to Sham and were significantly reduced after IR by ATL313 treatment (IR+ATL313) (*p<0.05 vs. Sham, ^#p<0.05 vs. IR, n=5/group).

FIGURE 4

Neutrophil infiltration and lung injury after reperfusion. Neutrophil infiltration was assessed by measuring MPO levels in BAL fluid (top). Lung injury was assessed by measuring pulmonary edema (wet/dry weight) (middle) and microvascular leak (Evans blue content) (bottom). A, Non-antibody-treated mice. B, Antibody-treated mice for IgG-treated control (IgG) and depletion of neutrophils (NE dep) or CD4+ T cells (CD4 dep). All antibody-treated mice underwent lung IR. ATL = ATL313 treatment (*p<0.05 vs. Sham, ^#p<0.05 vs. IR, ^§p<0.05 vs. IgG control, n=5/group).

FIGURE 5

Production of cytokines/chemokines after IR. Expression of TNF-α, IL-17, MCP-1, MIP-1, RANTES, and KC were measured in BAL fluid after reperfusion. Two separate statistical comparisons are shown: (A) non-antibody-treated mice, and (B) antibody-treated mice for depletion of neutrophils (NE dep) or CD4+ T cells (CD4 dep). All antibody-treated animals underwent lung IR. ATL = ATL313 treatment (*p<0.05 vs. Sham and IR+ATL, ^#p<0.05 vs. IgG control , ^§p<0.05 vs. NE dep, n=5/group).