Number of Circulating CD 73-Expressing Lymphocytes Correlates With Survival After Cardiac Arrest

Abstract

Background Patients resuscitated from cardiac arrest ( CA ) have highly variable neurological, circulatory, and systemic ischemia-reperfusion injuries. After the initial hypoxic-ischemic insult, a cascade of immune and inflammatory responses develops and is often fatal. The role of the immune response in pathophysiological characteristics and recovery is not well understood. We studied immune cell activity and its association with outcomes in a cohort of CA survivors. Methods and Results After informed consent, we collected blood samples at intervals over a week after resuscitation from CA . We examined the expression of CD 39 and CD 73 (alias 5'-nucleotidase), production of tumor necrosis factor-α, generation of reactive oxygen species, and secretion of vascular endothelial growth factor by circulating myeloid and lymphoid cells, in comparison to cells obtained from control subjects before coronary artery bypass grafting surgery. The number of circulating total and CD 73-expressing lymphocytes correlated with survival after CA . Incubation of immune cells, obtained from post- CA subjects, with AMP , a substrate for CD 73, resulted in inhibition of tumor necrosis factor-α production and generation of reactive oxygen species. This effect was blocked by adenosine 5'-(α, β-methylene) diphosphate, a specific inhibitor of CD 73 and ZM 241385, an A2 adenosine receptor antagonist. We also found that AMP -dependent activation of CD 73 induces production of vascular endothelial growth factor. Conclusions CD 73-expressing lymphocytes mediate cellular protection from inflammation after CA through inhibition of proinflammatory activation of myeloid cells and promotion of vascular endothelial growth factor secretion. The contribution of CD 73 lymphocytes in the regulation of acute inflammation and tissue injury after CA warrants further study.

Keywords: CD73; cardiac arrest; inflammation; lymphocytes.

Figure 1

Time‐dependent changes in subpopulations of white blood cells (WBCs) after cardiac arrest (CA). A, Representative flow cytometric contour plots demonstrating percentages of neutrophils (upper gate), monocytes (middle gate), and lymphocytes (lower gate) in peripheral blood of subjects who underwent preoperative coronary artery bypass grafting (control) and subjects with CA on different time points after return of spontaneous circulation (ROSC). B through E, Graphical representation of flow cytometry data showing total number of WBCs (B) and cells in neutrophil (C), monocyte (D), and lymphocyte (E) gates in groups of control subjects (n=30) and subjects with CA (n=48). Data are presented in standard percentile format (minimum value, 25th percentile; median, 75th percentile; and maximum value). Statistical significance was calculated using Kruskal‐Wallis test with Dunn's multiple‐comparisons posttest, and P values are indicated. F and G, The number of cells in neutrophil (F) and lymphocyte (G) gates in survivors (n=19) and nonsurvivors (n=29) after CA. Mann‐Whitney test was used, and P values are indicated. SSC indicates side scatter.

Figure 2

Number of CD3 T cells is significantly decreased after cardiac arrest (CA) and associated with survival. A and B, Representative flow cytometric plots demonstrating percentage of CD3‐positive (CD3^pos; upper left quadrant), CD19‐positive (CD19^pos; lower right quadrant), and CD3‐negative (CD3^neg)/CD19‐negative (CD19^neg; where negative indicates no expression; lower left quadrant) cells within the side scatter (SSC) ^low CD45^high lymphocyte gate (contour plots) in control subjects (A) and subjects with CA (B). C through E, Graphical representation of flow cytometry data showing total number of CD3^pos T cells (C), CD19^pos B cells (D), and CD3^neg/CD19^neg cells (E) in groups of control subjects (n=30) and subjects with CA (n=48). The number of cells was calculated using total number of white blood cells, percentage of cells in the lymphocyte gate, and percentage of cells corresponding to specific cell subpopulation. Statistical significance was calculated using Kruskal‐Wallis test with Dunn's multiple‐comparisons posttest, and P values are indicated. F and G, The number of CD3^pos T cells (F) and CD19^pos B cells (G) in survivors (n=19) and nonsurvivors (n=29) after CA. Mann‐Whitney test was used. ROSC indicates return of spontaneous circulation.

Figure 3

Higher number of CD73‐positive (CD73^pos) lymphocytes but not CD39‐positive (CD39^pos) cells is associated with survival after cardiac arrest (CA). A, Representative flow cytometric plots showing total percentage of CD39^pos cells (left and middle contour plots) and subsets of CD39‐expressing CD3‐positive (CD3^pos), CD19‐positive (CD19^pos), and CD3‐negative (CD3^neg)/CD19‐negative (CD19^neg) cells (where negative indicates no expression; right dot plots) in control subjects (top) and subjects with CA (bottom; 6 hours after return of spontaneous circulation [ROSC]). B, Graphical representation of flow cytometry data showing total number of CD39‐expressing cells in control subjects (n=30) and subjects with CA (n=48) at different times after ROSC. Kruskal‐Wallis test and Dunn's multiple‐comparisons posttest were used. C, The number of CD39^pos cells in survivors (n=19) and nonsurvivors (n=29) after CA. Mann‐Whitney test was used. D, Flow cytometric plots showing total percentage of CD73^pos cells in peripheral blood and subsets of CD73‐expressing CD3^pos and CD19^pos cells. E, Graphical representation of flow cytometry data showing total number of CD73‐expressing cells in control subjects (n=30) and subjects with CA (n=48) at different times after ROSC. Kruskal‐Wallis test and Dunn's multiple‐comparisons posttest were used. F, The number of CD73^pos cells in survivors (n=19) and nonsurvivors (n=29) after CA. Mann‐Whitney test was used. FITC indicates fluorescein isothiocyanate; SSC, side scatter.

Figure 4

CD73 mediates inhibition of lipopolysaccharide‐induced production of tumor necrosis factor‐α (TNF‐α) and generation of reactive oxygen species (ROS) by myeloid cells. A through C, E, and F, White blood cells (WBCs) were isolated 24 hours after return of spontaneous circulation (ROSC) from 5 subjects with cardiac arrest (CA) with a median value of 177 (interquartile range [IQR], 162–300) CD73‐expressing lymphocytes/μL of blood, which closely resembles the median value and IQR found in a group of survivors (median, 182; IQR, 150–275 CD73‐expressing lymphocytes/μL of blood) at 24 hours after ROSC. A, Representative flow cytometric plots showing cells with high levels of TNF‐α protein production in subpopulations of neutrophils (upper gate), monocytes (intermediate gate), and lymphocytes (lower gate) in the absence (basal) or presence of 10 ng/mL lipopolysaccharide alone or in combinations with 100 μmol/L AMP (lipopolysaccharide+AMP), 100 μmol/L of CD73 inhibitor, adenosine 5′‐(α, β‐methylene) diphosphate (APCP; lipopolysaccharide+AMP+APCP), and adenosine receptor inhibitor, 300 nmol/L ZM 241385 (lipopolysaccharide+AMP+ZM). Cells were incubated for 6 hours in the presence of brefeldin A to prevent secretion of TNF‐α from cells and 10 μmol/L erythro‐9‐(2‐hydroxy‐3‐nonyl)−adenine hydrochloride to prevent degradation of adenosine in cell culture. B, Graphical representation of flow cytometric data demonstrating percentage of cells with high expression of TNF‐α (n=5); 1‐way ANOVA was used, and P values from Tukey's multiple‐comparisons test are shown. C, Levels of TNF‐α protein in supernatant of WBCs measured by ELISA (n=5); 1‐way ANOVA was used with Tukey's multiple‐comparisons test. D, Levels of TNF‐α protein in the peripheral circulation of control subjects (n=30) and subjects with CA (n=48). Kruskal‐Wallis test and Dunn's multiple‐comparisons posttest were used. E, Representative flow cytometric histograms demonstrating basal (gray‐shaded) and 10 ng/mL lipopolysaccharide‐induced (open histogram) levels of ROS generation in major subpopulations of WBCs obtained from subjects with CA. F, Graphical representation of ROS generation in the absence (basal) or presence of lipopolysaccharide alone or in combinations with 100 μmol/L AMP (lipopolysaccharide+AMP) and 100 μmol/L of CD73 inhibitor, APCP (lipopolysaccharide+AMP+APCP) (n=5); 1‐way ANOVA with Tukey's multiple‐comparisons test was used. MFI indicates mean fluorescence intensity.

Figure 5

CD73 mediates upregulation of vascular endothelial growth factor (VEGF) protein secretion from white blood cells (WBCs) of subjects with cardiac arrest (CA). A, Level of VEGF protein in supernatant of WBCs obtained from subjects with CA and incubated for 6 hours in the absence (basal) or presence of 100 μmol/L AMP alone or in combination with 100 μmol/L adenosine 5′‐(α, β‐methylene) diphosphate (APCP; AMP+APCP). WBCs were isolated 24 hours after return of spontaneous circulation (ROSC) from 5 subjects with CA (n=5), with a median value of 177 (interquartile range [IQR], 162–300) CD73‐expressing lymphocytes/μL of blood, which closely resembles the median value and IQR found in a group of survivors (median, 182; IQR, 150–275 CD73‐expressing lymphocytes/μL of blood) at 24 hours after ROSC. Incubation medium contained 10 μmol/L erythro‐9‐(2‐hydroxy‐3‐nonyl)−adenine hydrochloride, an adenosine deaminase inhibitor, to decelerate adenosine catabolism in cell culture. Data presented as mean±SEM (n=5); 1‐way ANOVA with Tukey's multiple‐comparisons test was used. B, Levels of VEGF protein in peripheral circulation of control subjects (n=30) and subjects with CA (n=48); Kruskal‐Wallis test was used, and P values are indicated (Dunn's multiple‐comparisons test). C, The correlation between levels of VEGF protein and number of CD73‐positive (CD73^pos) lymphocytes in blood of subjects with CA at 6 and 12 hours after ROSC (n=48). Spearman's correlation coefficient and P values are as indicated.

Figure 6

Proposed role of CD73/5′‐nucleotidase–expressing lymphocytes in the control of immune response after cardiac arrest. Myeloid cells (neutrophils and monocytes) express CD39 ectonucleotidases that mediate hydrolysis of ATP, released from dead and apoptotic cells, to ADP and AMP. Then, AMP is hydrolyzed by CD73/5′‐nucleotidase, expressed on lymphocytes, to adenosine, which suppresses proinflammatory activation of myeloid cells, secretion of tumor necrosis factor‐α (TNF‐α), and production of reactive oxygen species (ROS) and promotes production of prosurvival vascular endothelial growth factor (VEGF).