Adenosinergic System Involvement in Ischemic Stroke Patients' Lymphocytes

Abstract

Adenosine modulates many physiological processes through the interaction with adenosine receptors (ARs) named as A₁, A_2A, A_2B, and A₃ARs. During ischemic stroke, adenosine mediates neuroprotective and anti-inflammatory effects through ARs activation. One of the dominant pathways generating extracellular adenosine involves the dephosphorylation of ATP by ecto-nucleotidases CD39 and CD73, which efficiently hydrolyze extracellular ATP to adenosine. The aim of the study is to assess the presence of ARs in lymphocytes from ischemic stroke patients compared to healthy subjects and to analyze changes in CD39 and CD73 expression in CD4⁺ and CD8⁺ lymphocytes. Saturation binding experiments revealed that A_2AARs affinity and density were significantly increased in ischemic stroke patients whilst no differences were found in A₁, A_2B, and A₃ARs. These results were also confirmed in reverse transcription (RT)-polymerase chain reaction (PCR) assays where A_2AAR mRNA levels of ischemic stroke patients were higher than in control subjects. In flow cytometry experiments, the percentage of CD73⁺ cells was significantly decreased in lymphocytes and in T-lymphocyte subclasses CD4⁺ and CD8⁺ obtained from ischemic stroke patients in comparison with healthy individuals. These data corroborate the importance of the adenosinergic system in ischemic stroke and could open the way to more targeted therapeutic approaches and biomarker development for ischemic stroke.

Keywords: A2A adenosine receptors; CD39; CD73; adenosine; adenosine receptors; ischemic stroke; lymphocytes.

Figure 1

(a) Relative adenosine receptors (ARs) mRNA levels determined by reverse transcription (RT)-polymerase chain reaction (PCR) in human lymphocytes from ischemic stroke patients (n = 50) and control subjects (n = 50). (b) Density of A₁, A_2A, A_2B, and A₃ARs, expressed as Bmax, in lymphocytes derived from ischemic stroke patients (n = 50) in comparison to control subjects (n = 50). (c) Saturation curve and (d) Scatchard plot of [³H]-ZM 241385 to A_2AARs in lymphocyte membranes derived from ischemic stroke patients (n = 50) and control subjects (n = 50). Data are expressed as the mean ± SEM. ** p < 0.01 vs control group.

Figure 2

(a) Exemplary flow cytometry density plots showing Fluorescence Minus One (FMO) control and CD4 stained cells in lymphocytes from healthy subjects and ischemic stroke patients. (b) Box and whiskers plot showing the percentage of CD4⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). (c) Representative density plots showing FMO control and CD8 stained cells in lymphocytes from healthy subjects and ischemic stroke patients. (d) Graphical representation of flow cytometry data showing the percentage of CD8⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). Data are shown as the median, interquartile range and the minimum and maximum values.

Figure 3

(a) Representative flow cytometry density plots showing FMO control and CD39 stained cells in lymphocytes from healthy subjects and ischemic stroke patients. (b) Graphical representation of flow cytometry data showing the percentage of CD39⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). (c) Representative density plots showing FMO control CD73 stained cells in lymphocytes from healthy subjects and ischemic stroke patients. (d) Box and whiskers plot showing the percentage of CD73⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). Data are shown as the median, interquartile range and the minimum and maximum values. * p < 0.05 vs healthy subjects.

Figure 4

(a) Representative flow cytometry density plots showing FMO control and CD39 stained cells in CD4⁺ lymphocytes from healthy subjects and ischemic stroke patients. (b) Graphical representation of flow cytometry data showing the CD39⁺ percentage of CD4⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). (c) Exemplary density plots showing FMO control and CD73 stained cells in CD4⁺ lymphocytes from healthy subjects and ischemic stroke patients. (d) Box and Whiskers plot showing the proportion of CD73⁺cells among CD4⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). Data are shown as the median, interquartile range and the minimum and maximum values. * p < 0.05 vs healthy subjects.

Figure 5

(a) Exemplary flow cytometry density plots showing FMO control and CD39 stained cells in CD8⁺ lymphocytes from healthy subjects and ischemic stroke patients. (b) Graphical representation of flow cytometry data showing the CD39⁺ percentage of CD8⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). (c) Representative density plots showing FMO control and CD73 stained cells in CD8⁺ lymphocytes from healthy subjects and ischemic stroke patients. (d) Box and Whiskers plot showing the proportion of CD73⁺cells among CD8⁺ lymphocytes in healthy subjects (n = 20) and ischemic stroke patients (n = 21). Data are shown as the median, interquartile range and the minimum and maximum values. ** p < 0.01 vs healthy subjects.