Resident Cardiac Immune Cells and Expression of the Ectonucleotidase Enzymes CD39 and CD73 after Ischemic Injury

BACKGROUND: The ectoenzymes CD39 and CD73 are expressed by a broad range of immune cells and promote the extracellular degradation of nucleotides to anti-inflammatory adenosine. This study explored the abundance of CD73 and CD39 on circulating and resident cardiac leukocytes and coronary endothelial cells under control conditions and in response to inflammation following myocardial ischemia and reperfusion (I/R). METHODS AND RESULTS: A method was elaborated to permit FACS analysis of non-myocardial cells (resident leukocytes, coronary endothelium and CD31(-) CD45(-) cells) of the unstressed heart. Under control conditions the murine heart contained 2.3 × 10(3) resident leukocytes/mg tissue, the most prominent fraction being antigen-presenting mononuclear cells (CD11b(+) CD11c(+) F4/80(+) MHCII(+)) followed by B-cells, monocytes and T-cells. CD73 was highly expressed on circulating and resident cardiac lymphoid cells with little expression on myeloid cells, while the opposite was true for CD39. Cardiomyocytes and erythrocytes do not measurably express CD39/CD73 and CD39 dominates on coronary endothelium. Three days after I/R, CD73 was significantly upregulated on invading granulocytes (2.8-fold) and T-cells (1.5-fold). Compared with coronary endothelial cells, CD73 associated with leukocytes comprised 2/3 of the total cardiac CD73. CONCLUSION: Our study suggests that extracellular ATP formed during I/R is preferentially degraded by CD39 present on myeloid cells, while the formation of immunosuppressive adenosine is mainly catalysed by CD73 present on granulocytes and lymphoid cells. Upregulated CD73 on granulocytes and T-cells infiltrating the injured heart is consistent with the existence of an autocrine adenosinergic loop which may promote the healing process

Leukocyte subpopulations in myocardial tissue under basal conditions.

<p><b>A:</b> Gating strategy used to identify different leukocyte subpopulations by multiparametric flow cytometry. CD45⁺ cells were gated and DAPI-staining was used to exclude dead and apoptotic immune cells. Living CD45⁺ cells were then divided in subleukocyte populations using a panel of cell-specific fluorochrome-labeld antibodies. Lymphocytes were gated into CD45R(B220)⁺ cells (B-cells) and CD3⁺ cells (T-cells). T-cells were subdivided in CD4⁺ cells (T-helper cells) and CD8⁺ cells (cytotoxic T-cells). Myeloid cells were characterized as CD11b⁺ cells and further subdivided in CD11c⁺ cells (APCs) and Ly6g⁺ cells (granulocytes). <b>B:</b> Leukocyte subpopulations in the unstressed heart. Values are means ± SD of n = 5 experiments.</p

Comparison of cardiac CD73⁺ leukocytes and coronary endothelial cells under basal conditions and after I/R.

<p>Values are calculated from data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034730#pone-0034730-t001" target="_blank">Table 1</a>. Values are means ± SEM of n = 5 experiments.</p

Calculated CD73/CD39 content per mg heart tissue under basal conditions and after I/R.

<p>The number of cell surface molecules was calculated by multiplying the absolute cell count/mg heart tissue with the percentage of CD73⁺/CD39⁺ cells and with the number of CD73/CD39 molecules on those cells. <b>A</b>: Calculated CD73 content per mg heart tissue. <b>B</b>: Calculated CD39 content per mg heart tissue. Values are means ± SD of n = 5 experiments. * P<0.05; n.d. = not detectable.</p

Analysis of the different cell fractions present within the unstressed heart.

<p>Fluorescence microscopy of intact murine cardiomyocytes (<b>A</b>) and non-cardiomyocytes (<b>B</b>) extracted from the murine heart. Red = CD45⁺ cells (fluorescence microscopy). Blue = nuclear stain with DAPI. <b>C</b>: Representative flow cytometry plot of non-cardiomyocytes. Black = CD45⁺ cells (lower right), light grey = endothelial cells (CD31⁺, upper left) and dark grey = CD31⁻ CD45⁻ (lower left). <b>D</b>: Representative Ter-119/CD45 plot of non-cardiomyocytes to derive the ratio of erythrocytes to leukocytes in myocardial tissue. Assuming a ratio of erythrocytes/leukocytes in peripheral blood to be 1000∶1, contamination of blood derived leukocytes was calculated to be <0.2% (n = 5).</p

Abundance of CD73 and CD39 on leukocytes in blood and heart under basal conditions.

<p><b>A</b>: CD73⁺ and CD39⁺ cells per leukocyte population in cardiac tissue. <b>B</b>: CD73/CD39 surface density on CD73⁺/CD39⁺ cells per leukocyte population in cardiac tissue. <b>C</b>: CD73⁺ and CD39⁺ cells per leukocyte population in blood. <b>D</b>: CD73/CD39 surface density on CD73⁺/CD39⁺ cells per leukocyte population in blood. Only positive gated CD73 or CD39 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034730#pone-0034730-g003" target="_blank">Fig. 3A, 3C</a>) were considered for the calculation of antigen density. Values are means ± SD of n = 5 experiments. n.d. = not detectable.</p

Expression of CD73 and CD39 on cardiac cells and circulating blood cells under basal conditions (n = 4–5).

*<p>Analysed by fluorescence microscopy.</p

Changes of total leukocytes, CD73⁺/CD39⁺ cells and CD73/CD39 surface density after I/R.

<p><b>A</b>: Representative flow cytometry plots of digested hearts under basal conditions compared to 3 days after I/R. <b>B</b>: Representative flow cytometry histograms of CD73 expression (red) under basal conditions compared to 3 days after I/R. Grey histograms = fluorescence minus one control (FMO). <b>C</b>: Representative flow cytometry histograms of CD39 expression (blue). Grey histograms = fluorescence minus one control (FMO). <b>D</b>: Increase of leukocyte populations within myocardial tissue after I/R. * P<0.05 for cells/mg heart tissue under basal conditions vs. I/R. CTC = cytotoxic t-cells, THC = T-helper cells, Treg = regulatory t-cells, BC = B-cells, NKC = NK cells, Gr = Granulocytes, Mo = Monocytes, APC = Antigen-presenting cells. <b>E</b>: Changes of CD73⁺/CD39⁺ cells per leukocyte population after I/R. <b>F</b>: Changes of CD73/CD39 surface density on CD73⁺/CD39⁺ leukocytes after I/R. Analysis was done 3 days after I/R. Values are means ± SD of n = 5 experiments. n.d. = not detectable.</p