Reactivity of peripheral blood lymphocytes to oxidized low-density lipoprotein: a novel system to estimate atherosclerosis employing the Cellscan.

Background: The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population. Hypothesis: In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls. Methods: The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases. Results: Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 mg/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 mg/ml). Maximal depolarization was shown upon triggering with 2 mg/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 ± 0.05 and 1.028 ± 0.055, respectively). Conclusion: These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.