Our laboratory previously identified an abnormally elevated CD161a+ immune cell population in splenocytes of the pre-hypertensive Spontaneously Hypertensive Rat (SHR) that was abnormally expanded following cholinergic activation with nicotine . In the present study, we tested the hypothesis that the expanded CD161a+ cell population represents an activated monocyte/macrophage population and are present in the bone marrow (BM). We isolated cells from the spleen and BM of pre-hypertensive (4-5 week old) SHR (n=3) and age-matched normotensive Wistar Kyoto (WKY, n=3) rats. Isolated cells were stained with a fluorochrome conjugated anti-rat CD161a monoclonal antibody and analyzed by flow cytometry. CD161a+ cells were more prevalent in splenocytes and BM of the pre-hypertensive SHR, compared to WKY (8.7 ± 1.4% vs 1.2 ± 0.6% and 12.6 ± 1.8% vs 1.7 ± 0.8%, respectively, p<0.001). BM cells and splenocytes were cultured for 36-48 hours in the presence or absence of nicotine (10μM) and then stained with fluorochrome conjugated anti-rat CD161a and CD68 antibodies. CD68 is a well accepted pan-macrophage marker that is up-regulated on activated monocytes/macrophages. The CD161+/CD68+ macrophages were comparable between the WKY and SHR in freshly isolated cells from the spleen (0.3 ± 0.2% vs 0.9 ± 0.5%, respectively) and BM (0.6 ± 0.4% vs 0.7 ± 0.2%, respectively). However, nicotine strongly expanded the CD161a+/CD68+ macrophage population in the BM of the SHR (1.1 ± 0.15% to 11.9 ± 3.45%, p<0.001) and WKY (1.7 ± 0.17% to 11.0 ± 1.9%, p<0.001). In contrast, the response of splenocytes to nicotine was strong in SHR (1.8 ± 0.40% to 10.7 ± 1.1%, p<0.001), but not significant in WKY (1.9 ± 0.4% to 3.1 ± 0.6%, p>0.05). Nicotine had no effect on the CD161a+/CD68- cell population in either splenocytes or BM cells of the SHR or WKY. Thus, an activated monocyte/macrophage population (CD161a+/CD68+) is expanded by cholinergic activation in both the BM and spleen of SHR, but only in the BM and not spleen of the WKY. Since, the pro-inflammatory nicotinic response present in the BM of the WKY is abrogated in the peripheral spleen, we conclude that the retention of this response in the SHR may contribute to the development of hypertension.
In situ production of innate immune cells in murine white adipose tissue
