Angiotensin II type 1 receptor expression on human leukocyte subsets: A flow cytometric and RT-PCR study
Introduction
Although it is well established that angiotensin II (Ang II), the main effector of the renin–angiotensin system (RAS), is involved in the regulation of blood pressure and plasma volume, vasoconstriction, hormone secretion, renal function and cellular growth [1], [2], accumulating evidence also supports a key role for Ang II in the inflammatory processes. Indeed, Ang II may induce the production of reactive oxygen species (ROS), inflammatory cytokines, and adhesion molecules in the vascular wall [3], thus playing a critical role in the modulation of the immune-mediated inflammatory process leading to atherosclerosis and its clinical manifestations [4], [5]. Many experimental studies have shown that Ang II triggers a variety of responses in human leukocytes, including cytosolic calcium changes in peripheral blood mononuclear cells [6], nuclear factor (NF)-κB activation [7], adhesion to endothelial cells (ECs) [8], chemotaxis [9] in monocytes, and modulation of intracellular redox status in neutrophils [10].
Ang II exerts its effects through the binding and activation of multiple receptors [11], however most of its known patho-physiological vascular functions depend upon the activation of the angiotensin II type 1 receptor (AT1R) [12]. The AT1R belongs to the superfamily of G protein-coupled receptors and it is expressed in several organs and tissues, including the kidney, adrenal glands heart, brain and blood vessels [13]. AT1R function has been widely investigated in ECs and vascular smooth muscle cells (VSMCs), where its activation results in the classical vascular effects of Ang II, including vasoconstriction, cell growth and matrix synthesis [14], [15].
Circumstantial evidence suggests that AT1Rs are also expressed in immune system cells, but available information is still fragmentary. AT1Rs have been investigated mainly on unfractionated human mononuclear leukocytes or platelets [16], [17]. It is of fundamental relevance to provide informations about the AT1R expression on the circulating leukocyte subsets, which are cell types differentially involved in the immune-mediated inflammatory responses in order to better clarify the relationship between angiotensin II and atherosclerosis. To this end, in the present study we developed a flow cytometric method to assay cell surface expression of AT1R on defined human leukocyte subpopulations in whole blood samples. In particular, by use of conventional immunofluorescence techniques together with a multiparametric flow cytometric analysis we examined AT1R expression on T- and B-lymphocytes, monocytes and polymorphonuclear leukocytes (PMNs). To confirm the presence of AT1R on the different leukocyte subpopulations we also investigated AT1R mRNA expression by reverse transcription (RT)-PCR analysis. Results show that the pattern of expression of AT1Rs clearly differs in the various subsets of immune cells, and provide a rational framework to study the role(s) of AT1R in the regulation of the immune response in health and disease.
Section snippets
Antibodies and other reagents
Rabbit polyclonal IgG anti-human AT1R (N-10) [anti-AT1 (N-10)] and its specific blocking peptide (sc-1173 P) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated F(ab′)2 fragment goat anti-rabbit IgG (H + L), (FITC-GAR), phycoerythrin (PE)-conjugated mouse anti-human CD45 (anti-CD45-PE), PE covalently linked to cyanin 5 (TC)-conjugated mouse anti-human CD3 (anti-CD3-TC), mouse anti-human CD19 (anti-CD19-TC), mouse anti-human CD16
Expression of AT1Rs
AT1Rs were identified on all the leukocyte subsets investigated, however their pattern of expression widely differed in the various subsets (Fig. 1, Fig. 2 and Table 1). In particular, the MFI value was higher in PMNs and B-lymphocytes with respect to both T-lymphocytes and monocytes (P < 0.05 for B-lymphocytes vs. T-lymphocytes; P < 0.01 in all the other cases), while the percentage of positive cells was highest in PMNs and slightly lower in monocytes and B-lymphocytes (P < 0.05 for PMNs vs.
Discussion
The results of the present study show that: a) AT1Rs are expressed on all the main human leukocyte subsets, i.e., T- and B-lymphocytes, monocytes and PMNs, and b) the expression pattern is peculiar to each subset. By use of a 3-color flow cytometric method and a commercially available anti-human AT1R (N-10) antibody in combination with leukocyte-specific antibodies, we were able to determine the expression of AT1Rs on selected cell populations in terms of both percentage of AT1R-positive cells
Acknowledgement
This work was supported in part by grants from the University of Insubria (FAR 2004) to SL and LG. The skilful assistance of Dr. Cristina Colombo in performing Western Blot experiments is also gratefully acknowledged.
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