Because of its hemodynamic effects, angiotensin II (AngII) plays a central role in the progression of chronic kidney diseases (CKD) and ischemic heart disease (1,2). AngII has been shown to be a potent proinflammatory molecule, and the beneficial effects of renin-angiotensin system (RAS) blockade are due not only to lowering BP but also to a reduction in inflammation (3). One of the main features of CKD is the accumulation of inflammatory cells, which plays a crucial role in disease progression (4), and recruitment of macrophages to the kidney through AngII infusion has been reported in various rodent models (5,6). In both diabetic nephropathy and atherosclerosis, monocytes/macrophages have been reported to play a key role (7–9). Monocytes have also been the primary focus of studies that have examined the interaction of AngII and inflammatory cells (10). However, the importance of T, natural killer (NK), and dendritic cells (DC) in inflammation and vascular disease has only recently begun to be appreciated. DC have been shown to present oxidized LDL to T cells, generating autoreactive T cells and promoting arterial injury (11). NK cells participate through the production of proatherogenic cytokines such as IFN-γ (12). Previous studies on AngII-induced inflammation and its role in kidney disease primarily focused on the induction of inflammatory molecules and the paracrine effects of AngII in vascular remodeling and tissue fibrosis (13,14). Despite these studies having examined the impact of AngII on various steps of inflammation, the direct regulatory effect of AngII on the function and cellular localization of specific immune cells remains to be fully explored in humans. The role of a mobile RAS present on the leukocyte is an important avenue of research that will contribute to a better understanding of the factors that control leukocyte recruitment and will potentially identify important therapeutic targets. In this report, we examine the status of the RAS at the cellular level and the effect of AngII on the function of T and NK cells.
Materials and Methods
Cells were isolated from healthy volunteers and were cultured as described previously (15).
The effect of AngII on NK cell function was tested using a cytotoxicity assay.
Culture Supernatant Analysis
The Beadlyte Human Multi-Cytokine Beadmaster Kit (Upstate, Lake Placid, NY) was used according to the manufacturer's protocol.
Real-Time PCR Analysis
Total RNA extraction, reverse transcription, and quantitative PCR (QPCR) were performed as described previously (16).
A Western blot was performed using detection antibodies for AngII type 1 (AT1) and type 2 (AT2) receptors.
Flow Cytometric Analysis
Experiments were performed with different antibodies to attempt to show effectively AT1 and AT2 surface receptor expression.
Fluorescence and calcium flux were measured in leukocytes as described previously (17).
Chemotaxis to AngII
Chemotaxis of specific cell subsets to AngII was assessed. Please refer to the online supplementary material for more details of Materials and Methods.
AngII and Its Precursors Augment Mitogenic T and NK Cell Proliferation
To study the mitogenic effects of AngII on human T and NK cells, we used nylon wool–purified cells that were cultured in the presence or absence of AngII. After isolation, cells were on average 87% CD3+ (T cells), 8% CD56+ (NK cells), and 2% CD11c+ (DC) by flow cytometric analysis. In all subjects studied, AngII alone was unable to stimulate cells in the absence of mitogen; however, AngII significantly increased phytohemagglutinin (PHA)-induced cell proliferation in a dosage-dependent manner (Figure 1A). T cell receptor (TCR)-specific proliferation by plate-bound anti-CD3 was also shown to increase in response to AngII, indicating that AngII can provide co-stimulatory signals when the TCR is specifically engaged (Figure 1B). To determine whether RAS enzymatic components are functionally present in our system, we also added AngI and angiotensinogen to mitogen-stimulated lymphocyte cultures; cells that were cultured with PHA in the presence of 10−6 mol/L AngI as well as angiotensinogen increased proliferation to a degree similar to AngII (data not shown), suggesting that these cells are able to convert RAS substrates. To determine whether the observed proliferative effect of AngII could be suppressed, we pretreated cells with 10−5 mol/L losartan and PD123319, antagonists for the AT1 and AT2 receptors, respectively, for 1 h before culturing them as described. Compared with 10−6 mol/L AngII treatment, both antagonists individually blocked proliferation significantly in response to AngII (Figure 1C), although the level of proliferation was higher than that of PHA. Interestingly, when both antagonists were added, proliferation was not significantly reduced compared with 10−6 mol/L AngII treatment and in fact increased when compared with either antagonist alone. This suggests that although AngII acts via both AT1 and AT2, AngII may exert its co-stimulatory effect through another AngII receptor. Cells were also labeled with carboxy fluorescein succinimidyl ester (CFSE) and cultured as described to determine which subsets proliferate in response to AngII. As shown in Figure 1, D and E, CD8+ T cells and NK cells showed a statistically significant, although <20%, increase in proliferation in response to AngII.
We also performed a proliferation assay in which purified cells were depleted of NK cells by bead-mediated removal of CD56+ cells. Under these conditions, the proliferative effect of AngII was blocked (Figure 1F), indicating that AngII may exert its function through NK cells. Upon examination of the direct effect of AngII on T cell proliferation by depleting NK and labeling the remaining T cell population with CFSE, as shown in Figure 1G, we show that CD8+ T cell proliferation in response to AngII is substantially diminished in the absence of NK. To address the direct effect of AngII on NK cell function, we assessed the cytotoxicity of NK cells in the presence and absence of AngII. No difference was noted in cell death when NK cells were treated with AngII (data not shown).
AngII Treatment Increases the Ratio of IFN-γ:IL-4 But Not Their Absolute Concentration
To determine whether AngII affects the cytokine immune response in this culture system, we measured signature cytokines in the cell supernatants. A Th1 immune response has been shown to be important in atherogenesis (12). Figure 2, A and B, shows the absolute concentration of IFN-γ and IL-4 after stimulation of nylon wool–purified cells with PHA and AngII at 10−6 mol/L. Although at 48 h a trend was observed in response to AngII treatment toward higher IFN-γ and lower IL-4 production, the difference between AngII treatment and PHA stimulation alone did not reach statistical significance, which could be due to considerable donor variability. We therefore calculated the IFN-γ:IL-4 (Figure 2C) for each donor for these two groups as has been done previously (18,19), which demonstrates a significant although modest shift toward greater IFN-γ production and lesser IL-4 production in response to AngII treatment at 48 h as compared with PHA alone. Of note, the ratio of IFN-γ:IL-4 is thought to be more important in bringing about changes in cytokine signaling, as Th1 and Th2 cytokines are mutually inhibitory (18,19). The induction of the inflammatory chemokines CCL2/monocyte chemoattractant protein-1 (MCP-1) and CCL5/RANTES by AngII was also assessed. Whereas no change occurred in CCL2/MCP-1 production (data not shown), RANTES levels increased almost two-fold with angiotensin treatment of T cells at 72 h (Figure 2D); at earlier time points, RANTES levels were too high to measure. This prolonged production of RANTES in response to AngII may serve to recruit cells to sites of inflammation, or RANTES may act via the CCR5 receptor to induce proliferation (20).
RAS Components Are Found on T Cells and NK Cells
Because both AngI and angiotensinogen had similar effects as AngII in augmenting PHA-induced proliferation, the presence of functional renin and angiotensin-converting enzyme (ACE) was suggested in T and NK cells. The presence of RAS elements on T and NK cells has not been comprehensively explored. mRNA was isolated from purified T and NK cells as well as from monocytes and DC, and QPCR was used to quantify the expression of angiotensinogen, ACE, renin, and the renin receptor. Of note, the renin receptor was recently reported to bind both circulatory renin and prorenin, promoting conversion of angiotensinogen to AngI (21). DC were derived from peripheral blood monocytes; their maturity was assessed by studying surface expression for CCR7, HLA-DR, and CD80. T and NK cells both were found to express ACE and the renin receptor, whereas only T cells expressed detectable levels of renin and only NK cells were found to express angiotensinogen (Figure 3). Monocyte and DC RAS expression was previously reported (22,23), and results of these studies are consistent with our data; expression for the renin receptor, however, is novel, and monocytes, immature DC (iDC), and mature DC (mDC) all were determined to express the renin receptor. Expression for renin and its receptor was highest in monocytes, with iDC expressing approximately one tenth the copy number of monocytes (Figure 3, A and B). Monocytes again showed substantially higher expression of ACE (Figure 3D), whereas monocytes and mDC expressed considerable mRNA levels of angiotensinogen (Figure 3C).
AngII Receptor Expression
At the level of the receptors for AngII, few data exist concerning the expression of AT1 and AT2 receptors in human T and NK cells. We examined the expression of AT1 and AT2 by Western blot and flow cytometric analysis after testing numerous anti-receptor antibodies for specificity. Western blotting demonstrated AT1 and AT2 receptor expression in cell lysate protein that was extracted from T and NK cells (Figure 4). Monocytes showed only AT1 expression by Western blot (Figure 4). The size of the AT1 protein that was expressed by all subsets was 41 kD, indicating that these cells possess the nonglycosylated form of the receptor; the positive control receptor, however, was glycosylated, resulting in a 50-kD protein. Glycosylation has been shown to increase the functional expression and stability of the AT1 receptor (24), which could suggest that monocytes, T cells, and NK cells may exhibit low AT1 surface expression. Indeed, flow cytometric analysis showed negligible AT1 and slight surface expression for AT2 on all three subsets compared with isotype control (Figure 5, A through C). T and NK cells showed higher levels of AT1 expression by Western blot and higher AT2 expression by FACS. We also assessed the expression of the AT1 and AT2 receptors in DC that were cultured from peripheral purified monocytes. Both iDC and mDC can be generated in vitro as described previously (25). Previous studies that examined AT1 and AT2 receptor expression on monocytes and DC were limited by the fact that either a leukemic cell line was used or proper isotype controls were not used (10,26,27). Interestingly, DC, whether immature or mature, showed a substantially higher level of AT2 receptor expression than all other subsets. DC maturation seemed to decrease AT2 expression, as iDC had twice that of mDC (Figure 5, D and E). Cells were also permeabilized before flow cytometry for ACE, AT1, or AT2 to determine whether these molecules are expressed intracellularly. Little to no difference in ACE and AT1 expression was found, but AT2 expression was markedly increased in all subsets after cells were permeabilized (Figure 5, F through H). These data indicate that the AT2 receptor is predominantly expressed intracellularly and raises the possibility that this receptor, like other chemoattractant G protein–coupled receptors, may be present in storage vesicles that can merge with the cell membrane upon activation (28).
AT1 and AT2 in Combination Fail to Block Completely AngII Signal Transduction
To determine whether the angiotensin receptor that we detected on leukocytes was functional, we tested for real-time Ca2+ flux in response to AngI and AngII in human peripheral blood mononuclear cells. Activators of phospholipase C, including AngII, release Ca2+ from intracellular stores and enhance Ca2+ influx. It is interesting that AngI (data not shown) and AngII (Figure 6A) both were able to generate calcium flux at levels comparable to ATP, a potent control stimulus. AT1 and AT2 antagonists alone and in combination were able to diminish calcium release in response to AngII significantly. The fact that both antagonists simultaneously were unable to suppress completely calcium release again suggests the involvement of another receptor that needs to be identified. Of note, treating cells with AngII receptor antagonists before assessing calcium flux to ATP had no effect, demonstrating that these antagonists do not nonspecifically suppress calcium flux. Leukocyte subset mRNA was also assessed for expression of the AT4 receptor, a recently identified AngII receptor, and the T and NK cell subsets were found principally to express this receptor (Figure 6B) (29).
T Cells, DC, and NK Cells Migrate to AngII
Recruitment of leukocytes to the site of inflammation is controlled primarily by chemokines. AngII, however, has been shown to have chemotactic properties with respect to monocytes (30), but no data exist on the chemotactic effect of AngII on other human leukocyte subsets; we therefore examined the chemoattractant effect of AngII on purified human T cells, DC, and NK cells. When AngII was added to both the upper and the lower chambers, no chemotaxis occurred, which excluded enhanced chemokinesis. In chemotaxis experiments with isolated T cells, cells migrated 3.5 times more to 10−12 mol/L AngII than to buffer (P = 0.0005; Figure 7A). The AT1 and AT2 antagonists losartan and PD123319 alone or in combination were unable to suppress T cell migration (data not shown), again suggesting the involvement of another angiotensin receptor. DC migrated to 10−8 mol/L AngII almost as much as to stromal-derived factor 1α (SDF-1α), a potent chemoattractant, and migrated three times more than to buffer (Figure 7B). Purified NK cells also exhibited directional migration to AngII at 10−7 mol/L (P = 0.002) and 10−8 mol/L (P = 0.001; data not shown) than to buffer alone; real-time videomicroscopy with NK cells was performed (see online supplementary movies).
The RAS plays a central role in the pathogenesis of cardiovascular and renal diseases. In addition to the hemodynamic-based injuries that are caused by the RAS, a growing body of evidence supports the idea that the RAS exerts its deleterious effects through the induction of inflammatory responses and oxidative stress in the vascular wall. In a model of immune complex nephritis, Ruiz-Ortega et al. (4) showed that AngII induces NF-κB activation and CCL2/MCP-1 expression in the renal cortex, an effect that is reduced by ACE inhibition. Others also showed that AngII increases the production of reactive oxygen species, the known activators of NF-κB, and other signal transducers that lead to the activation of proinflammatory cytokine genes (31,32). Because of the importance of T cells, NK cells, and DC in inflammation and in renal and vascular diseases, we explored whether RAS elements are expressed in these subsets, as well as how RAS expression affects their function. One of the first studies to link AngII and the immune system followed studies that showed the co-localization of ACE in alveolar macrophages and in human atherosclerotic plaques that were localized precisely in areas in which macrophages aggregate in atherosclerotic coronary segments (33,34). These data highlight the importance of monocytes/macrophages in the pathogenesis of vasculopathy. A local RAS also was shown to be present in many tissues, indicating that local systems exist and may serve to increase the effects of circulating AngII (29). In humans, the RAS at the cellular level has not yet been comprehensively studied in specific leukocyte subsets. Rat leukocytes have been found to secrete angiotensinogen (35), human neutrophils and mast cells were shown to convert AngI to AngII (36), and binding sites were found on human mononuclear cells by using radioactively labeled AngII (37). Although the contribution of monocyte-derived macrophages is established in the pathogenesis of kidney and vascular lesions, other leukocytes, such as NK cells, T cells, and DC, are shown to play crucial roles in renal and vascular diseases (16,38,39). AngII was previously shown to augment murine splenic lymphocyte proliferation by Nataraj et al. (40) and others (41), and angiotensin receptor blockers suppress murine T cell antigen responses (42). Our data demonstrate the co-stimulatory effect of AngII on human leukocytes; although AngII alone had no effect, NK and T cell proliferation in the presence of mitogen (either PHA or anti-CD3) was potently enhanced when AngII was added. Using CFSE in combination with subtyping antibodies before and after administration of AngII, we demonstrate that CD8+ T cells and CD56+ NK cells proliferate in response to AngII. In agreement with our data, IFN-γ mRNA levels and anti–CD3-stimulated lymphocyte proliferation were shown to be downregulated in renal transplant patients who were on long-term AT1 blockade (43). In our system, although no difference was evident in the absolute cytokine concentration in response to AngII, stimulating monocyte-depleted NK and T cells with AngII resulted in an increase in IFN-γ:IL-4 at 48 h. A Th1 immune response has been reported to be important in the pathogenesis of inflammation-induced vascular diseases (44). However, the interaction of AngII with Th1/Th2 cytokines and its importance in the development of inflammation remains to be fully explored.
When AngI and angiotensinogen were added to cells, a similar increase in proliferation resulted as in AngII-treated cells, indicating that cells in our system possess the necessary components to convert angiotensinogen to AngI and AngI to AngII. Indeed, we are also demonstrating the presence of RAS elements in human T and NK cells, which shows that they possess a complete enzymatic repertoire to synthesize their own AngII. Renin receptor mRNA was detected in T and NK cells, monocytes, iDC, and mDC. Renin could have cellular effects independent of AngII after binding to the renin receptor (45), and this could create an additional mechanism for renin-induced inflammation.
We also characterized AT1 and AT2 receptor expression in T cells, NK cells, and DC. We were able to demonstrate protein expression for both receptors in T and NK cells but found only slight amounts of AT1 in monocytes by Western blot. Expression of the AT1 and/or AT2 receptor was studied by Rasini et al. (27) and Nahmod et al. (10); however, interpretation of these results is problematic, given that optimal isotype controls were not used for polyclonal antibodies, which are likely to exhibit some nonspecific staining. By FACS, we could not convincingly demonstrate surface expression of AT1, and even with permeabilization, both AT1 and the isotype control gave comparable levels of expression. These results are most likely a consequence of the lack of availability of a suitable commercial flow cytometric antibody, given that we were able to detect AT1 protein by Western blot. Conversely, AT2 was clearly detectable on the surface of DC and intracellularly in T cells, monocytes, and NK cells. The AT2 receptor has been thought to antagonize the actions of the AT1 receptor and therefore to have beneficial effects in hypertension, cell growth, vascular remodeling, and proliferation (46). More recent studies, however, showed that the effects mediated through the AT2 receptor may in fact complement those mediated through the AT1 receptor (47,48), which is supported by our data indicating that the AT2 receptor is expressed on immunocompetent cells that undergo enhanced proliferation, calcium flux, and chemotaxis in response to AngII.
One interesting aspect of AngII in inflammation is its ability to recruit inflammatory cells; recruitment of inflammatory cells is one of the hallmarks of CKD. When AngII is released by inflamed tissue, a gradient is created, which could enhance the directional migration of inflammatory cells. In this regard, human monocytes and murine splenocytes were found to migrate to AngII (30,49). We are reporting for the first time that T cells, NK cells, and DC also migrate significantly to AngII. Given our chemotaxis data, AngII may orchestrate the collective recruitment of leukocyte subsets to sites of inflammation (e.g., atherosclerotic lesions). Our data also show that secretion of the chemokine CCL5/RANTES by the mixture of NK and T cells was upregulated in response to AngII, an effect that previously was noted in rat glomerular epithelial cells (50). Because CCL5/RANTES is known to be chemotactic for cells that bear the CCR1, CCR3, and CCR5 chemokine receptors, this may serve to further recruit leukocytes to sites of inflammation or perhaps to enhance directly T cell proliferation via the CCR5 receptor. The calcium flux elicited by AngII showed a comparable response to the calcium flux elicited by ATP. Both antagonists were able to block this receptor signal significantly, indicating the likely presence of both AngII receptors, which is consistent with our QPCR and Western blot data. Furthermore, the fact that the combination of both antagonists was unable to suppress completely calcium flux suggests the presence of another angiotensin receptor in leukocytes, such as AT4, which may have implications for differences in therapeutic responses.
We have demonstrated that AngII can have co-stimulatory effects on T and NK cells, that specific elements of the RAS exist on T cells, NK cells, and DC, and that DC, NK cells, and T cells migrate potently to AngII. The results of this study will allow us to comprehend better the mechanisms by which AngII leads to inflammation, as RAS antagonists may be used in conjunction with other anti-inflammatory agents as therapy for common diseases in which inflammation plays a major pathogenic role.
This work was funded in part by National Institutes of Health (NIH) grant P01 AI50157, DCI, and the Merck Medical School Award (R.A.). R.A. is the recipient of a Juvenile Diabetes Career Development Award. This work was supported in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.
Published online ahead of print. Publication date available at www.jasn.org.
M.J. and D.H.M. contributed equally to this work.
We dedicate this article to the memory of Joan Sechler, who died on July 5, 2006.
1. Ruiz-Ortega M, Lorenzo O, Suzuki Y, Ruperez M, Egido J: Proinflammatory actions of angiotensins. Curr Opin Nephrol Hypertens 10 : 321 –329, 2001
2. Long DA, Price KL, Herrera-Acosta J, Johnson RJ: How does angiotensin II cause renal injury? Hypertension 43 : 722 –723, 2004
3. Cooper ME, Tikellis C, Thomas MC: Preventing diabetes in patients with hypertension: One more reason to block the renin-angiotensin system. J Hypertens Suppl 24 : S57 –S63, 2006
4. Ruiz-Ortega M, Bustos C, Hernandez-Presa MA, Lorenzo O, Plaza JJ, Egido J: Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-kappa B activation and monocyte chemoattractant protein-1 synthesis. J Immunol 161 : 430 –439, 1998
5. Anders HJ, Ninichuk V, Schlondorff D: Progression of kidney disease: Blocking leukocyte recruitment with chemokine receptor CCR1 antagonists. Kidney Int 69 : 29 –32, 2006
6. Lombardi DM, Viswanathan M, Vio CP, Saavedra JM, Schwartz SM, Johnson RJ: Renal and vascular injury induced by exogenous angiotensin II is AT1 receptor-dependent. Nephron 87 : 66 –74, 2001
7. Cattell V: Macrophages in acute glomerular inflammation. Kidney Int 45 : 945 –952, 1994
8. Chow F, Ozols E, Nikolic-Paterson DJ, Atkins RC, Tesch GH: Macrophages in mouse type 2 diabetic nephropathy: Correlation with diabetic state and progressive renal injury. Kidney Int 65 : 116 –128, 2004
9. Nikolic-Paterson DJ, Lan HY, Hill PA, Atkins RC: Macrophages in renal injury. Kidney Int Suppl 45 : S79 –S82, 1994
10. Nahmod KA, Vermeulen ME, Raiden S, Salamone G, Gamberale R, Fernandez-Calotti P, Alvarez A, Nahmod V, Giordano M, Geffner JR: Control of dendritic cell differentiation by angiotensin II. FASEB J 17 : 491 –493, 2003
11. Angeli V, Llodra J, Rong JX, Satoh K, Ishii S, Shimizu T, Fisher EA, Randolph GJ: Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization. Immunity 21 : 5 61–74, 2004
12. Farag SS, Caligiuri MA: Human natural killer cell development and biology. Blood Rev 20 : 123 –137, 2005
13. Remuzzi G, Perico N, Macia M, Ruggenenti P: The role of renin-angiotensin-aldosterone system in the progression of chronic kidney disease. Kidney Int Suppl 99 : S57 –S65, 2005
14. Vaziri ND, Xu ZG, Shahkarami A, Huang KT, Rodriguez-Iturbe B, Natarajan R: Role of AT-1 receptor in regulation of vascular MCP-1, IL-6, PAI-1, MAP kinase, and matrix expressions in obesity. Kidney Int 68 : 2787 –2793, 2005
15. Voshol H, Dullens HF, Den Otter W, Vliegenthart JF: Human natural killer cells: A convenient purification procedure and the influence of cryopreservation on cytotoxic activity. J Immunol Methods 165 : 21 –30, 1993
16. Fiorina P, Ansari MJ, Jurewicz M, Barry M, Ricchiuti V, Smith RN, Shea S, Means TK, Auchincloss H Jr, Luster AD, Sayegh MH, Abdi R: Role of CXC chemokine receptor 3 pathway in renal ischemic injury. J Am Soc Nephrol 17 : 716 –723, 2006
17. McDermott DH, Fong AM, Yang Q, Sechler JM, Cupples LA, Merrell MN, Wilson PW, D'Agostino RB, O'Donnell CJ, Patel DD, Murphy PM: Chemokine receptor mutant CX3CR1–M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans. J Clin Invest 111 : 1241 –1250, 2003
18. Makhseed M, Raghupathy R, El-Shazly S, Azizieh F, Al-Harmi JA, Al-Azemi MM: Pro-inflammatory maternal cytokine profile in preterm delivery. Am J Reprod Immunol 49 : 308 –318, 2003
19. Panitsas FP, Theodoropoulou M, Kouraklis A, Karakantza M, Theodorou GL, Zoumbos NC, Maniatis A, Mouzaki A: Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response. Blood 103 : 2645 –2647, 2004
20. Taub DD, Turcovski-Corrales SM, Key ML, Longo DL, Murphy WJ: Chemokines and T lymphocyte activation: I. Beta chemokines costimulate human T lymphocyte activation in vitro. J Immunol 156 : 2095 –2103, 1996
21. Nguyen G, Delarue F, Burckle C, Bouzhir L, Giller T, Sraer JD: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 109 : 1417 –1427, 2002
22. Costerousse O, Allegrini J, Lopez M, Alhenc-Gelas F: Angiotensin I-converting enzyme in human circulating mononuclear cells: Genetic polymorphism of expression in T-lymphocytes. Biochem J 290 : 33 –40, 1993
23. Lapteva N, Nieda M, Ando Y, Ide K, Hatta-Ohashi Y, Dymshits G, Ishikawa Y, Juji T, Tokunaga K: Expression of renin-angiotensin system genes in immature and mature dendritic cells identified using human cDNA microarray. Biochem Biophys Res Commun 285 : 1059 –1065, 2001
24. Lanctot PM, Leclerc PC, Clement M, Auger-Messier M, Escher E, Leduc R, Guillemette G: Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor. Biochem J 390 : 367 –376, 2005
25. Elkord E, Williams PE, Kynaston H, Rowbottom AW: Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells. Immunology 114 : 204 –212, 2005
26. Okamura A, Rakugi H, Ohishi M, Yanagitani Y, Takiuchi S, Moriguchi K, Fennessy PA, Higaki J, Ogihara T: Upregulation of renin-angiotensin system during differentiation of monocytes to macrophages. J Hypertens 17 : 537 –545, 1999
27. Rasini E, Cosentino M, Marino F, Legnaro M, Ferrari M, Guasti L, Venco A, Lecchini S: Angiotensin II type 1 receptor expression on human leukocyte subsets: A flow cytometric and RT-PCR study. Regul Pept 134 : 69 –74, 2006
28. Gasser O, Missiou A, Eken C, Hess C: Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and upregulate it rapidly to the cell surface upon activation. Blood 106 : 3718 –3724, 2005
29. Chai SY, Fernando R, Peck G, Ye SY, Mendelsohn FA, Jenkins TA, Albiston AL: The angiotensin IV/AT4 receptor. Cell Mol Life Sci 61 : 2728 –2737, 2004
30. Kintscher U, Wakino S, Kim S, Fleck E, Hsueh WA, Law RE: Angiotensin II induces migration and Pyk2/paxillin phosphorylation of human monocytes. Hypertension 37 : 587 –593, 2001
31. Vaziri ND: Roles of oxidative stress and antioxidant therapy in chronic kidney disease and hypertension. Curr Opin Nephrol Hypertens 13 : 93 –99, 2004
32. Remuzzi G, Benigni A, Remuzzi A: Mechanisms of progression and regression of renal lesions of chronic nephropathies and diabetes. J Clin Invest 116 : 288 –296, 2006
33. Friedland J, Setton C, Silverstein E: Angiotensin converting enzyme: Induction by steroids in rabbit alveolar macrophages in culture. Science 197 : 64 –65, 1977
34. Diet F, Pratt RE, Berry GJ, Momose N, Gibbons GH, Dzau VJ: Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease. Circulation 94 : 2756 –2767, 1996
35. Gomez RA, Norling LL, Wilfong N, Isakson P, Lynch KR, Hock R, Quesenberry P: Leukocytes synthesize angiotensinogen. Hypertension 21 : 470 –475, 1993
36. Reilly CF, Tewksbury DA, Schechter NM, Travis J: Rapid conversion of angiotensin I to angiotensin II by neutrophil and mast cell proteinases. J Biol Chem 257 : 8619 –8622, 1982
37. Shimada K, Yazaki Y: Binding sites for angiotensin II in human mononuclear leucocytes. J Biochem 84 : 1013 –1015, 1978
38. Bobryshev YV, Lord RS: Co-accumulation of dendritic cells and natural killer T cells within rupture-prone regions in human atherosclerotic plaques. J Histochem Cytochem 53 : 781 –785, 2005
39. Burne-Taney MJ, Liu M, Ascon D, Molls RR, Racusen L, Rabb H: Transfer of lymphocytes from mice with renal ischemia induce albuminuria in naive mice: A possible mechanism linking early injury to progressive renal disease? Am J Physiol Renal Physiol 291 : F981 –F986, 2006
40. Nataraj C, Oliverio MI, Mannon RB, Mannon PJ, Audoly LP, Amuchastegui CS, Ruiz P, Smithies O, Coffman TM: Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. J Clin Invest 104 : 1693 –1701, 1999
41. Kunert-Radek J, Stepien H, Komorowski J, Pawlikowski M: Stimulatory effect of angiotensin II on the proliferation of mouse spleen lymphocytes in vitro is mediated via both types of angiotensin II receptors. Biochem Biophys Res Commun 198 : 1034 –1039, 1994
42. Sagawa K, Nagatani K, Komagata Y, Yamamoto K: Angiotensin receptor blockers suppress antigen-specific T cell responses and ameliorate collagen-induced arthritis in mice. Arthritis Rheum 52 : 1920 –1928, 2005
43. Weidanz JA, Jacobson LM, Muehrer RJ, Djamali A, Hullett DA, Sprague J, Chiriva-Internati M, Wittman V, Thekkumkara TJ, Becker BN: ATR blockade reduces IFN-gamma production in lymphocytes in vivo and in vitro. Kidney Int 67 : 2134 –2142, 2005
44. Ranjbaran H, Sokol SI, Gallo A, Eid RE, Iakimov AO, D'Alessio A, Kapoor JR, Akhtar S, Howes CJ, Aslan M, Pfau S, Pober JS, Tellides G: An inflammatory pathway of IFN-gamma production in coronary atherosclerosis. J Immunol 178 : 592 –604, 2007
45. Nguyen G: Renin/prorenin receptors. Kidney Int 69 : 1503 –1506, 2006
46. AbdAlla S, Lother H, Abdel-tawab AM, Quitterer U: The angiotensin II AT2 receptor is an AT1 receptor antagonist. J Biol Chem 276 : 39721 –39726, 2001
47. Steckelings UM, Kaschina E, Unger T: The AT2 receptor: A matter of love and hate. Peptides 26 : 1401 –1409, 2005
48. Reudelhuber TL: The continuing saga of the AT2 receptor: A case of the good, the bad, and the innocuous. Hypertension 46 : 1261 –1262, 2005
49. Weinstock JV, Kassab J: Chemotactic response of splenic mononuclear cells to angiotensin II in murine schistosomiasis. J Immunol 137 : 2020 –2024, 1986
50. Wolf G, Ziyadeh FN, Thaiss F, Tomaszewski J, Caron RJ, Wenzel U, Zahner G, Helmchen U, Stahl RA: Angiotensin II stimulates expression of the chemokine RANTES in rat glomerular endothelial cells. Role of the angiotensin type 2 receptor. J Clin Invest 100 : 1047 –1058, 1997