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The Journal of Immunology, 2006, 176: 5577-5586.
Copyright © 2006 by The American Association of Immunologists

Angiotensin II-Induced Mononuclear Leukocyte Interactions with Arteriolar and Venular Endothelium Are Mediated by the Release of Different CC Chemokines1

Teresa Mateo2,*, Yafa Naim Abu Nabah2,*, May Abu Taha*, Manuel Mata*, Miguel Cerdá-Nicolás{dagger}, Amanda E. I. Proudfoot{ddagger}, Rolf A. K. Stahl§, Andrew C. Issekutz, Julio Cortijo*,||, Esteban J. Morcillo*, Peter J. Jose# and Maria-Jesus Sanz3,*

* Department of Pharmacology and {dagger} Department of Pathology, Faculty of Medicine, University of Valencia, Valencia, Spain; {ddagger} Serono Pharmaceuticals Research Institutes, Geneva, Switzerland; § Division of Nephrology, Department of Medicine, University of Hamburg, Hamburg, Germany; Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada; || Research Foundation, University General Hospital Consortium, Valencia, Spain; and # Leukocyte Biology Section, Biomedical Sciences Division, Imperial College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1{alpha} generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80–90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1–1000 nM Ang-II, predominantly acting at its AT1 receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Atherosclerosis is one of the leading causes of morbidity and mortality in Western countries and the main contributor to the pathogenesis of myocardial and cerebral infarction, gangrene, and loss of function in the extremities. This process bears several histopathologic similarities to chronic inflammation. The early atherosclerotic lesion involves an inflammatory response consisting of intimal accumulation of T lymphocytes and lipid-laden macrophages, and these events occur continuously throughout the entire atherogenic process (1, 2, 3).

In many inflammatory diseases, leukocytes are recruited solely via postcapillary venules; however, leukocyte/arterial interactions are believed to be important for mononuclear leukocyte infiltration in atherogenesis. Recruitment of leukocytes requires the expression, on the endothelium and leukocytes, of various classes of cell adhesion molecules (CAMs)4 and the presence of counterreceptor molecules on the leukocyte/endothelial cell (4). Several CAMs such as selectins, ICAM-1, and VCAM-1 have all been implicated in atherogenesis (3). In this context, arterial endothelium has been shown to express the same CAMs as those expressed in venular endothelium (5, 6).

Angiotensin II (Ang-II), the main effector peptide of the renin-angiotensin system, is implicated in atherogenesis beyond its hemodynamic effects (7). We have demonstrated that 4 h of exposure to Ang-II in vivo causes arteriolar leukocyte adhesion in the rat mesenteric microcirculation, an effect mediated through interaction with its AT1 receptor subtype (8) and not observed under acute (1 h) stimulation with this peptide hormone (9). Furthermore, mononuclear cells were found to be the primary cells attached to the arteriolar endothelium, whereas the leukocytes interacting with the venular endothelium at the same time were predominantly neutrophils. Despite these findings, the same CAMs were expressed in both the arteriolar and venular endothelia in response to Ang-II (8), suggesting that other mechanisms were responsible for the differential cellular distribution within the microcirculation.

In addition to CAMs, chemoattractant molecules such as the chemokines are involved in the regulation of leukocyte trafficking (10). Although we have previously demonstrated the role of CXC chemokines in neutrophil recruitment induced by Ang-II (11), several CC chemokines such as MCP-1 (MCP-1/CCL2), MIP-1{alpha} (MIP-1{alpha}/CCL3), MIP-1beta/CCL4, RANTES/CCL5, and MCP-4/CCL13 seem to be involved in atherosclerotic lesion formation (12, 13). Interestingly, Ang-II induces increased expression of MCP-1 and RANTES in several animal models in vivo (14, 15), which might explain, in part, the mononuclear cell recruitment elicited by this peptide hormone.

Despite these findings, no extensive studies have been conducted to characterize the CC chemokines directly released by Ang-II and their possible consequences in the mononuclear leukocyte infiltration induced by this peptide hormone. Hence, the present study focuses on the ability of Ang-II to mediate mononuclear leukocyte accumulation in vivo and the mechanisms involved in this response. We first established that Ang-II provokes mononuclear cell recruitment in vivo following the release of MCP-1, RANTES, and MIP-1{alpha} and then investigated, by the use of intravital microscopy, the involvement of these chemokines in Ang-II-induced leukocyte interactions with both arteriolar and venular endothelial cells in vivo. Finally, we stimulated whole blood and cultures of human umbilical arterial endothelial cells (HUAECs) and HUVECs with Ang-II to determine the release of a wider spectrum of CC chemokines that may participate in the responses elicited by this peptide hormone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mononuclear leukocyte migration into the peritoneal cavity

All the studies were approved by the Institutional Ethics Committee. Male Sprague Dawley rats (200–250 g) were sedated with ether and injected i.p. with 5 ml of PBS or 1 nM Ang-II. After 1, 4, 8, and 24 h of the injection, animals were killed by an anesthetic overdose, and the peritoneal cavity was first lavaged with 5 ml of PBS and then with 30 ml of heparinized PBS (10 U/ml).

The 5- and 30-ml lavages were centrifuged separately to obtain the cell pellets that were then combined for total leukocyte counts in a hemocytometer and differential cell analysis of 500 cells/slide on cytospins stained with May-Grünwald and Giemsa stains. The results are expressed as the number of mononuclear leukocytes recovered from each cavity. The supernatants from the first (5 ml) lavage, after addition of carrier protein (0.5% BSA) and storage at –20°C, were used for chemokine ELISAs.

Intravital microscopy

The details of the experimental preparation have been described previously (8). Briefly, male Sprague Dawley rats (200–250 g) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and the trachea, right jugular vein, and carotid artery were cannulated. After performing a midline abdominal incision, a segment of the mid-jejunum was exteriorized and placed over an optically clear viewing pedestal maintained at 37°C. The exposed mesentery was continuously superfused with warmed bicarbonate buffered saline equilibrated with 5% CO2 in nitrogen. An orthostatic microscope (Nikon Optiphot-2, SMZ1) equipped with x20 objective lens (Nikon SLDW) and x10 eyepiece permitted tissue visualization. A video camera (Sony SSC-C350P) mounted on the microscope projected images onto a color monitor (Sony Trinitron PVM-14N2E), and these images were captured on a videotape (Sony SVT-S3000P) for playback analysis (final magnification of the video screen was x1300). Arterioles (20–30 µm in diameter) and single unbranched mesenteric venules (25–40 µm in diameter) were selected, and the diameters measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University). Centerline RBC velocity (Vrbc) was also measured on-line with an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University). Venular blood flow and wall shear rate ({gamma}) were calculated as described previously (16). The number of rolling, adherent, and emigrated leukocytes was determined off-line during playback analysis of videotaped images.

Experimental protocol

Animals were sedated and i.p. injected with 5 ml of PBS or Ang-II (1 nM), as described above. After 4 h, the mesentery was exposed for measurement of venular leukocyte rolling flux, velocity, adhesion, emigration, arteriolar leukocyte adhesion, mean arterial blood pressure (MABP), venular and arteriolar Vrbc shear rate, and diameter over a 5-min period.

To investigate the involvement of CC chemokines, rats were pretreated 15 min before Ang-II injection with Met-RANTES, a CCR1 and CCR5 antagonist (1 mg/kg i.v.), or with a rabbit anti-rat MCP-1 antiserum (1 ml/rat i.v.), or with a combination of both. These doses of Met-RANTES and anti-rat MCP-1 antiserum were effective in reducing inflammation in a rat model of colitis (17) and specifically neutralizing MCP-1 activity in vivo (18, 19), respectively. To determine the effect of these compounds on levels of circulating leukocytes, blood samples were taken from the rats when all the measurements were performed.

In another set of experiments, to determine whether platelets contribute to leukocyte recruitment, rats were administered with an anti-rat platelet serum (0.5 ml/kg i.v.) 24 h prior Ang-II i.p. injection.

Immunohistochemistry

After the completion of the intravital microscopy measurements, the mesentery was isolated, fixed in 4% paraformaldehyde, dehydrated using graded acetone washes at 4°C, and embedded in paraffin wax for localization of P-selectin, VCAM-1 MCP-1, and RANTES using a modified avidin and biotin immunoperoxidase technique as described previously (8). Tissue sections (4-µm thick) were incubated for 24 h with Ab at 200 µg/ml: anti-rat-P-selectin mAb RMP-1, or with anti-rat-VCAM-1 mAb 5F10, or their isotype-matched control Ab UPC 10 (murine IgG2a). For immunolocalization of MCP-1 and RANTES, tissue sections were incubated for 24 h with an Ab (100 µg/ml) against rat MCP-1 or rat-RANTES. Positive staining was defined as an arteriole or venule displaying brown reaction product.

Determination of surface expression of CD11b/CD18 integrins

The expression of CD11b/CD18 integrins was determined on rat monocytes in citrated whole blood obtained by cardiac puncture. Duplicate samples (100 µl) were incubated at 37°C with saline, Met-RANTES (0.1 µg), or anti-rat MCP-1 antiserum (10 µl) for 1 h before a 4-h incubation with Ang-II (100 nM) or saline as a control. Samples were then incubated for 20 min on ice in the dark with saturating amounts (10 µl) of the conjugated mAb anti-rat-CD11b/CD18-PE plus a specific monocyte/macrophage marker, anti-rat ED1-FITC mAb. RBCs were lysed and leukocytes fixed using an automated EPICS Q-PREP system (Coulter Electronics). Samples were run in an EPICS XL-MCL Flow cytometer (Beckman Coulter). The expression of the surface Ag (PE-fluorescence) was measured in monocytes identified by their FITC-fluorescence.

Cell culture

HUVECs and HUAECs were isolated by collagenase treatment (20) and maintained in human endothelial cells’ specific medium endothelial basal medium-2 supplemented with endothelial growth medium-2 and 10% FCS. Cells up to passage 2 were grown to confluence on 24-well culture plates. Before every experiment, cells were incubated for 16 h in medium containing 1% FCS and then returned to the 10% FCS medium at the start of all experimental incubations.

Cells were stimulated with 1–1000 nM Ang-II for 1, 4, 24, and 48 h. Selective antagonists of AT1 (10 µM losartan) or AT2 (10 µM PD123,319) receptors, or a combination of both, were added to some wells 1 h before Ang-II (100 nM) stimulation. At the end of the experiment, cell-free supernatants were stored at –20°C for ELISA.

Determination of chemokine release in human whole blood

Human whole blood (10 U ml/heparin, from healthy volunteers) was incubated with saline or 100 nM Ang-II for 4 h. Before centrifugation to obtain plasma, further heparin was added (to 100 U/ml) to promote the release of any chemokines bound to erythrocytes. Plasma samples were stored at –80°C for ELISA.

Determination of RANTES release in human platelets

Human whole blood was collected from normal volunteers in heparinized syringes and centrifuged at 120 x g for 15 min at room temperature. The platelet-rich plasma was resuspended in washing buffer (9 mM Na2 EDTA, 26.4 mM Na2HPO4, and 140 mM NaCl) and centrifuged at 800 x g for 10 min at room temperature. Then, the pellet containing the platelets was again resuspended in washing buffer and centrifuged at 200 x g for 10 min at room temperature. The supernatant obtained was centrifuged at 800 x g for 15 min at room temperature. The total yield of platelets was >1 x 108 (purity, >99%).The pelleted platelets were then resuspended in the assay buffer (HBSS, 10 mM HEPES, 1 mM Ca2+ and Mg2+, and 0.5% BSA) at a final concentration of 1 x 106 platelets/ml. Platelets were then incubated with saline or Ang-II 1 µM for 1 h at 37°C. After this time period, the samples were again centrifuged at 800 x g for 15 min, and the platelet-free supernatants were stored at –20°C for RANTES ELISA.

ELISAs

Rat MCP-1, RANTES, and MIP-1{alpha} levels were determined by conventional sandwich ELISAs. Results are expressed as pM chemokine in the supernatant from the 5-ml lavage. No cross-reactions were detected with any rat chemokines tested, other than that nominated in the specific assay: MIP-2, KC, MCP-1, RANTES or MIP-1{alpha}, all at 104 pM (cross-reaction < 0.005%).

Human CC chemokines (MCP-1, MCP-3/CCL7, MCP-4, RANTES, MIP-1{alpha}, MDC/CCL22, eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) were measured in plasma and cell culture supernatants from endothelial cells.

Statistical analysis

All values are mean ± SEM. Data between groups were compared using an ANOVA (one way-ANOVA) with a Newman-Keuls post hoc correction for multiple comparisons. Statistical significance was set at p < 0.05.

Materials

Ang-II, pentobarbital, UPC 10, and PD123,319 were purchased from Sigma-Aldrich. Losartan was donated by Merck Sharp & Dohme (Madrid, Spain). Endothelial basal medium-2 medium supplemented with endothelial growth medium-2 was acquired from Innogenetics. Human and rat chemokines and Abs for human eotaxin-3 and all rat chemokine ELISAs were purchased from PeproTech. The Ab pairs for all other human CC chemokine ELISAs were obtained from R&D Systems. Neutravidin-HRP was purchased from Perbio Science, and the K-Blue substrate was obtained from Neogen. The Abs RMP-1 and 5F10 were obtained as stated previously (8). Anti-rat CD11b/CD18-PE and anti-rat ED1-FITC mAbs were purchased from Serotec. The neutralizing anti-MCP-1 antiserum and Met-RANTES were produced as described previously (18, 21). Anti-rat platelet serum was obtained from Accurate Chemicals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intraperitoneal administration of 5 ml of 1 nM Ang-II in rats induced a significant mononuclear leukocyte recruitment that was maximal at 8–24 h (Fig. 1A). MCP-1, RANTES, and MIP-1{alpha} levels were elevated following Ang-II injection, peaking at 4 h (Fig. 1, B, C, and D), before significant mononuclear cell accumulation was detected. The amount of MCP-1 released by Ang-II was 40-fold higher than that of RANTES and MIP-1{alpha}. Significant levels of these CC chemokines were still present at 8 h but had declined to basal levels by 24 h. This time course is consistent with a contribution of CC chemokines to mononuclear cell recruitment.


Figure 1
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  		FIGURE 1. Time course of Ang-II-induced mononuclear leukocyte accumulation (A), MCP-1 (B), RANTES (C), and MIP-1{alpha} (D) generation. Rats were i.p. injected with 5 ml of PBS or 1 nM Ang-II. Results are mean ± SEM for n = 4–5 animals/group: *, p < 0.05 or **, p < 0.01 relative to values in the PBS-injected group.

 
Intravital microscopy was used to examine leukocyte trafficking in the mesentery 4 h after i.p. injection of Ang-II because these events would be expected to precede leukocyte accumulation in the peritoneal cavity. Exposure to 1 nM Ang-II for this time period induced a significant enhancement of arteriolar leukocyte adhesion (Fig. 2) without causing changes in the number of circulating leukocytes, MABP, arteriolar diameter, or shear rate (Table I). The enhancement of arteriolar leukocyte adhesion was inhibited by Met-RANTES (80% inhibition), anti-rat MCP-1 (90%), and by the combination of both inhibitors (93%). The number of circulating leukocytes, MABP, arteriolar diameter, or shear rate was unaffected by these treatments (Table I).


Figure 2
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  		FIGURE 2. Effects of Met-RANTES and anti-MCP-1 antiserum on subacute (4 h) Ang-II-induced leukocyte adhesion to rat mesenteric arterioles. Rats were treated i.p. with PBS (n = 7) or 1 nM Ang-II (n = 7). Some animals, were pretreated with Met-RANTES (1 mg/kg, i.v., n = 8), an anti-MCP-1 antiserum (1 ml/rat, i.v., n = 6), or combination of both (n = 5) 15 min before the administration of Ang-II. Results are mean ± SEM: **, p < 0.01 relative to the PBS group; ++, p < 0.01 relative to the Ang-II group.

 

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  		Table I. Hemodynamic parameters and systemic leukocyte countsa

 
In the postcapillary venules of the same animals, 4-h exposure to Ang-II induced a significant increase in venular leukocyte rolling flux, adhesion, and emigration, as well as a concomitant decrease in venular leukocyte rolling velocity (Fig. 3) without modifying venular diameter or venular shear rate (Table I). Pretreatment with Met-RANTES inhibited Ang-II-induced leukocyte rolling flux, adhesion, and emigration by 85, 96, and 86%, respectively, and reversed the decrease in leukocyte rolling velocity (Fig. 3). Interestingly, the neutralizing antiserum against MCP-1 did not significantly modify Ang-II-induced leukocyte responses in venules, although a combination of both inhibitors was slightly more effective than Met-RANTES alone, the leukocyte adhesion and emigration responses being virtually abolished (Fig. 3). None of the inhibitors affected the venular diameter or shear rate (Table I).


Figure 3
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  		FIGURE 3. Effects of Met-RANTES and anti-MCP-1 antiserum on subacute (4 h) Ang-II-induced leukocyte responses within rat mesenteric postcapillary venules. These responses were measured in the same rats as those described in the legend to Fig. 2: venular responses of leukocyte rolling flux (A), leukocyte rolling velocity (B), leukocyte adhesion (C), and leukocyte emigration (D) are mean ± SEM. *, p < 0.05 or **, p < 0.01 relative to the PBS group; +, p < 0.05 or ++, p < 0.01 relative to the Ang-II group.

 
These studies suggest a major role for MCP-1 together with CCR1 or CCR5 agonists, such as MIP-1{alpha} and RANTES, in leukocyte interactions with the arteriolar endothelium but a lesser role for MCP-1 in the responses at the postcapillary venules. In this context, immunohistochemistry studies revealed no expression of RANTES and MCP-1 in the arterioles and postcapillary venules of PBS-injected animals (Fig. 4, A–D). In contrast, while marked expression of RANTES was observed in the arterioles of Ang-II-treated animals, in the postcapillary venules this chemokine was present in moderate amounts (Fig. 4, E and F). In addition, in the arteriolar endothelium, after 4 h of Ang-II exposure, MCP-1 was also expressed, but its expression was lower than that encountered for RANTES (Fig. 4G). Consistent with our findings, MCP-1 was weakly expressed in the postcapillary venules exposed to Ang-II (Fig. 4H).


Figure 4
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  		FIGURE 4. Representative photomicrographs showing immunolocalization of RANTES and MCP-1 in rat mesenteric arterioles and postcapillary venules (A–H). Mesentery was fixed for staining with anti-RANTES (A, B, E, and F) or MCP-1 (C, D, G, and H) mAbs 4 h after the i.p. injection of PBS (A–D) or Ang-II (1 nM; E–H). Brown reaction product indicates positive immunoperoxidase localization on the vascular endothelium. All panels are lightly counterstained with hematoxylin. Results are representative of n = 4–5 experiments with each treatment.

 
To examine the potential contribution of CC chemokines to adhesion molecule up-regulation in response to Ang-II, we performed immunohistochemical studies on mesentery and flow cytometry on blood monocytes. In control PBS-injected animals, no arteriolar (Fig. 5), and only weak venular (data not shown), staining for P-selectin and VCAM-1 was detected. These adhesion molecules were clearly up-regulated 4 h after i.p. injection of Ang-II (Fig. 5). Similarly, incubation of rat whole blood with 100 nM Ang-II for 4 h up-regulated monocyte CD11b expression (Fig. 6). However, the effects of CC chemokine blockade on these responses were different. Met-RANTES had no detectable effect on the up-regulation of P-selectin or VCAM-1 in the mesentery but inhibited (72%) the Ang-II-induced CD11b up-regulation on blood monocytes, whereas anti-MCP-1 had no detectable effect on any of these adhesion molecules (Figs. 5 and 6).


Figure 5
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  		FIGURE 5. Representative photomicrographs showing immunolocalization of P-selectin and VCAM-1 in rat mesenteric arterioles (A–H). Mesentery was fixed for staining with anti-P-selectin (A–D) or VCAM-1 (E–H) mAbs 4 h after the i.p. injection of PBS (A and E) or Ang-II (1 nM; B and F). Some animals were pretreated with Met-RANTES (C and G) or anti-MCP-1 antiserum (D and H). Brown reaction product indicates positive immunoperoxidase localization on the vascular endothelium. All panels are lightly counterstained with hematoxylin. Results are representative of n = 4–5 experiments with each treatment.

 

Figure 6
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  		FIGURE 6. Effects of Met-RANTES and anti-MCP-1 antiserum on Ang-II-induced CD11b up-regulation on rat monocytes. Rat whole blood (pretreated for 1 h with saline, 0.1 µg of Met-RANTES or 10 µl of anti-MCP-1 antiserum) was incubated for 4 h with either saline or Ang-II (100 nM) and then stained with conjugated mAbs for CD11b and a specific monocytes/macrophage marker. Flow cytometry results are mean ± SEM of mean fluorescence intensity of n = 4 experiments. *, p < 0.05 relative to the saline group; +, p < 0.05 relative to the Ang-II group.

 
To extend these findings to man, we measured the production of different CC chemokines in response to Ang-II in arterial and venular endothelial cells in culture at 1, 4, 24, and 48 h and in whole blood at 4 h. There was a dose-dependent release of CC chemokines into the culture fluid of both HUAECs and HUVECs stimulated with Ang-II (1–1000 nM). Of those chemokines measured, by far the largest amounts released were of MCP-1. Significant increases in MCP-1 were detected as early as 1 h after stimulation with doses as low as 1 nM Ang-II in HUAECs and 10 nM in HUVECs, and levels continued to rise for up to 24 h (Fig. 7). RANTES was increased at 4 h in HUAECs, again at doses as low as 1 nM Ang-II, with less generation in HUVECs (Fig. 8, B and E). Small increases in MCP-3 were also detected at 4 h in HUAECs (data not shown) and in both cell cultures at 24 h (Fig. 8, C and F). After 48 h of stimulation with Ang-II, significant increases were detected only for MCP-1 and -3 (data not shown). In general, these responses appear to be mediated through interaction of Ang-II with its AT1 receptor since losartan, but not the selective AT2 receptor antagonist PD123,319, inhibited 100 nM Ang-II-induced CC chemokine release (Figs. 7 and 8). However, RANTES release was partly inhibited by PD123,319 (Fig. 8, B and E).


Figure 7
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  		FIGURE 7. Effects of Ang-II on MCP-1 release from endothelial cells. HUAECs and HUVECs were stimulated with Ang-II (1–1000 nM), or with 100 nM Ang-II + 10 µM losartan, + 10 µM PD1223,319, or + a combination of both antagonists. The release of MCP-1 (pM in the cell supernatant) at 1h (A, D), 4h (B, E) and 24h (C, F) is expressed as mean ± SEM of n = 5–6 experiments: *, p < 0.05 or **, p < 0.01 relative to values in the medium control group; +, p < 0.05 or ++, p < 0.01 relative to the 100 nM Ang-II group.

 

Figure 8
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  		FIGURE 8. Effects of Ang-II on Eotaxin-3, RANTES, and MCP-3 release from endothelial cells. HUAECs or HUVECs were stimulated with Ang-II (1–1000 nM), or with 100 nM Ang-II + 10 µM losartan, + 10 µM PD1223,319, or a combination of both antagonists. The release of eotaxin-3 at 1 h (A and D), RANTES at 4 h (B and E), and MCP-3 at 24 h (C and F) in response to Ang-II is expressed as mean ± SEM of n = 5–6 experiments: *, p < 0.05 or **, p < 0.01 relative to values in the medium control group; +, p < 0.05 or ++, p < 0.01 relative to the 100 nM Ang-II group.

 
We also investigated the release of CCR3-selective agonists because eotaxin-1 and -3 have been shown to antagonize responses to MCP-1 acting at the CCR2 receptor (22, 23). There was no detectable eotaxin-1 or -2 at any time point, but eotaxin-3 was secreted, mostly by arterial cell, within 1 h of stimulation with Ang-II (Fig. 8 A,D). We found no release of MIP-1{alpha}, MCP-4, or MDC from HUAECs or HUVECs, either in the presence or absence of Ang-II (data not shown).

On the other hand, when human whole blood was stimulated with Ang-II, we did detect small increases in the plasma levels of MIP-1{alpha} but not of MCP-1 or MCP-3 (Fig. 9). However, we could not ascertain whether RANTES was released by Ang-II because this chemokine was released in high and variable amounts during the 4-h incubation with saline (2.3–5.7 nM, presumably from platelets). To further investigate this possibility, human platelets were isolated and incubated with or without Ang-II (1 µM) for 1 h, and no differences in RANTES release were detected between control and stimulated platelets (41.9 ± 8.5 vs 35.6 ± 6.9 pM/106 platelets/ml). Interestingly, in our study, increased RANTES expression was observed in the mesenteric arterioles exposed to Ang-II, and there is evidence that the deposition and immobilization of platelet-derived RANTES can trigger enhanced recruitment of monocytes on activated endothelium (24, 25); therefore, some animals were pretreated with an anti-rat platelet serum 24 h prior Ang-II i.p. injection. The administration of the serum reduced the circulating platelet count by 98% without altering circulating leukocyte count. Although thrombocytopenia tended to reduce leukocyte-endothelial cell interactions in postcapillary venules, this attenuation did not reach statistical significance (data not shown). By contrast, Ang-II-induced leukocyte adhesion in mesenteric arterioles was diminished significantly (72% inhibition) by this pretreatment (Fig. 10).


Figure 9
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  		FIGURE 9. Effects of Ang-II (100 nM) on CC chemokine production in human blood. Human blood was incubated at 37°C with 100 nM Ang-II or saline for 4 h and the release of MIP-1{alpha}, MCP-1, and MCP-3 measured by ELISA. Results (pM in the plasma) are mean ± SEM of n = 5–6 experiments: *, p < 0.05 relative to values in the saline group.

 

Figure 10
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  		FIGURE 10. Effect of an anti-rat platelet serum on subacute (4 h) Ang-II-induced leukocyte adhesion to rat mesenteric arterioles. Rats were treated i.p. with PBS (n = 5) or 1 nM Ang-II (n = 5). Some animals were administered with an anti-rat platelet serum (0.5 ml/kg i.v.) 24 h prior Ang-II (n = 5) i.p. injection. Results are mean ± SEM: **, p < 0.01 relative to the PBS group; ++, p < 0.01 relative to the Ang-II group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Mononuclear leukocyte recruitment to the arterial wall is of importance in different pathophysiological states such as atherosclerosis and hypertension where Ang-II seems to play a critical role (12, 13, 26, 27). We have demonstrated recently that in vivo Ang-II promotes mononuclear leukocyte adhesion within the rat mesenteric arterioles (8). This is in contrast to the postcapillary venules where neutrophils are the major leukocyte subtype recruited by this peptide hormone (8). Similar CAMs are expressed in both the mesenteric arterioles and the postcapillary venules (8); thus, adhesion molecules are unlikely to explain the different leukocyte interactions with these endothelia. Although CXC chemokines are also involved in atherosclerosis and we have found a rapid release (1 h) of IL-8 from HUVECs stimulated with Ang-II (11), in vivo we could not find neutrophils interacting with the arteriolar endothelium (8). For this reason, we have now investigated whether the release of CC chemokines is responsible, in part, for the mononuclear leukocyte accumulation elicited by Ang-II.

Exposure to Ang-II, at physiologically relevant and subvasoconstrictor doses (1 nM), induced mononuclear leukocyte in-filtration into the rat peritoneal cavity within 8 h. This response was preceded by the generation of the CC chemokines MCP-1 > RANTES ~ MIP-1{alpha}. Although MCP-1 has been related to Ang-II-induced mononuclear cell infiltration (14, 28, 29), RANTES and MIP-1{alpha} have only been indirectly implicated in the inflammatory response elicited by this peptide hormone (15, 29, 30). However, we found that blockade of responses to RANTES and MIP-1{alpha} with Met-RANTES, a CCR1/CCR5 antagonist, was more effective than a neutralizing anti-MCP-1 antiserum at inhibiting the extravascular recruitment of mononuclear cells in response to Ang-II (data not shown). This extravascular cell recruitment is generally regarded as being dependent on rolling, adhesion, and diapedesis events at the postcapillary venules. Indeed, the effects of these CC chemokine inhibitors on mononuclear cell recruitment to the peritoneal cavity were similar to those detected when Ang-II-induced leukocyte-venular endothelial cell interactions were measured by direct observation of the mesentery (although some of these leukocytes are known to be neutrophils (8), which also express CCR1 (31, 32), and their responses may also be inhibited by Met-RANTES).

In contrast to the venular events, the leukocytes adhering to arterioles in response to Ang-II are known to be solely mononuclear (8), and this response was markedly inhibited by anti-MCP-1 and by Met-RANTES. This suggests a role for both MCP-1, which acts solely on CCR2, in addition to an agonist of CCR1 or CCR5. The inhibitory action of Met-RANTES may be related to its inhibition of monocyte CD11b up-regulation, as detected in Ang-II-stimulated blood, whereas the MCP-1 may mediate responses within the arteriolar wall. However, neither anti-MCP-1 nor Met-RANTES affected the Ang-II-induced expression of P-selectin and VCAM-1 in mesenteric arterioles or postcapillary venules. Indeed, an interesting finding derived from blockade of CCR1/CCR5 is that CC chemokines other than MCP-1 are prominent mediators of the arteriolar leukocyte adhesion induced by Ang-II.

To examine responses of human cells to Ang-II, the induction of several CC chemokines was investigated at the protein level in arterial and venular endothelial cells and in whole blood. In agreement with the in vivo experiments, the CCR2 agonist MCP-1 was the major CC chemokine detected in HUAECs and HUVECs, and its release into the culture medium was evident as early as 1 h after addition of Ang-II. Of the other CCR2 agonists, lesser amounts of MCP-3 were released at later time points, but no MCP-4 was detected. Of the CCR1/CCR5 agonists, RANTES was induced by 4 h of stimulation with Ang-II, mostly in arterial cells, but no MIP-1 {alpha} was detected in either endothelial cell culture. All of these responses were dependent on AT1 since they were blocked by pretreatment with losartan. The induction of RANTES was apparently also dependent on AT2 since PD123,319 inhibited the response in HUAECs and, to some extent, in HUVECs. These results are in agreement with other investigators: while a recent report (30) showed that serum RANTES levels in hypertensive patients with and without type 2 diabetes mellitus were decreased by losartan treatment, RANTES expression induced by Ang-II in rat glomerular endothelial cells was transduced by AT2 receptors (15).

Mononuclear cell responses induced by MCP-1 can be inhibited by the CCR3 agonists, eotaxin-1 and eotaxin-3, acting as antagonists of CCR2 (22, 23). Thus, we reasoned if eotaxins are released by Ang-II they may inhibit responses to MCP-1. An unexpected finding was that we detected the early release of eotaxin-3 from Ang-II-stimulated HUAECs and HUVECs. This release may be independent of de novo synthesis as Oynebraten et al. (33) recently reported that eotaxin-3 is stored in Weibel-Palade bodies. Because the release of eotaxin-3 was of greater magnitude in HUAECs than in HUVECs, it is tempting to speculate that its early release may delay MCP-1-induced mononuclear cell adherence to arteriolar endothelium. This question is not within the scope of the present study but may merit further investigation as the time required to deplete endothelial cell eotaxin-3 may partly explain why mononuclear cell responses often follow those of neutrophils.

No release of eotaxins 1 or 2, MIP-1{alpha}, MCP-4 or MDC was detected in the endothelial cell cultures although some of these chemokines have been encountered in atherosclerotic plaques (12, 13, 34). These results suggest that in vivo cells other than endothelial cells might release MIP-1{alpha} in response to Ang-II. Indeed, when human whole blood was stimulated with Ang-II for 4h, significant increases in MIP-1a plasma levels were encountered. Additionally, pretreatment of whole blood with Met-RANTES reduced the increased expression of CD11b in rat monocytes stimulated with Ang-II. In this context, Ang-II receptors are present on mononuclear cells (35) and neutrophils (36) and Ang-II promotes activation of these cells (36, 37). Since activated mononuclear cells and neutrophils produce MIP-1{alpha} (38, 39), these cells may be the source of this chemokine in our studies.

It is also well known that platelets store RANTES protein in their {alpha}-granules and release it during acute stages of inflammation (40, 41). In addition, different studies have shown that the deposition and immobilization of platelet-derived RANTES can trigger enhanced recruitment of monocytes and lymphocytes on activated endothelium (24, 25, 42). In the present study, we found no differences in RANTES release either in human whole blood or in isolated platelets when they were stimulated with Ang-II. In contrast, in vivo, increased RANTES expression was detected in the arterioles exposed to Ang-II (Fig. 4). Furthermore, when animals were depleted of platelets, the mononuclear cell recruitment elicited by Ang-II was reduced dramatically. These results suggest that other endogenous mediators released by this peptide hormone are responsible of RANTES endothelial deposition by platelets. In this regard, we demonstrated that a platelet-activating factor was involved in Ang-II-induced leukocyte-endothelial cell interactions, albeit in postcapillary venules (43). Thus, the effects of Met-RANTES in Ang-II-induced mononuclear cell recruitment in the arteriolar endothelium may be due to the blockade of both endothelial and platelet-derived RANTES. Indeed, Met-RANTES has been found to be effective in the inhibition of monocyte arrest in in vitro and in vivo models of atherosclerosis (24, 44).

In summary, we have demonstrated that Ang-II induces mononuclear cell recruitment in rat arterioles by the release of CC chemokines in vivo. The results suggest a requirement for both MCP-1, a selective CCR2 agonist, and a second CC chemokine, such as MIP-1{alpha} or more likely RANTES, acting at CCR1 or CCR5. Using human cells in culture and whole blood, we have demonstrated that Ang-II induces the synthesis of the mononuclear cell chemoattractants MCP-1, eotaxin-3, MIP-1{alpha}, and RANTES, followed by MCP-3. Although most of these responses are inhibited by AT1 receptor blockade, we conclude that CCR1, CCR2, or CCR5 receptor antagonists may become additional pharmacological tools to control the inappropriate attachment and migration of mononuclear cells into the subendothelial space of the arterial wall, a critical step in the development of several cardiovascular disease states where Ang-II generation is implicated.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants SAF 2002-01482, SAF 2002-04667, and SAF-2003-07206-C02-01 from the Comision Interministerial de Ciencia y Tecnologia, Spanish Ministerio de Educación y Ciencia; Research Group 03/166 of Conselleria de Cultura y Educación (Generalitat Valenciana); and awarded with the prize of the Spanish Pharmacological Society and Almirall-Prodesfarma Laboratories. Y.N.A.N., M.M., and P.J.J. were supported by a grant from Spanish Ministerio de Educacion y Ciencia. P.J.J. is primarily supported by Asthma U.K., T.M. by a grant from Conselleria de Cultura y Educación (Generalitat Valenciana), and M.A.T. by a grant from Spanish Ministerio de Asuntos Exteriores. A.C.I. was supported by Canadian Institutes of Health Research Grant MT-7684. Back

2 T.M. and Y.N.A.N. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Maria-Jesus Sanz, Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Avenida Blasco Ibañez, 15, E-46010 Valencia, Spain. E-mail address: maria.j.sanz{at}uv.es Back

4 Abbreviations used in this paper: CAM, cell adhesion molecule; Ang-II, angiotensin II; HUAEC, human umbilical arterial endothelial cell; MABP, mean arterial blood pressure. Back

Received for publication September 22, 2005. Accepted for publication February 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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