CXCR2 Blockade Impairs Angiotensin II–Induced CC Chemokine Synthesis and Mononuclear Leukocyte Infiltration
Objective— Angiotensin II (Ang-II) and mononuclear leukocytes are involved in atherosclerosis. This study reports the inhibition of Ang-II–induced mononuclear cell recruitment by CXCR2 antagonism and the mechanisms involved.
Methods and Results— Ang-II (1 nmol/L, i.p. in rats) induced CXC and CC chemokines, followed by neutrophil and mononuclear cell recruitment. Administration of the CXCR2 antagonist, SB-517785-M, inhibited the infiltration of both neutrophils (98%) and mononuclear cells (60%). SB-517785-M had no effect on the increase in CXC chemokine levels but reduced MCP-1, RANTES, and MIP-1α release by 66%, 63%, and 80%, respectively. Intravital microscopy showed that pretreatment with SB-517785-M inhibited Ang-II–induced arteriolar mononuclear leukocyte adhesion. Stimulation of human umbilical arterial endothelial cells (HUAECs) or whole blood with 1 μmol/L Ang-II induced the synthesis of chemokines. Ang-II increased HUAEC CXCR2 expression, and its blockade caused a significant reduction of MCP-1, -3, and RANTES release, as well as mononuclear cell arrest. Ang-II–induced MIP-1α release from blood cells was also inhibited.
Conclusion— Mononuclear leukocyte recruitment induced by Ang-II is, surprisingly, largely mediated by the CXC chemokines which appear to induce the release of CC chemokines. Therefore, CXC chemokine receptor antagonists may help to prevent mononuclear cell infiltration and the progression of the atherogenic process.
Inflammation has emerged as a crucial force driving the initiation and progression of atherosclerotic lesion formation.1 Common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking are associated with endothelial dysfunction, leading to a proinflammatory and prothrombotic phenotype of the endothelium.2 Experimental studies have demonstrated that leukocyte adhesion and infiltration into the arterial wall, regulated by leukocyte and endothelial cell adhesion molecules (CAMs) and chemokines, represent an essential step in atherosclerotic lesion formation.1,3–5
Angiotensin II (Ang-II), the main effector peptide of the renin-angiotensin system, is implicated in atherogenesis beyond its hemodynamic effects.6 We have demonstrated previously that 4-hour exposure to Ang-II in vivo induces arteriolar leukocyte adhesion in the rat mesenteric microcirculation, an effect mediated through interaction with its AT1 receptor subtype7 and not observed under acute (1 hour) stimulation with this peptide hormone.8 The leukocytes attached to the arteriolar endothelium are mononuclear, whereas those interacting with the venular endothelium are predominantly neutrophils.7 Despite these findings, the same CAMs are expressed in both the arteriolar and venular endothelia in response to Ang-II,7 suggesting that other mechanisms are responsible for the differential cellular distribution within the microcirculation.
Chemotactic cytokines, or chemokines, have the potential to recruit specific cell types.9 For example, ELR+ (Glu-Leu-Arg)-CXC chemokines such as interleukin (IL)-8 stimulate the recruitment of neutrophils that have high expression levels of the appropriate receptors (CXCR1 and CXCR2 in human, CXCR2 in rat.10 In contrast, mononuclear cell recruitment is associated with CC chemokines that stimulate appropriate receptors (for example, CCR1, CCR2, and CCR5) that are highly expressed on this cell type. In this context, we have found that Ang-II–induced mononuclear leukocyte recruitment is preceded by the generation and release of the CC chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), MCP-3 (CCL7), regulated on activation normal T cell expressed and secreted (RANTES/CCL5), and macrophage inflammatory protein-1α (MIP-1α/CCL3).11–13 We also demonstrated that either the blockade of CCR1 and CCR5 receptors with Met-RANTES, or the neutralization of MCP-1 activity, inhibited the attachment of mononuclear cells to the arteriolar endothelium.11 Similarly, Ang-II–induced neutrophil recruitment is preceded by the release of the CXC chemokines CINC/KC and MIP-214 (the rat homologues of IL-815,16).
CC chemokines have been strongly implicated in the prominent mononuclear cell accumulation at atherosclerotic lesions.4,5 To a lesser extent, CXC chemokines have also been implicated in atherogenesis. For example, IL-8, originally identified as a monocyte-derived factor that attracts neutrophils but not monocytes,9 has been detected in lesional macrophage-derived foam cells and other cell types at several different stages of human atherosclerotic lesions.17,18 However, neutrophils are rarely found in human atherosclerotic lesions. These observations suggest that IL-8 has a role in atherosclerosis that is independent of its capacity to recruit neutrophils, and possibly related to the presence of the relatively low expression of CXCR2 on mononuclear cells.19 In the present study we have investigated the effects of a CXCR2 receptor antagonist on Ang-II–induced mononuclear leukocyte adhesion and recruitment in rats in vivo and explored potential mechanisms of its action both in vivo and by use of human cells in vitro.
Materials and Methods
Leukocyte Migration Into the Peritoneal Cavity
All the studies were approved by the Institutional Ethics Committee. Male Sprague-Dawley rats (200 to 250 g) were sedated and injected intraperitoneally (i.p.) with 5 mL of PBS or 1 nmol/L Ang-II. After 1, 4, 8, and 24 hours, the total leukocyte and differential cell counts and the quantification of CINC/KC, MIP-2, MCP-1, RANTES, and MIP-1α in the peritoneal exudates were performed as previously reported.11,14
To evaluate the effect of a CXCR2 antagonist, rats were administered 1 hour before Ang-II injection with SB-517785-M (25 mg/kg, p.o., by gavage) and its effect on Ang-II–induced responses was evaluated 1, 4, 8, and 24 hours after the stimulus administration. SB-517785-M is an orally bioavailable N,N-diarylurea CXCR2 antagonist with a plasma half-life of 6.4 h.20 It is selective for CXCR2 over CXCR1 (200-fold selectivity by receptor membrane binding assays). The dose of SB-517785-M was effective in abolishing neutrophil infiltration in the post-capillary venules exposed to Ang-II for 1 h.14
In another set of experiments, after 1 hour of PBS or Ang-II i.p. administration in animals untreated or treated with an AT1 receptor antagonist (losartan gel, 10 mg/kg, i.v.) or with SB-517785-M (25 mg/kg, p.o.), a 100 mg sample of the mesenteric tissue was collected. Chemokine mRNA expression was determined by real-time quantitative RT-polymerase chain reaction (PCR) as previously described.14 Glyceraldehyide 3-phosphate dehydrogenase (GAPDH) was used as endogenous control gene.
The details of the experimental preparation have been described previously7 and can be found in the online data supplement (available online at http://atvb.ahajournals.org).
Animals were sedated and i.p. injected with 5 mL of PBS or Ang-II (1 nmol/L). After 4 hours, measurements of arteriolar leukocyte adhesion and venular leukocyte rolling flux, velocity, adhesion and emigration, and hemodynamic parameters of mean arterial blood pressure (MABP), arteriolar and venular Vrbc, shear rate, and diameter over a 5-minute period were determined.
One group of animals received losartan (10 mg/kg, i.v.), and another group received an AT2 receptor antagonist, PD123,319 (10 mg/kg, i.v.), both 15 minutes before the Ang-II i.p. administration. A third group of rats were pretreated 1 hour before Ang-II injection with SB-517785-M (25 mg/kg, p.o.).
HUAECs were isolated by collagenase as previously reported.11 Cells up to passage 2 were grown to confluence on 24-well culture plates.
For AT1 and AT2 receptor expression on HUAECs, total mRNA was isolated using TRIzol reagent. RT-PCR was performed using standard protocols using the primers described in the online data supplement. The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.
Cells were stimulated with 0.01 to 10 μmol/L Ang-II for 1 and 4 hours. Losartan (100 μmol/L) or PD123,319 (100 μmol/L) or a combination of both. Superoxide dismutase (SOD, 1000 U/mL) or cycloheximide (0.1 mg/mL) were added to some wells 1 hour before Ang-II (1 μmol/L) stimulation. Cell-free supernatants were stored at −80°C for IL-8 ELISA.
Ang-II affinity for CXCR2 receptor was determined by competition binding experiments with [125I]-IL-8 performed in CHO cell membranes expressing recombinant human CXCR2. Binding was conducted using a Scintillation Proximity Assay (SPA) using wheat germ agglutinin beads in a 96-well plate format. In one experiment the cold IL-8 competition was run in the presence and absence of Ang-II.
CXCR2 expression on HUAECs was determined by RT-PCR and immunofluorescence studies. CXCR2 mRNA levels were measured using the primers described in the online data supplement. For CXCR2 immunofluorescence studies on HUAECs, an anti-human murine monoclonal CXCR2 antibody and a secondary rabbit anti-mouse IgG polyclonal antibody fluorescein isothiocyanate (FITC)-labeled were used.
Neutralizing anti-CXCR1 (2.5 μg/mL) or anti-CXCR2 (10 μg/mL) mAbs, or a combination of both, were added to some wells 1 hour before stimulation with either Ang-II (1 μmol/L) for 4 and 24 hours or with IL-8 (10 nmol/L) for 4 hours. Cell-free supernatants were stored at −80°C for chemokine ELISAs. The results were expressed as pmol/L chemokine in the supernatants.
In additional experiments, HUAECs were stimulated with 1 μmol/L Ang-II for 4 hours. A neutralizing anti-CXCR2 (10 μg/mL) mAb was added to some plates 1 hour before Ang-II stimulation. Then, the Glycotech flow chamber was assembled, placed onto an inverted microscope stage, and freshly isolated human mononuclear cells (1×106/mL) were perfused across endothelial monolayers. Accumulation was determined after 5 minutes at 1 dyn/cm2 in all experiments. Arrested cells on the surface of the endothelium were visualized and recorded (×20 objective, ×10 eyepiece) using phase contrast microscopy.
Chemokine and MIF Release in Human Whole Blood
Human neutrophils and mononuclear cells from healthy donors (n=6) were isolated and purified by Ficoll Hypaque density gradient centrifugation. Human whole blood (10 U heparin/mL, from 6 different healthy volunteers) and leukocytes were incubated with saline or 1 μmol/L Ang-II for 4 hours. CXCR2, AT1, and AT2 mRNA expression on these cells was first determined. Then, losartan (100 μmol/L) was added to some samples 1 hour before Ang-II (1 μmol/L) stimulation. In another set of experiments, blood samples were preincubated with anti-CXCR1 (2.5 μg/mL) or anti-CXCR2 (10 μg/mL) mAbs, or a combination of both, 1 hour before the Ang-II stimulation. Plasma samples and leukocyte supernatants were stored at −80°C for IL-8, MIP-1α, and macrophage migration inhibitory factor (MIF) ELISAs.
All values are mean±SEM. Data between groups were compared using an analysis of variance (1-way ANOVA) with a Newman-Keuls post hoc correction for multiple comparisons. Statistical significance was set at P<0.05.
Ang-II, pentobarbital, neutralizing monoclonal anti-human CXCR1 antibody (clone 42705.111), neutralizing monoclonal anti-human CXCR2 antibody (clone 48311.211), PD123,319, ethidium bromide, SOD, and cycloheximide were from Sigma Chemical Co. Losartan was donated by Merck Sharp & Dohme. EBM-2 medium, supplemented with EGM-2 was from Innogenetics. Human and rat chemokines, and antibodies for and all rat chemokines were from PeproTech. The antibody pairs for all human chemokine and MIF ELISAs were from R&D Systems. Neutravidin-horseradish peroxidase was from Perbio Science, and the K-Blue substrate from Neogen. SB-517785-M was from GlaxoSmithKline. The polyclonal rabbit anti-mouse IgG antibody (FITC-labeled) was from Dako. TaqMan predevelopment and RT reagents were from PE Biosystems.
Intraperitoneal administration of Ang-II in rats induced a significant neutrophil recruitment which was preceded by increased CINC/KC and MIP-2 mRNA and protein expression (Figure 1). Pretreatment of the animals with a CXCR2 antagonist, SB-517785-M, virtually abolished Ang-II–induced neutrophil accumulation into the peritoneal cavity (Figure 1A) but had no effect on the mRNA and protein expression of CINC/KC and MIP-2 (Figure 1C, 1D, and 1E). Ang-II also induced a significant mononuclear leukocyte recruitment that was preceded by the generation of the CC chemokines, MCP-1, RANTES and MIP-1α (Figure 1B, 1C, 1F, 1G, and 1H). Interestingly, pretreatment with SB-517785-M resulted in a significant reduction of mononuclear leukocyte numbers after the Ang-II injection (Figure 1B). Furthermore, the CXCR2 antagonist also led to the inhibition of CC chemokine generation and release (Figure 1C, 1F, 1G and 1H). The peritoneal content of MCP-1, RANTES, and MIP-1α after 4-hour Ang-II exposure was reduced by 66%, 63%, and 80%, respectively in animals treated with SB-517785-M. By contrast, losartan administration inhibited all Ang-II–generated chemokines (Figure 1C). These results show that, in addition to inhibit neutrophil responses to CXC chemokines, antagonism of CXCR2 also inhibits the generation of CC chemokines and the subsequent mononuclear cell recruitment.
Exposure to Ang-II for 4 hours induced a significant enhancement of arteriolar leukocyte adhesion (Figure 2A). This effect was inhibited by losartan, SB-517785-M (85% inhibition) but not by PD123,319 (Figure 2A). The number of circulating leukocytes and the hemodynamic parameters were unaffected by these treatments (data not shown).
In the postcapillary venules of the same animals, 4-hour exposure to Ang-II induced a significant increase in venular leukocyte-endothelial cell interactions (Figure 2).
Pretreatment with losartan or SB-517785-M but not with PD123,3219, inhibited Ang-II–induced leukocyte responses (Figure 2).
To extend these findings to humans, we first investigated the expression of AT1 and AT2 receptors on HUAECs. Whereas the AT1 receptor was markedly expressed on HUAECs, the AT2 receptor was only weakly expressed (supplemental Figure IA). IL-8 secretion in HUAECs was unaffected in the first hour of incubation with Ang-II (data not shown) but was significantly increased after 4 hours (supplemental Figure IB). Losartan, but not PD123,319, inhibited 1 μmol/L Ang-II–induced IL-8 generation (supplemental Figure IB). Interestingly, IL-8 generation seems to be partly mediated through superoxide anion generation, because SOD inhibited Ang-II–induced IL-8 release by 50%. Furthermore, when the cells were pretreated with cycloheximide, IL-8 generation elicited by Ang-II was nearly abolished, indicating de novo protein synthesis.
To investigate CC chemokine generation in HUAECs and the effects of blockade of IL-8 receptors, we first determined whether Ang-II could directly interact with the CXCR2 receptor. Neither a competition for labeled IL-8 binding to human CXCR2-CHO cell membranes nor a displacement by cold IL-8 or Ang-II was seen (Figure 3A). Then, we next examined the expression of the CXCR2 receptor in HUAECs. HUAECs express the CXCR2 receptor (Figure 3B and 3C). Interestingly, after 1 hour stimulation with Ang-II, a clear increase in CXCR2 mRNA levels was observed which was inhibited by preincubation of the cells with losartan. These data were also confirmed by immunofluorescence (Figure 3C). When HUAECs were stimulated with 1 μmol/L Ang-II for 4 to 24 hours (Figure 4A and 4B), significant increases in MCP-1, RANTES, and MCP-3 were detected. Blockade of CXCR2 inhibited the secretion of these CC chemokines (76% to 80%, 86%, and 77% inhibition, respectively). Blockade of CXCR1 was less effective (a significant decrease was only observed for MCP-3, although this antibody concentration completely inhibited CXCR1-mediated neutrophil chemotaxis21) but the combination of both antibodies resulted in almost 100% inhibition of CC chemokine synthesis (Figure 4A and 4B). When HUAECs were stimulated with Ang-II for 4 hours, significant mononuclear leukocyte arrest was observed (Figure 4C). Interestingly, blockade of CXCR2 on HUAECs inhibited this response by 59% (Figure 4C). To add further evidence to these results, HUAECs were stimulated with IL-8 (10 nmol/L) for 4 hours. A significant increase in MCP-1 production was detected (Figure 4D) and although blockade of CXCR2 dramatically inhibited this response, it was not significantly affected by CXCR1 antagonism.
Finally, we investigated CXC, CC chemokine, and MIF generation in whole blood, neutrophils, and mononuclear leukocytes, because MIF has recently been identified as a novel ligand of CXCR2.22 Both leukocyte subtypes expressed CXCR2, AT1 but not AT2 receptors (supplemental Figure IIA). Human mononuclear leukocytes responded to Ang-II by producing IL-8 and MIP-1α but not MIF (supplemental Figure IIB, IIC, IIE, and IIF). Neutrophils were only capable of releasing small amounts of MIP-1α but not IL-8 (supplemental Figures IID and IB). These responses were AT1 receptor–mediated and blockade of CXCR2, but not of CXCR1, inhibited the MIP-1α release (supplemental Figures II and IB).
Monocyte arrest on the vascular endothelial lining is considered to be an initial step in the atherosclerotic process.2,23 Different studies have suggested that IL-8 and other members of the CXC chemokine subfamily have the potential to modulate atherogenesis. In this context, in situ hybridization and immunohistochemical staining of human coronary atheromas showed IL-8 expression primarily in the macrophage-abundant zones of the plaque.17 Further, one of the IL-8 receptors, CXCR2, was detected in the macrophage rich areas of human carotid atherosclerotic lesions.24 Despite these findings, neutrophils are scarce in most human atherosclerotic lesions.23
Ang-II is implicated in atherogenesis and endothelial dysfunction.6 Ang-II promotes the accumulation of both neutrophils and mononuclear cells into the peritoneal cavity, and these responses are preceded by the generation of CXC and CC chemokines.11,14 However, examination of the rat mesenteric microvasculature using intravital microscopy demonstrates important differences in the responses of these cell types. Ang-II promotes the adhesion of mononuclear leukocytes, but not of neutrophils, to arterioles.7 This is in contrast to responses in the postcapillary venules where neutrophils are the major leukocyte subtype recruited by this peptide hormone,7 and acute neutrophil responses were completely blocked by the preadministration of a CXCR2 antagonist, SB-517785-M14. In the present study we have investigated the less predictable effects of the CXCR2 antagonist on Ang-II–induced mononuclear cell arteriolar adhesion and recruitment to the peritoneal cavity, and identified potential mechanisms involved in these responses.
Pretreatment of rats with the CXCR2 antagonist significantly inhibited Ang-II–induced mononuclear cell arteriolar adhesion (85%) and recruitment to the peritoneal cavity (60% to 65%). These results were somewhat surprising because the CXC chemokines that interact with CXCR2 would be expected to promote neutrophil, rather than mononuclear cell, responses. However, our results are in broad agreement with those obtained in other animal models of atherosclerosis. Monocyte accumulation on early atherosclerotic endothelium in injured carotid arteries of apoE−/− mice is triggered by KC acting at CXCR2.25 In atherosclerosis-prone LDR−/− mice, a bone marrow deficiency in CXCR2 substantially reduces the severity of the atherosclerotic lesions.24 Further, we have found that CXCR2 blockade dramatically reduces the availability of the CC chemokines that are thought to mediate the mononuclear cell influx.
It is well documented that human and rodent circulating monocytes and T cells express functional IL-8 receptors.17,19,26,27 In contrast to neutrophils, there are substantially more CXCR2 than CXCR1 receptors on human mononuclear cells as found in this and a previous study,19 and rodent cells express CXCR2 but not CXCR1.10 Thus, CXCR2 appears to be the relevant receptor for the study of mononuclear cell responses to IL-8 and its rodent homologues. It is likely that at least some of the mononuclear cell responses to Ang-II are the direct result of recruitment of these cells by CXC chemokines acting on CXCR2. However, our results suggest that the major inhibitory effect of CXCR2 blockade on Ang-II–induced mononuclear cell accumulation is the reduction of CC chemokines that amplify and perpetuate the inflammatory response.
CXCR2 blockade has been shown to decrease the synthesis of MIP-1α and RANTES in other animal models of inflammation.28 We have previously reported that platelets contribute to the Ang-II–induced arteriolar adhesion of mononuclear leukocytes and that RANTES deposition is greater on the arteriolar than on the venular endothelium.11 Platelets are a major source of RANTES which can be released and deposited on the endothelium to trigger enhanced recruitment of mononuclear cells.25,29 Interestingly, a recent in vitro study has shown that platelet factor 4 (PF4/CXCL4, which activates a non–IL-8 receptor, CXCR3-B,30 and is costored with RANTES in platelet α-granules) enhances RANTES-induced mononuclear cell arrest but abrogates their IL-8–induced arrest on HUVECs.31 These differential responses might contribute to the selective recruitment of mononuclear cells by the arterial endothelium.
IL-8 is thought to be one of the major CXC chemokines involved in human myocardial inflammation.32 This chemokine in addition to GROα appears to be involved in triggering the firm arrest of monocytes on activated endothelium.26,33 To examine responses of human cells to Ang-II, the induction of IL-8 and CC chemokines was investigated at the protein level in HUAECs, whole blood, and mononuclear cells. Ang-II, acting at AT1 receptors, induced IL-8, MCP-1, RANTES, and MCP-3 secretion from HUAECs. We also show here for the first time that CXCR2 is expressed on HUAECs and its expression is upregulated by Ang-II. Of note, Ang-II–induced IL-8 generation is partly inhibited by pretreatment of the endothelial cells with SOD. In fact, superoxide anion generation can promote the synthesis and release of IL-8 from human endothelial cells.34 In agreement with the in vivo experiments, we found that the blockade of CXCR2 receptors on HUAECs inhibited the Ang-II–induced synthesis and release of the CC chemokines and the subsequent mononuclear cell arrest. It is unlikely that CXCR2 blockade inhibits MCP-1 or RANTES-induced mononuclear cell chemotaxis since previous studies have found that blockade of this receptor does not affect MCP-1–induced monocyte chemotaxis.26 On the other hand, when human whole blood was stimulated with Ang-II, the CC chemokine released was MIP-1α. We also found that exposure to Ang-II increased plasma IL-8 levels, as described previously.35 In contrast, Ang-II did not cause the release of MIF from isolated human mononuclear cells. In whole blood, we found that CXCR2 blockade inhibited the Ang-II–induced MIP-1α synthesis. These results show that blood elements as well as the vasculature are potential sources of CC chemokines on CXCR2 stimulation in vivo. They further suggest that CXC chemokines such as IL-8 can act in both a paracrine and juxtacrine manner, provoking the adhesion of mononuclear cells to the arterial endothelium and increasing the release of selective mononuclear cell chemoattractants.
In summary, we have demonstrated for first time that CXCR2 blockade inhibits Ang-II–induced mononuclear cell adhesion to the mesenteric arterioles, recruitment to the peritoneal cavity, and the synthesis and release of CC chemokines that can amplify the inflammatory cascade elicited by this peptide hormone. We suggest that AT1 or CXCR2 receptor antagonists may become valuable tools in the prevention of the vascular dysfunction which precedes atherosclerotic lesion formation.
Sources of Funding
This study was supported by grants SAF2005-01649, SAF2005-0669, and SAF2006-01002 from CICYT, Spanish Ministry of Education and Science. Y.N.A.N. and C.C. are supported by a grant from the Spanish Ministry of Education and Science. L.P. is supported by a Juan de la Cierva grant from the same Ministry. P.J.J. and T.M. are supported by a grant from Conselleria de Empresa Universidad y Ciencia, (Generalitat Valenciana) and M.L. by an AlBan Office grant and CDCH-UCV.
Y.N.A.N. and M.L. contributed equally to this study.
Original received April 25, 2007; final version accepted July 25, 2007.
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