Title: Up-Regulated Expression of the CXCR2 Ligand KC/GRO-α in Atherosclerotic Lesions Plays a Central Role in Macrophage Accumulation and Lesion Progression
Abstract: Macrophage-mediated inflammation is central to atherogenesis. We have determined previously that the CXC chemokine receptor CXCR2 is involved in advanced atherosclerosis. We sought to determine whether one of the ligands of CXCR2, KC/GRO-α, can also modulate atherogenesis. KC/GRO-α−/− mice were generated and mated with the atherosclerosis-prone LDLR−/− mice. There was a significant reduction in atherosclerosis in mice lacking KC/GRO-α; however, this reduction was only approximately half that seen previously in mice lacking CXCR2 in the leukocyte. To determine whether CXCR2 is involved in the early formation of atherosclerosis, leukocyte-specific CXCR2−/− chimeric mice on LDLR−/− background were generated. Early fatty streak lesion formation in these mice was not affected by leukocyte CXCR2 deficiency whereas lesions were less developed in mice lacking leukocyte CXCR2 when atherosclerosis was allowed to progress to the intermediate stage. Macrophages were relatively sparse in the lesions of leukocyte CXCR2−/− mice despite robust MCP-1 expression. These studies indicate that KC/GRO-α/CXCR2 does not play a critical role in recruitment of macrophages into early atherosclerotic lesions but both arterial KC/GRO-α and leukocyte-specific CXCR2 expression are central to macrophage accumulation in established fatty streak lesions. Macrophage-mediated inflammation is central to atherogenesis. We have determined previously that the CXC chemokine receptor CXCR2 is involved in advanced atherosclerosis. We sought to determine whether one of the ligands of CXCR2, KC/GRO-α, can also modulate atherogenesis. KC/GRO-α−/− mice were generated and mated with the atherosclerosis-prone LDLR−/− mice. There was a significant reduction in atherosclerosis in mice lacking KC/GRO-α; however, this reduction was only approximately half that seen previously in mice lacking CXCR2 in the leukocyte. To determine whether CXCR2 is involved in the early formation of atherosclerosis, leukocyte-specific CXCR2−/− chimeric mice on LDLR−/− background were generated. Early fatty streak lesion formation in these mice was not affected by leukocyte CXCR2 deficiency whereas lesions were less developed in mice lacking leukocyte CXCR2 when atherosclerosis was allowed to progress to the intermediate stage. Macrophages were relatively sparse in the lesions of leukocyte CXCR2−/− mice despite robust MCP-1 expression. These studies indicate that KC/GRO-α/CXCR2 does not play a critical role in recruitment of macrophages into early atherosclerotic lesions but both arterial KC/GRO-α and leukocyte-specific CXCR2 expression are central to macrophage accumulation in established fatty streak lesions. 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143: 205-211Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar Therefore, chemokines other than MCP-1 are likely contributing to progression of established lesions, a notion reinforced by the recent demonstration that CCR2 expression becomes down-regulated by monocyte differentiation into the macrophage or via activation of monocytes by a variety of inflammatory stimuli including oxidized low-density lipoprotein.25Han KH Chang MK Boullier A Green SR Li A Glass CK Quehenberger O Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator-activated receptor gamma.J Clin Invest. 2000; 106: 793-802Crossref PubMed Scopus (161) Google Scholar We previously observed that CXCR2, a receptor for interleukin (IL)-8 and several other CXC chemokines, has a major impact on macrophage accumulation in advanced lesions.26Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 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When fed an atherogenic diet for 16 weeks, the mice that received CXCR2−/− leukocytes (CXCR2−/− BMT) had significantly smaller lesions, with a smaller lipid core and less smooth muscle proliferation. Furthermore, at 16 weeks, the lesions of the CXCR2−/− BMT group were almost devoid of macrophages. On the other hand, at 16 weeks, the lesions from recipients of CXCR2+/+ leukocytes (CXCR2+/+ BMT) contained large numbers of CXCR2-positive macrophages. 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Because our previous study was limited to examination of only advanced lesions after 16 weeks of marked hyperlipidemia, the current study assessed the role of CXCR2 and GRO-α expression in early and intermediate lesions. Our results reveal that expression of KC/GRO-α in the vessel wall and lesion macrophage CXCR2 expression are central to the accumulation of macrophages in the progression of early atherosclerotic lesions. In contrast, our results also reveal that GRO-α and CXCR2 may not be essential for monocyte ingress into early atherosclerotic lesions. LDLR−/− mice backcrossed onto the C57BL/6 background were initially obtained from Jackson Laboratories (Bar Harbor, ME) and were bred at the Scripps Research Institute Animal Facility. Breeding pairs of the CXCR2+/− animals were initially obtained from Genentech (South San Francisco, CA) and bred to generate the CXCR2−/− and CXCR2+/+ mice used as bone marrow donors. These mice were genotyped by polymerase chain reaction using primers and conditions previously published.26Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-363Crossref PubMed Scopus (440) Google Scholar The KC/GRO-α-null mice (described below) were mated with LDLR−/− mice from our facility to generate double-mutant mice. Mice were weaned at 4 weeks of age and kept on a 12-hour light/dark cycle in a specific pathogen-free facility. They were fed a chow diet (diet no. 5015; Harlan Teklad, Madison, WI) ad libitum. Murine KC genomic clones were isolated from a 129-derived genomic library. A 3.8-kb XbaI-ApaI fragment from the 5′ end of the gene was subcloned into pGem 11zf (Promega Corp., Madison, WI). The fragment was rescued from the plasmid using XhoI/NotI digestion and inserted into the XhoI-NotI site from the pPNT vector, generating pPNT-XN. A 4.8-kb BamHI fragment from the 3′ end of the gene was cloned into the BamHI site of pPNT-XN, generating the final vector pPNTKC/ko. Orientation of the BamHI insertion was confirmed by digestion with XhoI/ClaI. The targeting vector was electroporated into CJ7 ES cells and neomycin-resistant clones were isolated. Targeted clones were identified by Southern blot of genomic DNA digested overnight with KpnI/EcoRV. Samples were fractionated in 0.7% agarose gels and transferred onto nylon membranes. The probe consisted of a 0.5-kb fragment located downstream of 3′ homologous region contained in the targeting vector. Heterozygous ES cells for the targeted locus were injected into C57BL/6J blastocysts, and chimeric mice were obtained from two different ES cell clones. Chimeric males were bred to C57BL/6J females. Progeny containing heterozygous genotype for the KC locus were intercrossed. KC−/− mice were obtained with the expected frequency of 25%. KC−/− mice were viable and fertile and did not show any gross abnormalities. KC/GRO-α−/− mice were mated with LDLR−/− mice (both on C57BL/6 background). Genotyping for KC/GRO-α was performed by reverse transcriptase-polymerase chain reaction. mRNA was isolated from the tail pieces from each animal and reverse-transcribed. Primers were designed and optimized to determine which region of the KC gene was disrupted because screening through the neo gene insertion would detect both the KC and LDLR altered alleles. The following primers were used: 5′-GAA GAC AGA CTG CTC TGA TGG CAC-3′ and 5′-CCC TTC TAC TAG CAC AGT GGT TGA-3′. When annealed at 52°C for 40 cycles, a 272-bp product (302 to 574 of the mRNA sequence) was yielded. A cohort of 14, 6- to 8-week-old male LDLR−/−,KC/GRO-α−/− mice and 14 age-matched male LDLR−/−,KC/GRO-α+/+ controls were fed a high-fat diet (HFD) that contained 15.8% fat, 1.25% cholesterol, and no cholate (diet no. 94059, Harlan Teklad) for 16 weeks to induce atherosclerosis. Blood was drawn immediately before the start of the HFD regimen and every 4 weeks thereafter. After 16 weeks the mice were sacrificed and processed exactly as described below for the CXCR2 bone marrow chimeric mice. For both sets of chimeric mice, 24 6-week-old male LDLR−/− mice were subjected to 1000 rads of total body irradiation to eliminate most of the endogenous bone marrow-derived cells as well as stem cells. For controls, half the irradiated mice were reconstituted with bone marrow cells isolated from KC/GRO-α+/+ (KC/GRO-α+/+ BMT) or CXCR2+/+ mice (CXCR2+/+ BMT mice) and the other half with marrow from KC/GRO-α−/− (KC/GRO-α−/− BMT) or CXCR2−/− mice (CXCR2−/− BMT mice) as experimental mice exactly as described previously.26Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-363Crossref PubMed Scopus (440) Google Scholar The mice consumed a chow diet for 4 weeks while they were allowed to be repopulated with the donor bone marrow. The KC/GRO-α chimeric mice were fed the HFD for 16 weeks to induce advanced atherosclerosis. To observe different degrees of atherosclerosis in the CXCR2 chimeric mice one group of six mice was fed the HFD for 3 weeks, whereas a second group of six mice was fed this diet for 6 weeks. At week 0 (before BMT) and at every 3 weeks (CXCR2 chimeric mice) or at every 4 weeks (KC/GRO-α chimeric mice), blood was drawn via the retro-orbital plexus after an 8-hour fast. Plasma was obtained by centrifuging the blood at 5000 × g for 10 minutes at 4°C. Total plasma cholesterol was measured with an enzymatic kit from Sigma (St. Louis, MO). Another group of 16 6- to 8–week-old male LDLR−/− mice was irradiated, and one half was reconstituted with marrow from CXCR2−/− mice and the other half with marrow from wild-type mice as described above. This was done to determine whether there were differences in the number of circulating leukocytes because of their CXCR2 status. Blood was taken at 4 weeks after BMT while the mice were consuming a chow diet. The mice were fed the HFD for an additional 3 weeks before being bled again. The neutrophils, monocytes, and lymphocytes were counted from Wright-stained blood smears at the Scripps Research Institute core pathology facility by a technician who was blinded to the identities of the mice. All procedures were in accordance with institutional guidelines. To assess if peripheral blood leukocyte expression of CXCR2 was enhanced by hyperlipidemia, eight 6- to 8-week-old male LDLR−/− mice were divided into two groups. One group was fed the chow diet, whereas the other group was given the HFD for 16 weeks. During this time the mice were bled at 0, 8, 12, and 16 weeks and their peripheral blood cells were analyzed for CXCR2 expression. The blood (0.1 ml) was centrifuged at 3000 × g for 5 minutes at 4°C and the cell pellet washed with phosphate-buffered saline containing 2% fetal bovine serum. The 0.1 ml cell suspension was incubated at 4°C with 1 ml of Fc receptor-blocking solution (Fc block:CD16/32, clone 2.4G2; PharMingen, La Jolla, CA) to prevent nonspecific Ig binding. The cells were stained for 30 minutes at 4°C with phycoerythrin-labeled Gr-1 (Pharmingen) and Cy-labeled anti-CD11b (Serotec, Oxford, UK) antibodies to identify neutrophils (CD11b+, Gr-1+ cells) and monocytes (CD11b+, Gr-1− cells). Subsequently the cells were incubated with rabbit anti-mouse CXCR2 antibody (a generous gift of Dr. N. Mukaida, Kanazawa University, Japan) for 30 minutes followed by incubation with fluorescein isothiocyanate-labeled rabbit IgG secondary antibody. The stained cells were analyzed on a Becton Dickinson (Mountain View, CA) fluorescence-activated cell sorting (FACS) system equipped with CellQuest software, and the percentages of neutrophils and monocytes staining positively for CXCR2 were determined. Extent of atherosclerosis in the mice was assessed by quantitative analysis of the lesions in the aortic valve as well as on the aortic surface of each mouse, as detailed previously.26Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-363Crossref PubMed Scopus (440) Google Scholar Briefly, the OCT-embedded, frozen aortic valves were sectioned serially at 10 μm thickness for a total of 300 μm beginning at the base of the aortic valve where all three leaflets were first visible. Every fourth section for a total of five sections from each animal was stained with Oil Red O to reveal the lipid-rich lesions. The stained areas were quantified using a computer-assisted video imaging system, and the mean area of the five sections from each animal was used to compare the lesion areas of the groups. Aortas were cleaned and stripped of fat on the adventitia before being excised from the animal. The longitudinally opened aortas were pinned on wax and stained with Sudan IV to reveal the lesions on the surface. The lesions were quantitated by calculating the percentage of the total surface area that was covered with lesion using computer-assisted morphometry. Detailed staining methods are described in our previous publication.26Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-363Crossref PubMed Scopus (440) Google Scholar The mouse aortic valve lesions were analyzed with the following antibodies: anti-MOMA-2 (Serotec) for the detection of intimal macrophages; anti-mouse CXCR226Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.J Clin Invest. 1998; 101: 353-363Crossref PubMed Scopus (440) Google Scholar for detection of leukocyte CXCR2; and anti-KC/GRO-α (R&D Systems, Minneapolis, MN) and anti-mouse MCP-1 (R&D Systems) for the detection of specific murine chemokines. The frozen tissue sections were blocked with 5% normal sera and incubated overnight at 4°C with the primary antibody (1 to 10 μg/ml). The sections were blocked for endogenous peroxidase activity with Peroxo-Block (Zymed, South San Francisco, CA) and incubated with the appropriate secondary antibody (5 μg/ml) for 1 hour. The washed sections were incubated for 30 minutes with Vectastain ABC Elite solution (Vector Laboratories, Burlingame, CA). At this point, the staining protocol for the chemokines was enhanced by incubating the sections in 1:100 Tyramide signal amplification solution (New England Nuclear, Beverly, MA), followed by another 30-minute incubation with Vectastain ABC solution. All sections were developed with 9-amino-3-ethylene-carbazole (AEC) (Vector Laboratories) and counterstained with hematoxylin. Negative control sections for KC/GRO-α and MCP-1 were prepared by using appropriate dilutions of the normal serum of the species in which the primary antibodies were made. Results are given as mean ± SD unless otherwise noted. Student's t-test was used to compare the percentage of CXCR2+ cells obtained by FACS analysis as well as plasma cholesterol levels and the leukocyte counts between the treatment groups. Mann-Whitney U-test was used to compare the Oil Red O-stained aortic valve lesion areas. To test the role of KC/GRO-α in atherosclerosis, KC/GRO-α−/−