Interleukin-8 Production by Macrophages From Atheromatous Plaques
Interleukin-8 (IL-8) is a chemotactic peptide produced by macrophages that may be involved in the recruitment of inflammatory cells into atherosclerotic plaques. In vitro, IL-8 production by macrophages isolated from carotid plaques (1240±510 pg·105 cells−1·24 h−1, mean±SEM, n=6) and noncarotid plaques (4312±1588 pg·105 cells−1·24 h−1, n=9) was significantly greater than IL-8 production by blood monocytes isolated from the same patients (526±278 pg·105 cells−1·24 h−1, n=6, P<.05 and 726±384 pg·105 cells−1·24 h−1, n=9, P<.01, respectively). IL-8 produced by atherosclerotic macrophages was demonstrated to be biologically active in a neutrophil chemotaxis assay. IL-8 mRNA was detectable in plaque macrophages and blood monocytes from these patients, but blood monocytes from normal donors did not exhibit detectable IL-8 mRNA. IL-8 mRNA was localized in macrophage-rich areas of atherosclerotic plaques by in situ hybridization. These studies demonstrate that macrophages from atherosclerotic plaques show an enhanced capacity to produce IL-8 compared with normal and patient blood monocytes and that macrophages are a major site of IL-8 mRNA production in atherosclerotic plaques. These results provide further evidence for a proinflammatory role for macrophages in atherosclerosis.
- Received October 13, 1995.
- Revision received March 5, 1996.
Atherosclerotic plaques demonstrate many features of chronic inflammatory lesions, including recruitment of monocytes and T cells, proliferation of adjacent mesenchymal cells, fibrosis, and scarring. Accumulation of monocyte-derived macrophages is particularly prominent, and due to their numbers alone, these cells would be expected to have an important role in the development of an atherosclerotic plaque. Macrophages secrete a broad spectrum of substances in addition to performing their typical scavenger and phagocytic roles.1 Many of these products may have a potential role in the atherogenic process. IL-8, a chemotactic and activating cytokine for neutrophils, is produced by smooth muscle cells,2 endothelial cells,3 4 5 epithelial cells,6 and monocytes and macrophages.7 8 9 10 11 In addition, IL-8 is an attractant with in vitro activity for neutrophils, lymphocytes, and basophils12 and may contribute to recruitment of inflammatory cells in atherosclerotic plaques. IL-8 also has potent angiogenic activity.3 13 To investigate the contribution of plaque macrophages to IL-8 production, we isolated macrophages from carotid and noncarotid atherosclerotic plaques and measured IL-8 protein and bioactivity and demonstrated IL-8 protein in situ. In addition, we detected IL-8 mRNA expression in plaque macrophages.
Collection of Tissue and Blood
Atheromatous plaques were collected at surgery. Six endarterectomy specimens were collected from 5 male patients (age, 56 to 71 years) undergoing carotid endarterectomy; 1 patient had bilateral procedures. Noncarotid material was obtained from six abdominal aortic specimens from 2 women and 4 men (age, 57 to 82 years) and three femoral artery specimens from 1 woman and 2 men (age, 61 to 80 years). Tissue was handled by using sterile techniques and placed in sterile normal saline. Blood was collected simultaneously from a peripheral vein in buffered sodium citrate anticoagulant.
Isolation of Macrophages From Atheromatous Plaque
Atherectomy material was processed within 1 hour of collection. Tissue was washed in 0.9% normal saline to remove macroscopic amounts of blood, placed in Eagle's minimal essential medium (Flow Laboratories) without added serum, chopped finely with a scalpel blade, and teased apart with sterile hypodermic needles. This material was washed through stainless steel meshes to remove particulate debris.
The filtered material was allowed to settle for 1 hour at 37°C in 50-mm-diameter plastic Petri dishes (Disposal Products), and nonadherent cells and other material were removed by washing three times with warm culture medium. Adherent cells were removed by exposure to trypsin-versene solution (Commonwealth Serum Laboratories) for 5 minutes. The trypsin was then neutralized by adding fetal calf serum (final concentration, 10%; Commonwealth Serum Laboratories), and the cells were washed twice with serum-free culture medium.
The cell yield, determined by counting in a Neubauer hemocytometer, was approximately 5×105 cells/g tissue.14 These cells were >95% mononuclear, nonspecific esterase positive (range, 85% to 95%),14 and expressed CD14 antigen as shown by immunofluorescence using a monoclonal anti-CD14 antibody (FMC 33; range, 89% to 95%); >90% of these cells were viable.
T cell contamination was assessed by staining with an anti-CD3 monoclonal antibody (Becton Dickinson) and was <5%. Smooth muscle cell contamination was <1% as assessed by staining with a monoclonal anti–muscle–specific actin antibody (HHF 35, provided by Dr A. Gown) after permeabilization with acetone at 4°C for 30 minutes.
Isolation of Blood Monocytes
Mononuclear cells were prepared by using density centrifugation15 on Ficoll Paque (Pharmacia LKB Biotechnology). These cells were allowed to adhere to 100-mm-diameter plastic tissue-culture Petri dishes for 1 hour. Adherent mononuclear cells were harvested in the same manner as plaque macrophages and characterized by their nuclear morphology and nonspecific esterase histocytochemistry as described above; >90% of these cells were viable.
Culture of Plaque Macrophages and Blood Monocytes
Freshly isolated macrophages and monocytes were always treated under the same conditions. They were cultured at a concentration of 5×104 cells/mL in serum-free Eagle's medium for 24 hours at 37°C in a 5% CO2/air atmosphere. After this the culture medium was collected, centrifuged at 3000g for 10 minutes to remove any particulate debris, and stored at −70°C. Cells were harvested as described above and frozen at −70°C in serum-free Eagle's medium at a concentration of 5×104 cells/mL.
Measurement of IL-8 Antigen
IL-8 antigen was measured by using an enzyme-linked immunosorbent assay (Biotrack, Amersham) that employs a monoclonal anti-human IL-8 capture antibody and a polyclonal detecting antibody with a peroxidase detection system. The assay has a sensitivity of <5 pg/mL and has no detectable cross-reactivity with other cytokines. Immediately before the assay, cell lysates were prepared from the frozen macrophage and monocyte samples by sonication with 10 1-second pulses with a Sonifier cell disrupter (Branson Sonic Power Co). Cell lysates, conditioned medium, and standards (medium and recombinant human IL-8) were all assayed in duplicate.
Measurement of IL-8 Bioactivity
IL-8 bioactivity in conditioned medium from plaque macrophages and blood monocytes was measured by using a neutrophil chemotaxis bioassay based on the Boyden chamber technique.16 The technique was adapted by using a 96-well microtiter plate (Flow Laboratories) as the lower chamber and cutoff 6-mm-diameter polypropylene microfuge tubes with cellulose filters (3.0 μm pore size, 120 μm thickness; Membra-fil, Costar) glued on the end as the upper chamber. Test samples and human recombinant IL-8 (final volume, 110 μL; Biosource) in a final were placed in the lower chamber. Blood neutrophils were isolated from normal donors by density centrifugation on Polymorphprep (Nycomed) and suspended in Eagle's medium at a concentration of 3×106 cells/mL; 60 μL was placed in the upper chambers. The upper chambers were suspended in the lower chambers, ensuring that the fluid levels in both chambers were at the same height. The chambers were placed at 37°C in a humidified 5% CO2/air atmosphere for 45 minutes, after which the upper chambers were washed and the filters fixed in 70% alcohol. The alcohol also dissolved the glue fixing the filters to the polypropylene tubes. The filters were then stained with Harris hematoxylin (Gurr, BDH Chemicals), mounted on microscope slides under coverslips, and viewed by microscopy. The extent of neutrophil migration was determined by the difference in focal distance between the surface of the filter and the leading front of neutrophils migrating into the filter. A minimum of five neutrophils per high-power field in the focal plane was used to determine the leading front of neutrophil migration. Three high-power fields were examined randomly per filter, and the mean migration distance was determined. All samples and standards were assayed in triplicate. Conditioned medium from representative samples was diluted 1:2, 1:4, and 1:8 to demonstrate that the bioactivity was titratable and treated at 100°C for 5 minutes to demonstrate that it was heat labile. Experiments were performed to inhibit neutrophil chemotactic activity in conditioned medium by using a neutralizing rabbit polyclonal anti-human IL-8 antibody (final concentration, 25 μg/mL; Endogen). Normal rabbit serum was used as a control. At a concentration of 10 μg/mL, this antibody neutralizes 50% of the bioactivity of 10 ng/mL of human recombinant IL-8 in a Boyden chamber assay and completely neutralizes the bioactivity of IL-8 at 1.0 ng/mL. Conditioned medium (100 μL) was preincubated with anti–IL-8 or nonimmunized serum (10 μL) for 30 minutes at 37°C before the assay for neutrophil chemotactic activity.16
Demonstration of IL-8 Antigen In Situ
Endarterectomy tissue was embedded in optimal cutting temperature compound (Miles), frozen immediately in liquid nitrogen–cooled isopentane, and stored at −70°C. Cryostat-cut tissue sections (6 μm thickness) were incubated with rabbit anti-human IL-8 antibody (Endogen) followed by sheep anti-rabbit Ig-AP conjugate (Silenus). Serial sections were then stained with a monoclonal Ham 56 antibody (Dako) by using a three-layer alkaline phosphatase/anti–alkaline phosphatase (APAAP) technique to demonstrate macrophages. Cytospin preparations of blood monocytes cultured with lipopolysaccharide (10 ng/mL) were used as a positive control.
Detection and Quantification of IL-8 mRNA
Human PBMs and plaque macrophages were isolated as described above. Total RNA was prepared according to the method of Chomczynski and Sacchi.17 RNA (20 μg per lane) was subjected to electrophoresis performed in a 1.2% agarose gel containing formaldehyde, transferred to nitrocellulose, and baked in a vacuum oven at 80°C for 2 hours. The nitrocellulose was then prehybridized, hybridized, washed,18 and analyzed for human IL-8 mRNA by probing with a 23-mer oligonucleotide probe (5′-GGG GTC CAG ACA GAG CTC TCT TC-3′) designed by reference to human IL-8 cDNA.19 Oligonucleotides were 3′ end labeled,20 and cDNA was labeled by the random priming method.21 Differences in mRNA expression were quantified by measuring radioactivity bound to nitrocellulose filters. This was achieved by exposing the filter to radiosensitive imaging plates (Fuji) for 4 to 24 hours and scanning the plates in a Fuji Bio-Imaging Analyser BAS-1000 (Berthold Australia P/L). Expression of GAPDH mRNA was quantified by using a rat GAPDH cDNA probe and used to correct for variations in RNA loading.
In Situ Demonstration of IL-8 mRNA in Arterial Sections
To colocalize macrophages and IL-8 mRNA, 3% paraformaldehyde–fixed 5-μm cryostat tissue sections were double stained for macrophages with HAM 56 (Dako) and detected with APAAP complex (Silenus) and fuchsin red (Biogenex). This was followed by in situ hybridization using a digoxigenin-11–dUTP 3′-tailed human IL-8 oligonucleotide probe (as described above) on the same sections. Sections were prehybridized for 1 hour at 42°C and then hybridized with the digoxigenin-labeled probe at 45°C overnight. Detection of specific mRNA was accomplished by using an anti-digoxigenin AP antibody (Boehringer Mannheim) at a dilution of 1:3000 followed by incubation with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate for 3 to 4 hours.
All results are expressed as mean±SEM. Statistical significance was assessed by using the Mann-Whitney U test and paired testing. Blood monocytes were collected from six normal donors and isolated and cultured as described above to determine basal IL-8 production.
IL-8 Production by Plaque Macrophages and Blood Monocytes
IL-8 was detectable by enzyme-linked immunosorbent assay in conditioned medium and cell lysates from plaque macrophages and PBMs of patients (Fig 1⇓). Total production of IL-8 by plaque macrophages from patients undergoing carotid endarterectomy procedures (1240±510 pg·105 cells−1·24 h−1, n=6) was significantly different (P<.05) from PBMs from these patients (526±278 pg·105 cells−1·24 h−1, n=6) and from normal patient PBMs (406±113 pg·105 cells−1·24 h−1, n=6, P<.05; normal patient PBM data not shown in graph). Macrophages from noncarotid atheromatous plaques also had augmented IL-8 production (4312±1588 pg·105 cells−1·24 h−1, n=9) compared with PBMs from the same patients (726±384 pg·105 cells−1·24 h−1, P<.01, n=9). IL-8 produced by plaque macrophages was found mainly in the culture medium (noncarotid 65%, carotid 89%) rather than being cell associated. IL-8 production in plaque macrophages and PBMs was also examined relative to total cell protein (data not shown). The same results were found as above, indicating that the increased IL-8 production was specific and not due merely to an increase in the total protein content of the cell (ie, increased size of mature macrophages).
IL-8 Bioactivity in Plaque Macrophage– and Blood Monocyte–Conditioned Media
Macrophage- and blood monocyte–conditioned media from six noncarotid lesions were tested for neutrophil chemotactic activity. Recombinant IL-8 induced dose-dependent neutrophil migration that was maximal at 1 nmol/L (Fig 2⇓). Migration in response to control medium was 4.3±1.5 μm. Neutrophil migration in response to noncarotid macrophage–conditioned medium (25.2±3.5 μm, n=6) was significantly greater than migration in response to PBM-conditioned medium (15.0±2.0 μm, n=6, P<.01). The addition of IL-8 antibody reduced the neutrophil chemotactic activity in both macrophage- and PBM-conditioned media. Maximum inhibition of chemotactic activity was 86%. Chemotactic activity in representative macrophage-conditioned medium was titratable and heat labile.
Expression of IL-8 mRNA and Antigen
Fig 3⇓ shows Northern blot analyses of total RNA from representative samples of normal PBMs before and after 24-hour serum-free incubations (lanes 1 and 2, respectively), macrophages isolated from noncarotid (lane 3) and carotid (lanes 4 and 5) atheroma, patient PBMs at time 0 (lanes 6 and 7), and patient PBMs after a 24-hour incubation (lane 8). A single band at 1.8 kb was demonstrated that corresponded to the size for the IL-8 mRNA.19 Slot-blot analysis of RNA showed that PBMs from normal donors did not express detectable levels of IL-8 mRNA before or after a 24-hour serum-free incubation (n=6). However, after incubation with 10 ng/mL lipopolysaccharide there was induction of IL-8 mRNA as observed by Northern blot analysis (data not shown).7 In PBMs isolated from patients undergoing surgery for repair of noncarotid and carotid atheroma, IL-8 mRNA levels were 1591±45 and 1728±281 arbitrary units (n=4), respectively, after a 24-hour incubation. In macrophages isolated from noncarotid and carotid atheroma, IL-8 mRNA levels increased to 2103±86 and 1768±44 arbitrary units (n=4, P<.01). Culture of atheroma-derived macrophages or PBMs from these patients for 24 hours in serum-free conditions had no effect on the expression of IL-8 mRNA compared with unincubated samples, indicating that during the various separation and incubation procedures macrophages were not activated ex vivo. IL-8 mRNA expression by atheromatous macrophages and PBMs was studied in four patients before and after incubation in serum-free culture. In these samples IL-8 mRNA levels were also unaffected by the culture.
In situ hybridization demonstrated that IL-8 mRNA is expressed predominantly in macrophage-rich regions of the plaques (Fig 4⇓). IL-8 mRNA was not detected in sections of histologically normal renal artery from a renal transplant donor (data not shown). There was no signal in control tissue hybridized with an IL-8 sense probe (Fig 4B⇓). In support of these findings, immunohistochemistry in serial sections of atherosclerotic tissue also showed that IL-8 antigen was expressed predominantly by macrophages (Fig 5⇓). Some regions of macrophage staining showed no corresponding staining for IL-8, suggesting that plaque macrophages are not entirely homogeneous with respect to their activation. Koch et al, 13 who also report this observation, suggest that some endothelial cells are IL-8 positive.
The current studies demonstrate that macrophages isolated from human atheromatous plaques produce IL-8 in short-term serum-free culture without exogenous stimuli. IL-8 antigen was secreted and neutrophil chemotactic activity (neutralizable by IL-8 antibody) was detected, indicating bioactivity of the secreted IL-8. Blood monocytes isolated concurrently from the patients undergoing atherectomy produced significantly lower but detectable amounts of IL-8 antigen than plaque macrophages from the same patients. However, IL-8 production by monocytes isolated from normal individuals was undetectable when cultured under identical conditions. IL-8 levels in blood monocytes are similar to those of resident tissue macrophages, such as pulmonary alveolar macrophages, and tissue macrophages resemble PBMs in their IL-8 production under certain conditions.11 22 In addition, our previous studies23 indicate that isolation procedures do not activate macrophages with respect to TNF and IL-1 production.
These data suggest a systemic activation of IL-8 production by blood monocytes of this group of patients undergoing vascular surgery24 and further augmentation of IL-8 production by monocyte-derived macrophages in plaques. Macrophages from distal aortic and femoral lesions tended to produce the greatest amounts of IL-8. This is supported by qualitative immunohistochemical analysis10 which shows that the majority of IL-8–positive cells in abdominal aortic aneurysm sections are macrophages, while only a minority of aortic endothelial cells are IL-8 positive. Why noncarotid macrophages produce more IL-8 than carotid macrophages is not clear from the current study. Abdominal aortic atheromas removed at surgery are usually more heterogeneous and complicated in nature, and different local conditions may affect the levels of IL-8 in these lesions compared with carotid lesions.
Production of IL-8 antigen by plaque macrophages was accompanied by expression of IL-8 mRNA both in vitro and in vivo. The levels of IL-8 mRNA in freshly isolated uncultured or cultured PBMs from normal donors were below detectable limits.11 In PBMs and macrophages isolated from carotid and noncarotid plaques, however, there was a strong signal for IL-8 mRNA. The IL-8 mRNA levels were higher in noncarotid than carotid macrophages, consistent with the greater IL-8 antigen production in noncarotid macrophages. High levels of IL-8 mRNA were also detected in blood monocytes from patients undergoing atherectomy. The difference in IL-8 mRNA expression between blood monocytes and plaque macrophages was not as pronounced as the differences in IL-8 antigen production. This may suggest posttranscriptional or translational control of IL-8 production.
The pattern of gene expression and the functional status of cells in different regions of atherosclerotic plaques may differ considerably.25 They are probably regulated by local factors present in the lesions. One such regulatory factor may be oxidized LDL, which affects the expression of cytokines and chemotactic factors by arterial walls26 and in adherent human PBMs.27 Other local regulatory factors may include TNF-α and IL-1, which are produced by macrophages, smooth muscle cells, and endothelial cells.2 5 19 Both of these cytokines augment transcription of the IL-8 gene in blood monocytes, macrophages, and endothelial cells.2 11 Carotid plaque macrophages produce more TNF than macrophages from distal aortic plaques,23 but these lesions do not show enhanced IL-1 production. Thus, in aortic atherosclerotic plaques, it would appear unlikely that either TNF-α or IL-1 is a major local regulator of IL-8 secretion by plaque macrophages. The factors responsible for regulation of IL-8 production in atherosclerotic plaques remain to be elucidated.
Many cell types have been reported to have the potential to produce IL-8 mRNA. The current study demonstrates that macrophages realize this potential in atherosclerotic plaques. Previous studies have suggested that the predominant cell producing IL-8 protein within atheroma is the macrophage.10 This study shows for the first time that IL-8 mRNA is localized mostly to invading macrophages in the regions of the plaques.
Atherosclerotic lesions contain few neutrophils, despite local production of IL-8. The lesions we studied are at an advanced/chronic stage, and neutrophil invasion is not a characteristic feature of chronic inflammation. Although IL-8 may attract neutrophils,28 29 under certain conditions it can inhibit neutrophil adhesion to cytokine-activated endothelium. In a complex inflammatory lesion, the net accumulation of neutrophils is probably determined by the interaction of a variety of factors, including IL-8 chemoattractant activity.
The functional contribution of IL-8 to the development of atherosclerotic lesions remains to be defined. IL-8 has similar chemotactic activity for lymphocytes and neutrophils in vitro30 and promotes neovascularization in the cornea.3 13 These properties may be important for lymphocyte recruitment into plaques and for neovascularization of the intima.
In summary, these studies demonstrate upregulation of IL-8 protein, bioactivity, and mRNA in macrophages from atheromatous plaques. They also indicate macrophage activation and suggest a mechanism by which they may recruit T lymphocytes into plaques.
Selected Abbreviations and Acronyms
|PBM||=||peripheral blood monocyte|
|TNF||=||tumor necrosis factor|
This work was supported by a grant-in-aid from the National Heart Foundation of Australia.
Wang JM, Sica A, Peri G, Walter S, Padura IM, Libby P, Ceska M, Lindley I, Colotta F, Mantovani A. Expression of monocyte chemotactic protein and interleukin-8 by cytokine-activated human vascular smooth muscle cells. Arterioscler Thromb. 1991;11:1166-1174.
Streiter RM, Kunkel SL, Elner VM, Martony CL, Koch AE, Polverini PJ, Elner SG. Interleukin-8: a corneal factor that induces neovascularization. Am J Pathol. 1992;141:1270-1284.
Schroder J-M, Christophers E. Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells. J Immunol. 1989;142:244-251.
Sica A, Wang JM, Colotta F, Dejana E, Mantovani A, Oppenheim JJ, Lousen CG, Zachariae COC, Matsushima K. Monocyte chemotactic and activating factor gene expression induced in endothelial cells by IL-1 and TNF. J Immunol. 1990;144:3034-3038.
Thelen M, Peveri P, Kernan P, Von Tscharner V, Walz A, Baggiolini M. Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist. FASEB J. 1988;2:2702-2706.
Baggiolini M, Walz A, Kunkel SL. Neutrophil activating peptide-1/interleukin-8, a novel cytokine that activates neutrophils. J Clin Invest. 1989;84:1045-1049.
Larsen CG, Anderson AO, Apella E, Oppenheim JJ, Matsushima K. Neutrophil activating protein (NAP-1) is also chemotactic for T-lymphocyte. Science. 1989;243:1464-1466.
Koch AE, Polverini PJ, Kunkel SL, Harlow CA, Di Pietro LA, Elner VM, Elner SG, Streiter RM. Interleukin-8 is a macrophage-derived mediator of angiogenesis. Science. 1992;258:1798-1801.
Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest. 1968;21(suppl 97):77-109.
Apostolopoulos J, Howlett GJ, Fidge N. Effects of dietary cholesterol and hypothyroidism on rat apolipoprotein metabolism. J Lipid Res. 1987;28:642-648.
Matsushima K, Morishita K, Yoshimura T, Lavu S, Kobayashi Y, Lew W, Appella E, Kung HF, Leonard EJ, Oppenheim JJ. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med. 1988;167:1883-1893.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Strieter RM, Kunkel SL, Showell HJ, Remick DG, Phan SH, Ward PA, Marks RM. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-α, LPS, and IL-1β. Science. 1989;243:1467-1469.
Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260-1266.
Terkeltaub R, Banka CL, Solan J, Santoro D, Brand K, Curtiss LK. Oxidized LDL–induced monocytic cell expression of interleukin-8, a chemokine with T lymphocyte chemotactic activity. Arterioscler Thromb. 1994;14:47-53.
Gimbrone MA, Obin MS, Brock AB, Luis EA, Hass PE, Hebert CA, Yip YK, Leung DW, Lowe DG, Kohr WJ, Darbonne KB, Bechtol KB, Baker JB. Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions. Science. 1989;246:1601-1602.
Luscinskas FW, Kiely JM, Ding H, Obin MS, Hebert CA, Baker JB, Gimbrone MA. In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions. J Immunol. 1992;149:2163-2171.