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An Inflammatory Pathway of IFN- Production in Coronary Atherosclerosis¹

Hooman Ranjbaran^2,*, Seth I. Sokol^2,, Amy Gallo^*, Raymond E. Eid^*, Alexander O. Iakimov^*, Alessio D’Alessio, John R. Kapoor, Shamsuddin Akhtar, Christopher J. Howes, Mihaela Aslan, Steven Pfau, Jordan S. Pober and George Tellides^3,*

^* Department of Surgery, Department of Medicine, Department of Pathology, and Department of Anesthesiology, Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, CT 06510

	Abstract

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Inflammation is associated with the pathogenesis of coronary atherosclerosis, although the mechanisms remain unclear. We investigated whether cytokine secretion by innate immune responses could contribute to the production of proarteriosclerotic Th1-type cytokines in human coronary atherosclerosis. Cytokines were measured by ELISA in the plasma of patients with coronary atherosclerosis undergoing cardiac catheterization. IL-18 was detected in all subjects, whereas a subset of patients demonstrated a coordinated induction of other IFN--related cytokines. Specifically, elevated plasma levels of IL-12 correlated with that of IFN- and IFN--inducible chemokines, defining an IFN- axis that was activated independently of IL-6 or C-reactive protein. Systemic inflammation triggered by cardiopulmonary bypass increased plasma levels of the IFN- axis, but not that of IL-18. Activation of the IFN- axis was not associated with acute coronary syndromes, but portended increased morbidity and mortality after 1-year follow-up. IL-12 and IL-18, but not other monokines, elicited secretion of IFN- and IFN--inducible chemokines in human atherosclerotic coronary arteries maintained in organ culture. T cells were the principal source of IFN- in response to IL-12/IL-18 within the arterial wall. This inflammatory response did not require, but was synergistic with and primed for TCR signals. IL-12/IL-18-stimulated T cells displayed a cytokine-producing, nonproliferating, and noncytolytic phenotype, consistent with previous descriptions of lymphocytes in stable plaques. In contrast to cognate stimuli, IL-12/IL-18-dependent IFN- secretion was prevented by a p38 MAPK inhibitor and not by cyclosporine. In conclusion, circulating IL-12 may provide a mechanistic link between inflammation and Th1-type cytokine production in coronary atherosclerosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Atherosclerosis, the leading cause of death and disability in the developed world, is considered an inflammatory disease (1). Circulating mediators of inflammation, such as IL-6 and C-reactive protein (CRP),⁴ are risk factors for death from coronary thrombosis. In addition, clinical and experimental studies have demonstrated that leukocyte populations within the arterial wall are involved in the initiation, progression, and complications of coronary atherosclerosis. However, both the relationship of systemic mediators of inflammation to the local arterial process and the precise nature of inflammatory responses within the artery wall remain unclear. Various microbes, such as Chlamydia pneumoniae, and certain noninfectious, "altered-self" endogenous molecules, e.g., oxidized low-density lipoprotein (oxLDL), have been proposed as specific antigenic stimuli for local intra-arterial immune responses. An alternative, but not mutually exclusive hypothesis is that extravascular inflammation initiates or exacerbates adaptive immune responses within the arterial plaque. In support of the concept that systemic inflammation contributes to atherogenesis, patients with diverse inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, periodontal disease, and influenza, have an increased incidence of coronary atherosclerosis with higher cardiovascular mortality. Additionally, the aggregate inflammation from multiple different infections influences the course of coronary atherosclerosis (2), and nonspecific activation of the immune system may also contribute to the total inflammatory load (3). Epidemiologic data suggest that the life-time exposure to infectious diseases and other sources of inflammation, particularly in childhood, represents a "cohort morbidity phenotype" that contributes to the adult onset of advanced coronary atherosclerosis (4). Thus, besides possible direct infection of the diseased artery, microbes may indirectly contribute to arterial inflammation by circulating pathogen-derived (e.g., LPS) and pathogen-stimulated (e.g., cytokines) proinflammatory factors (2).

Cytokines represent a possible link between systemic inflammation and local inflammatory processes. These molecules are a diverse group of proteins that are produced during host defense and serve to mediate and regulate innate and adaptive immunity (5). In innate immune responses, the effector cytokines are predominantly produced by activated dendritic cells or macrophages and are sometimes called monokines. Principal members of this group are IL-1, IL-6, IL-12, IL-18, IFN-, and TNF. A different set of cytokines are produced by activated T lymphocytes in adaptive immune responses and are therefore referred to as lymphokines. IFN- is the prototypic cytokine produced by CD4⁺ Th1 lymphocytes and is also produced by CD8⁺ cytolytic T cells and by NK cells. In contrast, IL-4, IL-5, and IL-13 are the signature cytokines for Th2 effector cells. IL-10 may be produced either by regulatory Th3 lymphocytes or by mononuclear phagocytes. Lastly, chemokines are an extended family of structurally homologous, small cytokines that share the ability to stimulate leukocyte chemotaxis and are also produced by many nonhemopoietic cell types principally in response to both monokines and lymphokines. Chemokines that may perpetuate IFN--producing immune responses include the IFN--inducible chemokines CXCL10/(IP-10), CXCL11/(I-TAC), and CXCL9/(Mig) by the recruitment of Th1 cells that selectively express their cognate receptor, CXCR3. The separation of cytokines into mediators and regulators of innate vs adaptive immunity must be qualified by the bidirectional interactions of these components of host defense (5). Innate immune responses stimulate and influence the nature of adaptive immune responses, and, in turn, adaptive immune responses enhance and focus the effector mechanisms of innate immune responses. These interactions may occur as paracrine mechanisms within the local inflammatory microenvironment, or the cytokines may circulate in the bloodstream and act on their target cells as hormonal agents.

This study consists of two related components to test our hypothesis that inflammation may result in the production of proarteriosclerotic Th1-type cytokines in human coronary atherosclerosis. First, we examined the relationships of circulating monokines to lymphokines and chemokines in patients with coronary atherosclerosis. Second, we examined the effects of monokines on local production of lymphokines and chemokines within organ cultures of human atherosclerotic coronary arteries. We find evidence for a pathophysiological pathway we call the IFN- axis, which is activated independently of IL-6 and CRP, is not dependent on TCR signaling although it may exacerbate Ag-driven T cell responses within coronary arteries, and is a novel prognosticator of adverse outcomes in coronary atherosclerosis.

	Materials and Methods

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Patients

Research protocols were approved by the institutional review board of the West Haven VA Hospital and informed consent was obtained. Blood samples were collected in EDTA tubes before cardiac catheterization, before and during coronary artery bypass graft (CABG) surgery, or at a primary care clinic. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4°C and stored at –80°C until analysis. Patients were followed up at 6 mo and at 1 year by telephone interview and by review of electronic medical records. Primary endpoints were all cause mortality, myocardial infarctions, stroke/transient ischemic attacks, and the composite of these complications.

Reagents

Biochemicals included recombinant human cytokines (R&D Systems), PMA, ionomycin (Sigma-Aldrich), and neutralizing Abs to TNF, IFN- (R&D Systems), class I (clone W6/32) and II (clone LB3.1) MHC molecules, and CD1d (BD Biosciences). Pharmacological agents included rapamycin, cyclosporin A (CsA) (Sigma-Aldrich), PD 98059, JNK inhibitor II, SB 203580 (Calbiochem), and atorvastatin (Pfizer).

Organ and cell culture

Atherosclerotic coronary arteries were obtained from explanted hearts of transplant recipients or cadaver organ donors. Research protocols were approved by the institutional review boards of Yale University and the New England Organ Bank, and informed consent was obtained. The coronary arteries were dissected free from epicardial fat immediately after cardiac explantation within the operating room and transported to the laboratory after 10–20 min of warm ischemic time and 1–2 h of cold ischemic time. Artery segments were divided into 3-mm rings and cultured in 24-well plates in M-199 medium supplemented with 20% FBS, 2.8 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). The medium was changed after 6 h to remove any basal cytokine production before commencing the experiments. Coronary artery-infiltrating leukocytes were isolated without collagenase digestion by mincing the arteries into 1- to 2-mm pieces in supplemented RPMI 1640 medium at 4°C, sequentially passing the tissue and supernatant through 0.5- and 0.1-mm stainless steel sieves, followed by density gradient centrifugation on lymphocyte separation medium (Invitrogen Life Technologies) twice. With this isolation protocol, >95% of the recovered cells wereviable mononuclear leukocytes. PBMCs were isolated by leukapharesis of healthy donors, purified by density gradient centrifugation, and depleted of monocytes by incubation on fibronectin-coated plastic for 1 h. CD4⁺ and CD8⁺ T cells were isolated by positive selection from which recently activated HLA-DR⁺ T cells and naive CD45RA⁺ or memory CD45RO⁺ T cells were depleted using Ab-coated magnetic beads (Dynal Biotech). Enrichment efficacy was confirmed by flow cytometry. T cells were cultured in 24-well plates at 3 x 10⁵ cells/well in supplemented RPMI 1640 medium. For TCR stimulation of isolated leukocytes, the plate was preincubated with anti-human CD3 (Clone OKT3; eBioscience) at 10 µg/ml at 37°C for 2 h, followed by the addition of cells with anti-human CD28 (BD Biosciences) at 1 µg/ml for 48 h.

Immunohistochemistry and immunofluorescence microscopy

Immunohistochemical staining was performed using isotype-matched, nonbinding control Abs or mouse anti-human CD3, CD4, CD8, CD45, CD68 (DakoCytomation), CD45RA, CD45RO (eBioscience), and CD-56 (BD Biosciences). Binding of secondary Ab (Jackson ImmunoResearch Laboratories) was detected with peroxidase/3-amino ethyl carbazole kits (Vector Laboratories). For immunofluorescence analysis, the cells were washed, fixed with 4% paraformaldehyde for 15 min, then permeabilized with 0.1% saponin for 5 min, and blocked with buffer containing 1% BSA/5% mouse and goat serum for 1 h at room temperature. Immunostaining was performed by overnight incubation with control Abs or mouse anti-human CD3 (DakoCytomation), CD56 (BD Pharmingen), and goat anti-human IFN- (R&D Systems) in blocking buffer at 4°C. Detection of bound primary Ab was visualized by the addition of Alexa Fluor 488- or 594-conjugated secondary Abs (Invitrogen Life Technologies) for 30 min at room temperature and imaged with a Zeiss LSM510 confocal, laser-scanning microscope.

ELISA

Cytokine plasma and supernatant levels were determined using sandwich ELISA kits (R&D Systems). The lower limit of detection (i.e., the lowest concentration of cytokine standard used to construct a reference line) was 0.13 pg/ml for IL-1, 0.16 pg/ml for IL-6, 0.25 pg/ml for TNF, 6.25 pg/ml for IFN-, 11.7 pg/ml for IL-5, 15.6 pg/ml for IL-12 p70, IFN-, CXCL10, and CXCL11, 23.4 pg/ml for IL-10, 25.6 pg/ml for IL-18, and 62.5 pg/ml for CXCL9. High-sensitivity CRP assays were performed by the clinical laboratory of Yale-New Haven Hospital.

ELISPOT assay

ELISPOT assays were performed on 2 x 10⁵ CD45RO⁺/CD8⁺ T cells per well in 96-well plates according to the manufacturer’s instructions (BD Biosciences).

Flow cytometry

Cells were labeled with isotype-matched control Ab or mouse anti-human perforin, granzyme B (BD Pharmingen), CD25, CD69 (Beckman Coulter Immunotech System), and CXCR3 (R&D Systems) and analyzed using a FACSort (BD Biosciences). For intracellular staining, the cells were first fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin. Alternatively, cells were incubated with CFSE at 500 µM for 20 min before treatment or with propidium iodide (PI) at 25 µg/ml for 5 min before FACS analysis.

Statistical analysis

The distribution of plasma cytokine levels was skewed, even after log transformation, and comparisons were performed by the nonparametric Wilcoxon Mann-Whitney U test. Relationships were also analyzed by nonparametric Spearman correlations. Continuous clinical variables were compared between groups using the two-sample Student’s t test, and categorical clinical variables, including outcomes, were analyzed by Fisher’s exact test. Relative risks with 95% confidence intervals for outcomes were calculated. In vitro experimental groups were compared by ANOVA. All p values were two-tailed and values <0.05 were considered to indicate statistical significance, except in correlation comparisons between multiple cytokines where two-tailed p values <0.001 were considered statistically significant for individual tests to ensure an overall study significance level of 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Heterogeneous pattern of circulating monokines

Plasma levels of cytokines were measured in 108 patients presenting with symptomatic coronary atherosclerosis before cardiac catheterization. The concentrations of monokines varied considerably (Fig. 1A). IL-12 was detected in 47 patients and was induced to relatively high levels. In contrast, circulating IL-18 was found in all patients within a relatively narrow range. Low levels of IFN-, TNF, IL-1, and IL-6 were measured in the plasma of most patients. Patients presenting with acute coronary syndrome (ACS) had increased IL-6 and CRP, decreased IFN-, but similar levels of IL-12, IL-18, TNF, and IL-1 compared with patients with chronic stable angina (Table I). Few patients had multiple monokines elevated within the highest quartile of plasma concentrations (Fig. 1C). Moreover, there was poor correlation among these markers of innate immune responses with the exception of IL-6 vs TNF and CRP (Table II), and systemic activation of this particular aspect of innate immunity has been previously described in patients with symptoms of, or at risk for, ACS.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Plasma levels of innate and adaptive immunity cytokines. The concentrations of circulating cytokines typically produced by macrophages/dendritic cells (A) or T lymphocytes (B) and IFN--inducible chemokines (B) were determined in 108 patients with coronary atherosclerosis undergoing cardiac catheterization. The number of cytokines that were elevated (>75th percentile of the concentration range) in each individual subject were plotted to express the distribution of inflammatory marker induction within the study population (C). The data are log transformed: y = log(cytokine plasma levels (pg/ml) + 1) and individual data are shown.

Coordinated induction of plasma lymphokines and IFN--inducible chemokines

The signature lymphokines IFN-, IL-5, and IL-10, representative of Th1, Th2, and Th3 immune responses, respectively, were detected in the plasma of a minority of patients with coronary atherosclerosis (Fig. 1B) and were not associated with ACS (Table I). The CXCR3-binding chemokines, CXCL10, CXCL11, and, in particular, CXCL9, were more readily detectable in the circulation than the lymphokines (Fig. 1B). The induction of lymphokines and chemokines occurred in a more limited subgroup of patients compared with the distribution of monokines (Fig. 1C). Additionally, there were significant correlations between the plasma levels of IFN-, IL-5, IL-10, CXCL10, CXCL11, and CXCL9 (Table II). In other words, a subset of patients showed systemic activation of adaptive immunity at the time of presentation.

Circulating IL-12 correlates with plasma lymphokines and IFN--inducible chemokines

We next analyzed our data for relationships between the cytokines characteristic of innate and adaptive immune responses. We found significant correlations between IL-12 vs IFN-, IL-5, IL-10, CXCL10, CXCL11, and CXCL9 plasma levels (Table II). IFN- was detected in the circulation of 27 patients with high plasma levels of IL-12, CXCL10, CXCL11, or CXCL9. We refer to this related group of inflammatory markers (the IFN--inducer, IL-12; the prototypical Th1-type cytokine, IFN-; and the IFN--inducible chemokines, CXCL10, CXCL11, and CXCL9) as the IFN- axis. There were no clear correlations between the other circulating monokines and any of the lymphokines or chemokines we analyzed (Table II), thus the systemic activation of adaptive immunity appeared unrelated to the IL-6/CRP axis. Detectable or elevated plasma levels of the IFN- axis were not related to patient clinical characteristics at the time of presentation (Table III). Specifically, potential immunomodulatory factors such as smoking or HMGCoA reductase inhibitors (statins) were not related to circulating markers of the IFN- axis. Further analysis of specific statins, e.g., simvastatin or lovastatin, similarly showed no correlation to plasma levels of the IFN- axis (data not shown). We also found significant correlations between the cytokines of the IFN- axis in 59 age/gender/race-matched patients without a diagnosis of coronary atherosclerosis (data not shown), although the plasma levels of IFN- and IFN--inducible chemokines, but not of IL-12 or IL-18, were less in the referent subjects (Table IV). In contrast, the IFN- axis was undetectable, despite similar basal levels of circulating IL-18, in 18 healthy young adults (data not shown).

View this table: [in this window] [in a new window]   Table IV. Plasma levels of IFN--related cytokines in patients with and without coronary atherosclerosisa

Systemic inflammation elicits IL-12 and IFN--inducible chemokine production

We assessed whether an inflammatory stimulus, namely cardiopulmonary bypass (CPB), would increase systemic levels of the IFN- axis. Plasma cytokine levels were measured in a separate group of 38 patients with coronary atherosclerosis undergoing CABG surgery. IL-12 increased after CPB, whereas IL-18, which was persistently detected perioperatively, was unchanged by CPB (Fig. 2). There was an associated increase in plasma levels of CXCL10 and CXCL9 after CPB, with minor elevations in the concentrations of the less abundant proinflammatory factors, IFN- and CXCL11, that did not reach statistical significance. A 2-fold or greater increase in IL-12 plasma levels occurred in the same 15 patients with a doubling or more of circulating CXCL10, whereas induction of IFN- was detected in only 8 of those subjects. In the subgroup of patients with increased circulating IL-12, there were significant increases in plasma levels of IFN-, CXCL10, CXCL11, and CXCL9, but not of IL-18 (Fig. 2). Induction of the IFN- axis was associated with a greater duration of CPB (188 ± 14 vs 153 ± 5 min; p = 0.0128), but not to patient clinical characteristics before surgery (Table V).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. CPB activates the IFN- axis. Cytokine plasma levels were determined in 38 patients (solid lines) at various times after CABG surgery. The patients were also divided into two groups: those with >2-fold elevations in IL-12 (dashed lines; n = 15) and those without increased IL-12 (dotted lines; n = 23). Data are means ± SEM. *, p < 0.05 post-CPB vs 0 h and +, p < 0.05 IL-12 induction vs no IL-12 induction (ANOVA).

Increased plasma levels of the IFN- axis is associated with worse outcome

We determined the incidence of serious complications in our patient population. There were no deaths within the first month of study enrollment. After 1 year follow-up, there was a greater occurrence of death and composite endpoints in the patients with detectable or elevated plasma concentrations of the IFN- axis (Table VI). There was a similar predictive effect for complications at 1 year when using three IFN- axis markers in the analysis as compared with any two such inflammatory factors (data not shown). A more simple evaluation using CXCL10 alone, a single downstream marker of the cytokine cascade, provided similar prognostic value as compared with multiple markers (Table VI).

View this table: [in this window] [in a new window]   Table VI. Plasma levels of the IFN- axis and clinical outcomesa

IL-12/IL-18 induce IFN- and IFN--dependent chemokine secretion

We used an organ culture system to determine whether cytokines produced by innate immune responses could affect adaptive immune responses within human coronary arteries. We first characterized the cytokine polarization of T cells in atherosclerotic artery rings using polyclonal lymphocyte activators. PMA and ionomycin stimulated secretion of predominantly IFN- and IL-10, with minimal IL-5, into the culture supernatant (Fig. 3A). CXCL10, CXCL11, and CXCL9 were produced in parallel with IFN- secretion (Fig. 3B). The monokines IL-12 and IL-18 induced the secretion of low amounts of IFN- by atherosclerotic coronary arteries (Fig. 3C), whereas IFN-, TNF, IL-1, and IL-6 did not (data not shown). The effects of IL-12 and IL-18 on IFN- production were strongly synergistic (Fig. 3C). IL-12 and IL-18 also had synergistic interactions for the secretion of CXCL10, CXCL11, and CXCL9 (Fig. 3, D--F). The production of CXCR3 ligands in response to IL-12/IL-18 was IFN--dependent (Fig. 3G). In certain of these experiments, the IFN--inducible chemokines were secreted in the absence of, or preceding, detectable IFN- in the supernatant; however, the production of CXCL10, CXCL11, and CXCL9 was consistently prevented by IFN--neutralizing Abs. IL-12/IL-18 did not stimulate IL-5 secretion, although they resulted in minimal IL-10 production from the arteries of some donors (data not shown). Both IFN- and IFN- resulted in robust secretion of CXCL10, CXCL11, and CXCL9, but TNF, IL-1, and IL-6 did not induce CXCR3 ligand production (Fig. 3H). IL-12/IL-18 did not result in IFN- production, and there was no positive feedback of IL-12 or IL-18 secretion in response to IFN- or CXCL10 (data not shown). Nonatherosclerotic coronary arteries had qualitatively similar inflammatory responses, although the secretion of IFN-, CXCL10, CXCL11, and CXCL9 in response to PMA/ionomycin or IL-12/IL-18 was 10–30% of that seen from atherosclerotic artery segments (data not shown). The production of CXCR3-binding chemokines in response to IL-12/IL-18 was also IFN--dependent in nonatherosclerotic arteries (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 3. IL-12/IL-18 induces IFN- and IFN--inducible chemokines in vitro. Atherosclerotic coronary artery segments from at least three donors in separate experiments were treated with PMA at 1 µg/ml and ionomycin at 1 µM for 3–72 h (A and B), IL-12 and/or IL-18 at various doses for 24 h (C–F), IL-12 at 5 ng/ml and IL-18 at 25 ng/ml with neutralizing Ab to IFN- or control IgG at 10 µg/ml for 24 h (G), and IFN- at 40 ng/ml, IFN- at 100 ng/ml, TNF at 10 ng/ml, IL-1 at 10 ng/ml, or IL-6 at 10 ng/ml for 24 h (H). Cytokine levels in the supernatant were measured by ELISA. Data are means ± SEM.

Separate inflammatory and cognate pathways of IFN- production

The secretion of IFN- from coronary artery rings secondary to IL-12/IL-18 was relatively rapid and was detectable in the supernatant within 24 h (Fig. 4A). CXCL10 secretion due to IL-12/IL-18 was detected even earlier by 12 h and only lagged slightly behind the kinetics of chemokine production in response to exogenous IFN- (Fig. 4B). The magnitude of cytokine responses varied in atherosclerotic arteries from different subjects, with calcified segments generally showing lesser responses. Coronary arteries with a normal appearance from individuals without coronary atherosclerosis produced 6.7-fold less IFN- than diseased vessels (Fig. 4C). We were unable to normalize IFN- secretion to leukocyte protein markers measured by ELISA; however, nonatherosclerotic arteries had a 7.7-fold less CD3:GAPDH transcript ratio than atherosclerotic arteries in our experiments (data not shown), indicating that artery-infiltrating T cells were not more primed or responsive to IL-12/IL-18 stimulation in the presence of disease. Consistent with a direct inflammatory signal for cytokine production, inhibition of Ag presentation by blocking Abs to class I and II MHC molecules did not prevent IFN- secretion in response to IL-12/IL-18 (Fig. 4D). We also excluded a role for Ag presentation to NKT cells with a neutralizing Ab to CD1d. IL-12/IL-18 provided a robust signal for cytokine production and induced similar, and in certain donors even greater, IFN- secretion from atherosclerotic coronary arteries than polyclonal TCR activation using a combination of agonistic Abs to CD3 and CD28 (Fig. 4E). Although cytokine production in response to IL-12/IL-18 did not require Ag presentation, there was strong synergy between the inflammatory and cognate stimuli for IFN- secretion (Fig. 4F). CXCL10 production was also augmented in response to the combination of IL-12/IL-18 and CD3/CD28 signals (Fig. 4G). Additionally, there was considerable priming of cytokine responses between the inflammatory and cognate signals. Coronary artery rings exposed to IL-12/IL-18 followed by CD3/CD28 Ab treatment, or vice versa, secreted more IFN- and CXCL10 than artery segments that received no initial treatment or sequential identical treatments (Fig. 4, H and I).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Inflammatory and cognate stimuli of IFN- and CXCL10 secretion. Coronary artery segments from at least three donors in separate experiments were treated with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml or IFN- at 40 ng/ml for various times (A and B), with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml for 48 h using atherosclerotic (diseased) and nonatherosclerotic (normal) arteries (C), with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml for 48 h in the presence of neutralizing Abs to class I and II MHC Ags, CD1d, or control IgG at 10 µg/ml (D), and with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml or agonistic Abs () to CD3 at 0.1 µg/ml and CD28 at 1 µg/ml for 48 h (E). To determine synergistic and priming interactions, atherosclerotic coronary arteries were treated with IL-12 at 20 ng/ml and IL-I8 at 100 ng/ml and/or anti-CD3 at 0.1 µg/ml and anti-CD28 at 1 µg/ml for 48 h (F and G), incubated in medium alone for 4 days, and then treated again for a further 48 h (H and I); this experiment is representative of three different donors. Cytokine levels in the supernatant were measured by ELISA. Data are means ± SEM or individual values in the priming experiments.

Coronary artery-infiltrating T cells secrete IFN- in response to IL-12/IL-18

We performed immunohistochemical analyses to determine the coronary artery cell type(s) that produce IFN- in response to IL-12/IL-18. As has been previously described, there were numerous CD3⁺ T cells (predominantly CD45RO⁺ memory cells with more CD4⁺ Th cells than CD8⁺ T cytolytic cells), rare, if any, CD56⁺ NK or NKT cells, and many CD68⁺ macrophages within the intima and adventitia of atherosclerotic coronary arteries (Fig. 5). There were fewer, nevertheless invariably present, leukocytes in nonatherosclerotic coronary arteries, even in young children (Fig. 5). We were unable to reliably detect IFN- in situ by immunohistochemistry after treating coronary artery rings with IL-12/IL-18 and/or CD3/CD28 Abs. More sensitive techniques of immunofluorescence microscopy of arteries or flow cytometric analysis of extracted leukocytes were also not sufficient to identify IFN- under our experimental conditions. In contrast, immunofluorescence microscopy of extracted leukocytes was very sensitive in distinguishing intracellular cytokines. Using this technique, IFN- was localized exclusively to coronary artery-infiltrating CD3⁺ T cells after treatment with IL-12/IL-18 (Fig. 6, A and B). There were no detectable CD56⁺ NK or NKT cells in the atherosclerotic coronary arteries before or after cytokine treatment (Fig. 6, C and D). Furthermore, depletion of leukocytes extracted from atherosclerotic coronary arteries with CD56 Ab-coated magnetic beads did not diminish IL-12/IL-18-induced IFN- secretion (data not shown). Additionally, IL-12/IL-18 did not induce cytokine or chemokine production in cultured endothelial or vascular smooth muscle cells (data not shown).

View larger version (77K): [in this window] [in a new window]   FIGURE 5. Phenotype of artery-infiltrating leukocytes. Atherosclerotic coronary arteries (OCT-embedded) were analyzed by immunohistochemistry using control IgG (A) or Abs to CD3 (B), CD4 (C), CD8 (D), CD45RA (E), CD45RO (F), CD56 (G), and CD68 (H). Nonatherosclerotic coronary arteries (paraffin-embedded) from a child (I) and an adult (J) were stained with an Ab to CD45. Positive immunostaining is a brown-crimson color. Results are representative of three different artery donors. Scale bar represents 300 µm and photomicrographs were at x200 magnification.

View larger version (70K): [in this window] [in a new window]   FIGURE 6. Coronary artery-infiltrating T cells produce IFN- in response to IL-12/IL-18. Leukocytes were extracted from atherosclerotic coronary arteries and were treated (right panels) or not (left panels) with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml for 48 h. Immunostaining was performed with fluorescent-labeled Abs to CD3 (top panels, green), CD56 (bottom panels, green), and IFN- (all panels, red), and the cells were visualized by confocal, laser-scanning microscopy at x630 magnification. The results are representative of three independent experiments.

To further characterize T cell responses to inflammatory stimuli, we studied lymphocyte subtypes purified from PBMCs because insufficient numbers of leukocytes were recovered from coronary arteries for these types of studies. IL-12/IL-18 induced IFN- secretion predominantly from CD45RA⁺ (naive) and CD45RO⁺ (memory) CD8⁺ T cells, whereas all T cell subsets responded to CD3/CD28 Abs (Fig. 7, A and B). CD45RO⁺/CD4⁺ T cells were activated by higher doses of IL-12/IL-18 or in combination with CD3/CD28 Abs, and even CD45RA⁺/CD4⁺ T cells produced modest amounts of IFN- under these conditions (data not shown). We focused further investigations on CD45RO⁺/CD8⁺ T cells because they constitute a significant proportion of coronary artery-infiltrating lymphocytes and they acquire other effector mechanisms, besides cytokine production, upon activation. Direct and synergistic effects of IL-12/IL-18 on IFN- production by CD45RO⁺/CD8⁺ T cells were confirmed by ELISPOT assay (Fig. 7C). However, IL-12/IL-18-treated cells did not undergo size changes characteristic of activated lymphoblasts following TCR activation (Fig. 7D). CD45RO⁺/CD8⁺ T cells did up-regulate surface CXCR3 expression in response to either IL-12/IL-18 or CD3/CD28 Abs compared with controls (Fig. 7E). In contrast, inflammatory, unlike cognate, stimuli did not increase surface expression of the activation markers, CD25 and CD69, or intracellular expression of the effector molecules, perforin and granzyme B. Only CD3/CD28 Abs stimulated T cell proliferation measured by CFSE fluorescence dilution, and neither signal resulted in cellular death as detected by PI uptake. IL-12/IL-18 did not significantly augment the CD3/CD28 Ab-mediated changes in CD45RO⁺/CD8⁺ T cell phenotype (data not shown).

View larger version (44K): [in this window] [in a new window]   FIGURE 7. T cell inflammatory and cognate stimuli. CD45RA+ (naive) vs CD45RO+ (memory) CD4+ (helper) and CD8+ (cytolytic) T cells isolated from PBMCs of healthy donors were treated with IL-12 at 20 ng/ml and IL-18 at 100 ng/ml and/or agonistic Abs to CD3 at 0.1 µg/ml and CD28 at 1 µg/ml for 48 h, and IFN- supernatant levels were measured by ELISA (A and B). CD45RO+/CD8+ T cells were similarly treated and analyzed by IFN- ELISPOT assay (C) or flow cytometry for forward scatter (FSC) vs side scatter (SSC) size characteristics (D) and cellular fluorescence intensity using fluorescent-labeled control IgG or Abs to CXCR3, CD25, CD69, perforin, and granzyme B, or after CFSE labeling or PI incubation (E). ELISA data are means ± SEM from three different experiments (n = 12), and IFN- was not detected from untreated cells. ELISPOT and flow cytometry data are representative of three independent experiments.

IFN- secretion by IL-12/IL-18 is dependent on p38 MAPK

Finally, we investigated whether there were differences in the signaling pathways between inflammatory and cognate stimuli of IFN- production in atherosclerotic coronary arteries. We did not detect differences in the expression of transcriptional factors that induce IFN-, such as T-bet, GADD45, and GADD45, after IL-12/IL-18 or anti-CD3/CD28 treatment (data not shown). Initial dose response experiments documented the efficacy of a p38 MAPK inhibitor, SB 203580, but not of inhibitors of other MAPKs, in preventing IFN- secretion secondary to IL-12/IL-18 (Fig. 8A). The mTOR inhibitor, rapamycin, partially inhibited cytokine production under these conditions, whereas the calcineurin inhibitor, CsA, and the HMGCoA reductase inhibitor, atorvastatin, were ineffective (Fig. 8B and data not shown). These drug effects were confirmed in additional experiments from multiple donors (Fig. 8C). In contrast, only CsA effectively prevented IFN- production by CD3/CD28 Abs (Fig. 8D).

View larger version (17K): [in this window] [in a new window]   FIGURE 8. Pharmacological differences between inflammatory and cognate pathways of IFN- production. Vehicle (0.1% DMSO) and different doses of ERK, JNK, and p38 MAPK inhibitors, rapamycin (Rapa), and CsA were added to coronary artery rings (n = 2) from single donors (A and B), or atorvastatin at 10 µM, CsA at 300 ng/ml, rapamycin at 30 ng/ml, and p38 MAPK inhibitor at 10 µg/ml were added to coronary artery rings (n = 8) from three donors in separate experiments (C and D). The arteries were treated with IL-12 at 10 ng/ml and IL-18 at 50 ng/ml (A–C) or agonistic Abs to CD3 at 0.1 µg/ml and CD28 at 1 µg/ml (D) for 48 h. IFN- supernatant levels were measured by ELISA. Data are means ± SEM. *, p < 0.05 vs vehicle alone (ANOVA).

	Discussion

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IFN-, a key proatherogenic cytokine, is expressed in arterial plaques (12, 13) and by circulating T cells (8, 14) in patients with coronary atherosclerosis. However, plasma IFN- is difficult to detect, possibly due to limited systemic secretion, a short half-life, or quenching by endothelial cytokine receptors and low-affinity heparan sulfate binding sites. Previous studies have not found a correlation between increased plasma levels of IL-12 and circulating IFN- and IFN--inducible chemokines. In the case of IFN-, it is likely related to limited assay sensitivity because one report did not detect this cytokine in the plasma (7) and another found very low levels (0.2 pg/ml) of IFN- (8). For IFN--inducible chemokines, prior studies measured leukocyte intracellular CXCL10 and CXCL9 production and did not find an association with plasma IL-12 (8). We have found that vascular cells produce 2–3 orders of magnitude greater quantities of CXCR3 ligands than do T cells (15), and thus systemic chemokine levels may be a better indicator of Th1-type immune responses than leukocyte chemokine expression. We cannot exclude the fact that the circulating IFN- detected in our patients was derived from leukocytes other than T cells, viz NK cells. However, IFN- is usually produced in greater amounts by clonally expanded effector lymphocytes than after initial innate immune responses, and CD4⁺ T cells predominate as IFN- producers in chronic infections (16). In addition to the relationship between the IFN- axis markers, we also found an association between IL-12 vs IL-10 and a weaker correlation between IL-12 vs IL-5 plasma levels. The coexpression of IL-12 and IL-10 transcripts has been described in atherosclerotic plaques (13) and may represent a common proinflammatory stimulus rather than a causal role of IL-12 in Th2 and Th3 immune responses. Our study did not find serologic evidence for the IFN- axis in patients with ACS compared with those with chronic stable angina. This does not exclude local activation of T cells within the vulnerable arterial plaque, or it may imply that plaque rupture is not mediated by an Ag-driven Th1-type immune response. Although controversial, other investigators have also failed to find systemic signs of adaptive immune responses in patients with unstable angina despite evidence of inflammation and activation of innate immunity (17, 18, 19). Our findings substantiate the extensive experimental data on the role of the IFN- axis in the pathogenesis of atherosclerosis. Administration of IL-12, IL-18, or IFN- exacerbates plaque formation, whereas the genetic absence of these cytokines or CXCR3 inhibits the development of atherosclerosis in apolipoprotein E-deficient mice (20, 21, 22, 23, 24, 25, 26).

We cannot determine whether increased plasma levels of the IFN- axis in a subset of our patients undergoing cardiac catheterization derived from their coronary atherosclerotic disease or whether it reflected subclinical extracardiac inflammation. A similar dilemma exists in interpreting whether inflammation, as measured by circulating IL-6 and CRP, preceded or was caused by ACS, and there are numerous studies that demonstrate both mechanisms can occur (27). Longitudinal observations of our patients undergoing CABG surgery showed that innate immune responses secondary to CPB increased plasma levels of IL-12 and IFN--inducible chemokines, demonstrating that systemic inflammation can contribute to induction of the IFN- axis in patients with coronary atherosclerosis. We found that patients with coronary atherosclerosis have higher levels of circulating IFN- and IFN--inducible chemokines than referent patients and that the IFN- axis is not detected in healthy young adult subjects. These data indicate that activation of the IFN- axis, similar to that of the IL-6/CRP axis, is not unique to patients with coronary atherosclerosis. We found that activation of the IFN- axis in patients with coronary atherosclerosis is associated with worse clinical outcomes after 1 year, suggesting pathobiological relevance. This is the first report to identify plasma levels of IL-12, IFN-, or IFN--inducible chemokines as predictors of future cardiovascular events. Circulating IL-18 has been previously reported to have prognostic value in a longer follow-up period of 4 years (28). Our relatively small cohort of patients constitute a homogeneous group of middle-aged/elderly, male Caucasians with advanced coronary atherosclerosis and many unfavorable cardiovascular events. Our findings support further seroepidemiologic analysis of the IFN- axis in prospective trials with large numbers of patients.

We investigated whether monokines induce Th1-type immune responses in atherosclerotic coronary arteries using an organ culture model because similar mechanistic experiments are not possible in patients. Direct extrapolation from in vivo to in vitro studies is generally complex, although there was overlap between the highest IL-12 plasma levels in patients and the lowest IL-12 concentrations in organ culture experiments. We observed a sequential cytokine cascade from IL-12/IL-18 to IFN- to IFN--inducible chemokines. Similar to our parallel patient studies, CXCL10 was a more optimal marker of IFN- responses than other CXCR3 ligands, because CXCL11 was less abundant and CXCL9 had higher background levels. There was no evidence for feedback stimulation of the pathway, which suggests that the association of increased plasma levels of IL-12 with other IFN- axis markers in patients may be causal rather than due to macrophage activation with consequent IL-12 secretion in response to T cell production of IFN-. This is in keeping with our clinical observation that circulating IFN- is rarely detected in the absence of IL-12.

We identify T cells as the major IL-12/IL-18 responding cell type within atherosclerotic coronary arteries and, similar to others, found few NK or NKT cells within arterial plaques (29). Unlike other studies (30, 31), we did not find that vascular cells produce IFN- in response to IL-12 and/or IL-18. The ability of IL-12 and IL-18 to promote the Ag-driven development of Th1 immunity is well established (32). More recently, it has become clear that IL-12 and IL-18 can also activate T cells in an Ag-independent fashion (33). Studies of IFN- production by murine CD4⁺ T cells have characterized separate IL-12/IL-18 and TCR signaling pathways that are dependent on p38 MAPK activation and calcineurin activity, respectively (34). Interactions between innate and adaptive stimuli for cytokine responses by murine CD8⁺ T cells have been investigated in a viral infection model in which both IL-12/IL-18 and Ag result in IFN- production, but only TCR signaling induces IL-2 secretion that is pivotal for T cell clonal expansion (35). Our experiments demonstrate that IL-12 and IL-18 may nonspecifically activate IFN- production by artery-infiltrating T cells and/or exacerbate Ag-driven T cell responses within the arterial wall. We have found an invariable, but relatively sparse, infiltrate of leukocytes in coronary arteries without evidence of atherosclerosis, as has been previously described in children and young adults (29) and which may play a role in immune surveillance (36). Our data indicates that T cells in nonatherosclerotic coronary arteries are similarly responsive to IL-12/IL-18 stimulation as in atherosclerotic coronary arteries. We speculate that episodic circulating IL-12 secondary to acute infectious diseases may initiate the overrecruitment of arterial leukocytes, such as CXCR3-bearing Th1 cells, through the production of IFN--inducible chemokines by vascular cells. Sustained exposure to circulating IL-12 in chronic inflammatory conditions, such as autoimmune diseases or atherosclerosis itself, may further contribute to the progression of arterial leukocytic infiltrates and vascular injury. Moreover, IFN- may be directly proarteriosclerotic in the absence of inflammation (37). It is noteworthy that the phenotype of IL-12/IL-18-stimulated lymphocytes in our study is consistent with the characterization of T cells within stable plaques as IFN--producing (12, 13), nonproliferating (38, 39) with a polyclonal distribution of T cell receptors (40, 41, 42), and noncytolytic (43, 44, 45). T cell oligoclonal expansion (46, 47, 48) and acquisition of cytolytic effector function (49, 50, 51) occurs in unstable plaques and may involve additional immunologic triggers that are associated with the production of IL-6 and CRP.

Nonspecific mechanisms for T cell activation in atherosclerosis have been previously reported. oxLDL induced production of IL-12 (but not IFN-) from human carotid plaque cells, and IL-12 enhanced IFN- secretion in PBMCs (52). This study suggested that a specific T cell epitope in oxLDL is not necessary for oxLDL-induced T cell activation. More recently, CD4⁺/CD28^– T cell clones derived from unstable plaques or the peripheral blood of patients with ACS were described to constitutively express IL-12Rs in the absence of antigenic stimulation (53). These T cell clones responded to IL-12 by up-regulation of CCR5 (a chemokine receptor also preferentially expressed by Th1 cells in addition to CXCR3) and increased transendothelial migration and atheroma recruitment in vitro and in vivo, respectively. An important implication of these nonspecific mechanisms of T cell activation is that they may evade normal tolerance control, such as natural regulatory T cells that inhibit atherosclerosis in mouse models (54). Strategies to inhibit Ag-independent activation of T cells may be of fundamental importance in preventing the pathogenesis of atherosclerosis. Our organ culture experiments indicate that a p38 MAPK inhibitor is more effective than the mTOR inhibitor, rapamycin, which has also been described to inhibit IL-12-induced production of IFN- (55).

In conclusion, we provide evidence for an inflammatory pathway regulated by inducible plasma levels of IL-12, and possibly facilitated by basal levels of circulating IL-18, that is associated with Th1-type immune responses and worse outcomes in patients with coronary atherosclerosis. Our findings suggest that the systemic IFN- axis contributes to the pathogenesis of coronary atherosclerosis similar to, but independent of, the extensively studied IL-6/CRP cytokine cascade. We demonstrate that inflammatory stimuli may nonspecifically activate IFN- production by artery-infiltrating T cells and/or exacerbate Ag-driven T cell responses within coronary arteries. Thus, circulating IL-12 and IL-18 may constitute a mechanism for the bystander activation of T cells in the pathogenesis of atherosclerosis.

	Acknowledgment

 
We thank Liping Zhao for expert technical assistance.

	Disclosures

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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 the National Institutes of Health Grant PO1 HL70295.

² H.R. and S.I.S. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. George Tellides, 295 Congress Avenue, Boyer Center for Molecular Medicine 454, New Haven, CT 06510. E-mail address: george.tellides{at}yale.edu

⁴ Abbreviations used in this paper: CRP, C-reactive protein; oxLDL, oxidized low-density lipoprotein; CABG, coronary artery bypass graft; CsA, cyclosporin A; PI, propidium iodide; CPB, cardiopulmonary bypass; CAD, coronary artery disease.

Received for publication June 5, 2006. Accepted for publication October 23, 2006.

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