Interleukin-7–Mediated Inflammation in Unstable Angina
Possible Role of Chemokines and Platelets
Background— Atherogenesis and plaque destabilization involve immune-mediated mechanisms, but the actual mediators have not been fully clarified. Interleukin (IL)-7 is a regulator of T-cell homeostasis but also may be involved in inflammation. We hypothesized that IL-7 could be involved in the inflammatory processes observed in atherosclerosis and acute coronary syndromes.
Methods and Results— To study the role of IL-7 in coronary artery disease, we analyzed IL-7 levels and the effect of this cytokine on inflammatory mediators in patients with stable and unstable angina and in healthy control subjects. Our major findings were (1) Plasma levels of IL-7 were significantly increased in patients with stable (n=30) and unstable angina (n=30) comparing healthy control subjects (n=20), particularly in those with unstable disease. (2) Increased release from activated platelets appeared to be a major contributor to the raised IL-7 levels in patients with angina. (3) IL-7 enhanced the expression of several inflammatory chemokines in peripheral blood mononuclear cells from both healthy control subjects and patients with angina, particularly in those with unstable disease. Similar effects were seen in monocytes but not in T cells. (4) MIP-1α further increased the release of IL-7 from platelets in a dose-dependent manner. (5) Aspirin reduced both the spontaneous and the SFLLRN-stimulated release of IL-7 from platelets, and when administered to healthy control subjects for 7 days (160 mg qd), it reduced plasma levels of IL-7.
Conclusions— Our findings suggest a role for IL-7-driven inflammation in atherogenesis and the promotion of clinical instability in coronary artery disease involving interactions between platelets, monocytes, and chemokines.
Received February 5, 2003; accepted February 28, 2003.
Atherosclerotic plaques are characterized by prominent monocyte and T-cell infiltration and are widely recognized as inflammatory lesions.1 Besides being involved in atherogenesis, monocytes/macrophages and T cells also may contribute to plaque rupture, with subsequent thrombosis resulting in acute coronary syndromes.1–3
The precise mechanisms leading to recruitment and activation of immune cells into atherosclerotic lesions are not completely understood. Advanced human plaques demonstrate a polyclonal T-cell composition suggesting that several plaque-derived antigens (eg, modified autoantigens such as oxidized lipoproteins, heat shock proteins, Chlamydia pneumoniae, and other microbial antigens) may be involved in this process.2 However, alternative immune activation pathways independent of antigen stimulation has recently been described in this disorder.2,4
Interleukin (IL)-7 is a major regulator of T-cell homeostasis.5 In addition to its capacity to enhance antigen-induced T-cell proliferation, IL-7 has recently also been shown to induce T-cell activation independent of T-cell receptor stimulation.5,6 Moreover, IL-7 may also induce activation of monocytes and natural killer cells, leading to enhanced production of inflammatory cytokines.5,7 In fact, enhanced IL-7 activation has recently been implicated in the pathogenesis of some inflammatory disorders such as rheumatoid arthritis and inflammatory bowel disease.8,9 We hypothesized that IL-7 also could be involved in the inflammatory processes observed in atherosclerosis possibly promoting plaque progression and instability. In the current study, this hypothesis was investigated in patients with stable and unstable angina by using different experimental approaches.
Patients and Control Subjects
Patients with angina were consecutively registered and recruited to the study.10 All patients with unstable angina (n=30) had had ischemic chest pain at rest within the preceding 48 hours (ie, Braunwald class IIIB) with transient ST-T–segment depression and/or T-wave inversion (Table). All patients with stable angina (n=30) had stable effort angina of >6 months’ duration and a positive exercise test (Table). Exclusion criteria were myocardial infarction or thrombolytic therapy within the previous month, ECG abnormalities invalidating ST-segment analyses, and concomitant disease likely to be associated with inflammation (eg, infections and autoimmune disorders). The diagnosis of coronary artery disease (CAD) was confirmed in all patients by coronary angiography showing at least 1 vessel disease (>75% narrowing of luminal diameter). When performing the substudies [ie, the in vitro experiments and the percutaneous coronary intervention (PCI) study (see below)], the patients were randomly selected from our patient population. Control subjects in the study were 20 sex- and age-matched healthy blood donors (15 men and 5 women, 53±15 years). Plasma (EDTA), platelet-free plasma, and serum were collected and stored as previously described.10,11
Cell Culture Experiments
Peripheral blood mononuclear cells (PBMC), CD14+ monocytes, and CD3+ T cells were isolated as previously described10,12 and incubated in 96- or 24-well trays (2×106 cells/mL, Costar) in medium alone [RPMI 1640 with 2 mmol/L l-glutamine, and 25 mmol/L HEPES buffer (Gibco) supplemented with 5% human AB+ serum] or stimulated with phytohemagglutinin (PHA; Murex; final dilution, 1:100) or different concentrations of IL-7 (R&D Systems) or a combination thereof. In some experiments, PBMC were also stimulated with lipopolysaccharide from Escherichia coli O26:B6 (LPS; final concentration, 10 ng/mL; Sigma). Cell pellets and cell-free supernatants were harvested after 6 and 20 hours, respectively, and stored in liquid nitrogen (cell pellets) or at −80°C (supernatants). In a separate experiment, we also analyzed IL-7 levels in unstimulated and trimeric CD40 ligand-stimulated (CD40L, R&D Systems) human umbilical vein endothelial cells (HUVEC)13 supernatants after culturing for 20 hours.
Stimulation of Platelets in Platelet-Rich Plasma
Preparation and stimulation of citrated platelet-rich plasma (PRP) was performed as previously described.11 Briefly, PRP was incubated for 30 minutes at room temperature after addition of different concentrations of the thrombin receptor agonist SFLLRN or Tris-buffered saline only. In some experiments, different concentrations of macrophage inflammatory protein-1α (MIP-1α, R&D Systems), aspirin (Sigma), or prostaglandin E1 (PGE1, Sigma) were also added to PRP either alone or in combination with SFLLRN. At baseline and after 30 minutes, PRP was centrifuged at 10 000g for 10 minutes, and platelet-free plasma and platelet pellets (with Tris-buffered saline) were stored separately at −80°C. Platelet pellets were lysed by freezing and thawing 3 times, and IL-7 levels were analyzed in the lysates. The increase in IL-7 levels (ng/108 platelets) was expressed as the concentration in platelet-free plasma at the end of the experiments minus the concentration at baseline.
RNase Protection Assay
Total RNA was extracted from PBMC, T cells, and monocytes by using RNeasy columns (Qiagen) and stored in RNA storage solution (Ambion) at −80°C. RNase protection assay was performed with chemokine receptor (hCR5 and hCR6) and chemokine (hCK5) multiprobes (Pharmingen).14 The mRNA signal was normalized to the signal from the housekeeping gene GAPDH.
IL-7, IL-8, and monocyte chemoattractant protein (MCP)-1 were measured by enzyme immunoassay (R&D Systems). The prothrombin fragments F1+2 were determined in citrate plasma (Diatube-H) by an enzyme immunoassay from Dade Behring GmbH.
When comparing 3 groups of individuals, 1-way ANOVA was followed by Scheffé’s post hoc test for statistical significance. The data were subjected to logarithmic transformation before performing the ANOVA analysis. For comparisons within the same individuals, the Wilcoxon matched pairs test was used. Probability values (2-sided) were considered significant at levels <0.05.
IL-7 Levels in Patients With Angina
Both patients with stable (n=30) and unstable angina (n=30) had raised plasma levels of IL-7 compared with control subjects (n=20) with particularly high levels in those with unstable disease (Figure 1). The same pattern with high levels in patients with angina was also found in platelet-free plasma and serum (data not shown), but, notably, IL-7 levels were in general markedly higher in serum than in plasma (≈8.3-fold), suggesting release during coagulation. Plasma levels of IL-7 were also analyzed in 20 patients with stable and in 20 patients with unstable angina before and 16 hours after PCI. Whereas this procedure markedly enhanced IL-7 levels in stable angina, no such increase was seen in unstable disease (Figure 1).
IL-7 in Relation to Clinical and Thrombotic Parameters
IL-7 was significantly correlated with plasma levels of prothrombin fragments within the total patient group (r=0.65, P<0.01). Moreover, patients with unstable angina had a mean troponin I levels of 0.3±0.1 ng/mL, significantly correlated with IL-7 (r=0.57, P<0.05). Furthermore, although there was no difference between single-vessel and multivessel (>2 vessel affected) disease in stable angina, those with multivessel disease tended to have higher IL-7 levels (P=0.06) in the unstable angina group. Finally, within the total patient group, IL-7 was significantly correlated with serum levels of high-sensitivity C-reactive protein (r=0.61, P<0.01). In contrast, no correlation was found between IL-7 and either platelet or leukocyte counts.
Platelets: An Important Cellular Source of IL-7 Levels in Plasma
As suggested by the high serum levels, platelets could be an important cellular source of IL-7 in circulation. We therefore next examined the release of IL-7 in unstimulated and SFLLRN-stimulated platelets in PRP from 7 patients with unstable angina, 7 with stable angina, and in 7 healthy control subjects. The platelets provided large amounts of IL-7 after lysis and released large amounts of IL-7 into the supernatants after SFLLRN stimulation (Figure 2). However, although platelets from patients with unstable angina had decreased intracellular IL-7 levels and showed decreased release on SFLLRN stimulation comparing control subjects, platelets from these patients spontaneously released significantly higher levels of IL-7 (Figure 2). In contrast, platelets from patients with stable angina showed enhanced release of IL-7 both spontaneously and after SFLLRN stimulation, although the spontaneous release was significantly lower than in unstable disease (Figure 2). Finally, preincubation with PGE1, a stimulator of platelet adenylyl cyclase with inhibiting effects on platelet granule secretion, inhibited the release of IL-7 from SFLLRN-stimulated (100 μmol/L) platelets in a dose-dependent manner, suggesting that this cytokine is secreted from α-granules during activation.
In contrast to the release from platelets, IL-7 was not detectable in unstimulated, PHA-stimulated or LPS-stimulated PBMC supernatants from neither patients with angina nor control subjects. Moreover, HUVEC did not release detectable amounts (<0.1 pg/mL) of IL-7 either spontaneously or after CD40L stimulation.
Effect of IL-7 on Chemokine Expression in PBMC
To elucidate any possible pathogenic consequences of IL-7 in unstable angina, we examined the effect of IL-7 on chemokine expression in PBMC from 7 patients with unstable angina, 7 with stable angina, and 7 healthy control subjects. IL-7 markedly influenced the gene expression of a wide range of chemokines in PBMC from patients with unstable angina (Figure 3A). First, IL-7 induced gene expression of MCP-1 (3.3-fold, P<0.01), IL-8 (2.9-fold, P<0.05), MIP-1α (4.3-fold, P<0.01), and MIP-1β (4.5-fold, P<0.01) in unstimulated PBMC and enhanced the PHA-stimulated mRNA levels of these chemokines. Second, although no effect was seen when IL-7 was given alone, it enhanced the PHA-stimulated gene expression of lymphotactin (3.4-fold, P<0.01), interferon-γ–inducible protein (IP)-10 (2.9-fold, P<0.01), and inducible (I)-309 (2.3-fold, P<0.05).
Although a similar pattern was seen in PBMC from healthy control subjects and patients with stable angina, some significant differences were found. First, unstimulated cells from patients with unstable angina expressed higher levels of MCP-1, IL-8, MIP-1α, and MIP-1β, comparing both control subjects (>2.0-fold, P<0.05) and patients with stable angina (≈1.6-fold, P<0.05), suggesting that PBMC from patients with unstable angina are markedly preactivated in vivo. Second, IL-7 had a more marked effect on chemokine gene expression in unstable angina, with a particularly enhancing effect on the PHA-stimulated expression of MCP-1, IL-8, MIP-1α, and MIP-1β. For MCP-1 and IL-8, these patterns were also verified at the protein level (Figure 3, B and C).
Effect of IL-7 on Chemokine Receptor Expression in PBMC
IL-7 also markedly enhanced the mRNA level of CXCR4 (2.4-fold, P<0.05) and CCR7 (2.0-fold, P<0.05), representing chemokine receptors involved in both leukocyte homeostasis and inflammation, with similar effect in both unstable and patients with stable angina and control subjects. In contrast, although unstimulated PBMC from patients with unstable angina expressed high mRNA levels of CCR1, CCR2, and CCR5 (ie, receptors for MCP-1, MIP-1α, MIP-1β, and RANTES), comparing both patients with stable angina and control subjects (>1.6-fold, P<0.05 for all), IL-7 did not influence the expression of these receptors in either patients or control subjects.
Effects of IL-7 on Chemokine Expression in T Cells and Monocytes
We next examined the effect of IL-7 on chemokine expression when T cells and monocytes were cultured separately. Several significant findings were revealed: (1) Monocytes but not T cells from patients with unstable angina spontaneously expressed enhanced mRNA levels of several chemokines, comparing both control subjects and patients with stable angina (ie, MCP-1, IL-8, MIP-1α, MIP-1β; >2.0-fold and ≈1.6-fold, respectively, P<0.05). (2) IL-7 induced a significant increase in chemokine gene expression in both patients and control subjects, with a particularly marked response in patients with unstable disease (Figure 4). (3). In contrast, IL-7 had no effect on chemokine gene expression in T cells from either patients or control subjects.
Effects of Chemokines on IL-7 Release From Platelets
We found that IL-7 enhances the expression of MIP-1α in PBMC, and because platelets express the receptor for MIP-1α (ie, CCR1),15 we next examined if this “IL-7-inducible” chemokine could further enhance the release of IL-7 from platelets in healthy control subjects (n=5). We found that MIP-1α in a dose-dependent manner (0.01 to 1.0 μg/mL) induced a significant IL-7 release from platelets (Figure 5A). Moreover, MIP-1α (0.1 μg/mL) showed additive effect on the SFLLRN-stimulated release of this cytokine when given simultaneously with SFLLRN (10 μmol/L) (Figure 5B).
IL-7 Release From Platelets After Aspirin Administration In Vitro and In Vivo
Because platelets appear to be a major contributor to IL-7 release into circulation and potentially also within atherosclerotic plaques, we finally examined whether aspirin could inhibit the release of IL-7 from platelets in 5 healthy control subjects not receiving aspirin. Aspirin reduced both the spontaneous (data not shown) and the SFLLRN-stimulated (10 μmol/L) release of IL-7 from platelets in a dose-dependent manner (Figure 6A). However, no such effect was observed when platelets were stimulated with high SFLLRN concentrations (ie, 100 μmol/L). Finally, we examined the effect of aspirin in the in vivo situation, and when given for 7 days, this medication (160 mg qd) markedly reduced IL-7 plasma levels in healthy control subjects (≈60% reduction, n=8) (Figure 6B).
Several studies have shown that IL-7 functions as a regulator of both antigen-independent and antigen-dependent T-cell proliferation, playing an important role T-cell homeostasis.5,6 In the current study, we showed that IL-7 also increases the production of several inflammatory chemokines in PBMC, with a particularly enhancing effect in unstable angina. Moreover, although most studies have focused on the effect of IL-7 on T cells, we show that monocytes rather than T cells produce chemokines on IL-7 stimulation. These findings indicate that in addition to its role in T-cell homeostasis, IL-7 may also have an inflammatory potential involving chemokine- and monocyte-mediated mechanisms. Our results suggest that such a mechanism may be operating in unstable angina possibly promoting clinical instability.
Herein we report raised IL-7 levels in patients with angina with particularly high levels in unstable disease. Moreover, although IL-7 promoted chemokine production in PBMC from both patients with angina and healthy control subjects, a particularly enhancing effect was seen in unstable disease. If such an enhancing effect also exists within an atherosclerotic plaque, it may suggest a pathogenic role for IL-7 in both atherogenesis and in the promotion of acute coronary syndrome. Thus, chemokines such as IL-8, MCP-1, MIP-1α, and IP-10, all upregulated by IL-7 in PBMC, are expressed within atherosclerotic plaques with particularly high levels in advanced lesions.16,17 Moreover, recent in vivo studies have shown that targeted disruption of the genes for MCP-1, CCR2 (ie, MCP-1 receptor), and CXCR2 (ie, IL-8 receptor) significantly decrease atherosclerotic lesion formation in mice prone to development of atherosclerotic-like lesions.18,19 Furthermore, chemokines such as IL-8 and MCP-1 have also been implicated in the promotion of clinical instability in patients with CAD, at least partly by enhancing oxidative stress and apoptosis within the atherosclerotic lesions.16,20 Our findings in the current study suggest that IL-7 could contribute to such chemokine-mediated pathophysiology in patients with angina representing a “new” mediator of inflammation in these patients.
A major finding in the current study was that platelets appear to be an important contributor to raised plasma/serum levels of IL-7 in patients with angina. Thus, we report that platelets release significant amounts of this cytokine on activation and the inhibiting effect of PGE1 suggest that IL-7 is secreted from α-granules. Moreover, although platelets from patients with unstable angina had decreased intracellular IL-7 levels and showed decreased release on SFLLRN stimulation, platelets from these patients spontaneously released high levels of IL-7. In contrast, platelets from patients with stable angina showed enhanced release of IL-7 both spontaneously and after SFLLRN stimulation, although the spontaneous release was significantly lower than in unstable disease. Although such a pattern as in stable angina may reflect a moderate degree of platelet activation, possibly caused by priming effects of inflammatory and thrombogenic mediators, the pattern in unstable angina appears to reflect a more marked activation of these cells, with an increased proportion of degranulated platelets in vivo resulting in a decreased response on further stimulation ex vivo.10,11 Our results during PCI showing enhanced plasma levels of IL-7 after this mechanically induced plaque rupture in stable but not unstable angina further support an increased proportion of degranulated platelets in unstable angina. Nevertheless, our findings add IL-7 to the list of cytokines that are released from activated platelets, further underscoring an inflammatory potential of these cells. Moreover, whereas IL-7 induced the production of MIP-1α in monocytes, this chemokine further stimulated the release of IL-7 from platelets possibly representing an inflammatory loop operating in unstable angina.
Recent studies suggest that the progression of acute coronary syndromes are unrelated to the initial culprit lesion but arise from complication in other segments of the coronary arteries, reflecting widespread coronary inflammation.21 Our findings in the current study may suggest that IL-7 may be involved in this promotion of clinical instability. First, although there was no difference between those with single-vessel and multivessel disease in stable angina, those with multivessel disease tended to have higher IL-7 levels in patients with unstable disease, suggesting a link between IL-7 and widespread CAD in these patients. Second, we found that IL-7 was significantly correlated with the levels of prothrombin fragments and troponin I, further supporting a link between IL-7 and the degree of thrombotic events and clinical instability in CAD. The marked rise in IL-7 after mechanically induced plaque rupture by PCI in stable angina, but not during intervention on culprit lesions in unstable angina, may further support such a notion. Thus, although it may be argued that the PCI-induced rise in IL-7 could reflect that high IL-7 level is a consequence rather than a cause of unstable angina, recent studies describe acute coronary syndromes as a continuous process involving multiple ruptured atherosclerotic plaques involving not only the culprit lesion.21 Hence, although not specific for atherosclerosis and not the initial event, IL-7–driven inflammation may contribute to the progression of patient instability, representing an amplification factor in this process. Interestingly, low-dose aspirin significantly decreased plasma levels of IL-7 in healthy control subjects, and our in vitro experiment shows that this medication inhibit IL-7 release from platelets in a dose-dependent manner. However, although nearly all patients with angina received low-dose aspirin, they still had raised IL-7 levels, and aspirin did not inhibit IL-7 release when platelets were activated by high concentrations of SFLLRN. Thus, although our findings further underscore the anti-inflammatory potential of aspirin even at low dosage, it also illustrates the need for more potent platelet inhibition in these patients.
Although the current study suggests an association between IL-7 and acute coronary syndromes, we have no prognostic data relating high IL-7 levels to the occurrence of adverse clinical events during follow-up. Moreover, our findings in cells isolated from the circulation may not necessarily reflect the situation within an atherosclerotic plaque. Nevertheless, although the exact role of IL-7 will have to be confirmed in future studies, our findings suggest that IL-7–driven inflammation may play a role in the promotion of clinical instability in patients with CAD involving interactions between platelets, monocytes, and chemokines.
Hansson GK, Libby P, Schönbeck U, et al. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res. 2002; 91: 281–291.
Libby P. Current concepts of the pathogenesis of acute coronary syndromes. Circulation. 2001; 104: 365–372.
Houtkamp MA, van der Wal AC, de Boer OJ, et al. Interleukin-15 expression in atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2001; 21: 1208–1213.
Wong P, Pamer EG. Antigen-independent CD8 T cell proliferation. J Immunol. 2001; 166: 5864–5868.
Watanabe M, Ueno Y, Yajima T, et al. Interleukin 7 transgenic mice develop chronic colitis with decreased interleukin 7 protein accumulation in the colonic mucosa. J Exp Med. 1998; 187: 389–402.
Aukrust P, Müller F, Ueland T, et al. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes. Circulation. 1999; 100: 614–620.
Holme PA, Müller F, Solum NO, et al. Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection. FASEB J. 1998; 12: 79–90.
Clemetson KJ, Clemetson JM, Proudfoot AEI, et al. Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets. Blood. 2000; 96: 4046–4054.