Interleukin-1 receptor antagonist gene polymorphism and coronary artery disease.
BACKGROUND: Cytokine gene variations are contributory factors in inflammatory pathology. Allele frequencies of interleukin (IL)-1 cluster genes [IL-1A(-889), IL-1B(-511), IL-1B(+3953), IL-1RN Intron 2 VNTR] and tissue necrosis factor (TNF)-alpha gene [TNFA(-308)] were measured in healthy blood donors (healthy control subjects), patients with angiographically normal coronary arteries (patient control subjects), single-vessel coronary disease (SVD), and those with multivessel coronary disease (MVD). METHODS AND RESULTS: Five hundred fifty-six patients attending for coronary angiography in Sheffield were studied: 130 patient control subjects, 98 SVD, and 328 MVD. Significant associations were tested in an independent population (London) of 350: 57 SVD, 191 MVD, and 102 control subjects. IL-1RN*2 frequency in Sheffield patient control subjects was the same as in 827 healthy control subjects. IL-1RN*2 was significantly overrepresented in Sheffield SVD patients (34% vs 23% in patient control subjects); IL-1RN*2 homozygotes in the SVD population (chi2 carriage=8.490, 1 df, P=0.0036). This effect was present though not quite significant in the London population (P=0. 0603). A summary trend test of the IL-1RN SVD genotype data for Sheffield and London showed a significant association with *2 (P=0. 0024). No significant effect of genotype at IL-1RN was observed in the Sheffield or London MVD populations. Genotype distribution analysis comparing the SVD and MVD populations at IL-1RN showed a highly significant trend (P=0.0007) with the use of pooled data. No significant associations were seen for the other polymorphisms. CONCLUSIONS: IL-1RN*2 was significantly associated with SVD. A difference in genetic association between SVD and MVD was also apparent. (+info
Constitutive expression of interleukin-1alpha precursor promotes human vascular smooth muscle cell proliferation.
Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the failure of vascular surgeries and contributes to the development of atherosclerotic lesions. Evidence that interleukin-1 (IL-1) is a mitogen for cultured VSMC has implicated its release by activated macrophages in the development of atherosclerosis. VSMC also produce IL-1, including the precursor form of IL-1alpha. However, it is not known whether IL-1alpha precursor is processed to mature IL-1alpha or released from VSMC, nor is it known whether either precursor or mature IL-1alpha functions as an autocrine growth factor. The goals of the present study were to establish whether proliferation is enhanced in human VSMC transfectants producing IL-1alpha constitutively at levels comparable to those produced after activation, and to determine which domains of IL-1alpha are important for its activity. Human VSMC were stably transfected with expression vectors directing constitutive expression of either full-length IL-1alpha precursor [IL-1alpha-(1-271)], its NH2-terminal domain [IL-1alpha-(1-112)], or mature IL-1alpha [IL-1alpha-(113-271)]. Both IL-1alpha-(1-271) and IL-1alpha-(113-271) stable transfectants produced moderate levels of IL-1alpha (0.2-1.0 ng/10(6) cells) and released low levels of IL-1alpha into the supernatant (<20 pg/ml). VSMC stably transfected with either IL-1alpha-(1-271) or IL-1alpha-(113-271) expression plasmids proliferated rapidly compared with nontransfected or vector-transfected VSMC and displayed a distinct morphology characterized by elongated, spindle-shaped cells. Stable transfection with IL-1alpha-(1-271) was somewhat more effective than transfection with IL-1alpha-(113-271). Interestingly, VSMC transfected with IL-1alpha-(113-271) expression plasmids also expressed IL-1alpha-(1-271) mRNA, suggesting that IL-1alpha-(113-271) activates an IL-1-induced IL-1 autocrine loop. In contrast, neither proliferation rates nor morphology was affected by stable transfection with IL-1alpha-(1-112) expression plasmids. Exogenous IL-1 receptor antagonist partially reversed the enhanced DNA synthesis in VSMC transfected with either IL-1alpha-(1-271) or IL-1alpha-(113-271) expression plasmids, suggesting that the pro-proliferative effect of VSMC-derived IL-1alpha is at least partially mediated by signaling via the type I IL-1 receptor. These results demonstrate that IL-1alpha precursor is an autocrine growth factor for human VSMC and further indicate that amino acids 113-271 play a crucial role in its actions. (+info
Acute-phase responses in transgenic mice with CNS overexpression of IL-1 receptor antagonist.
The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels. (+info
Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.
Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies. (+info
IL-1 up-regulates osteopontin expression in experimental crescentic glomerulonephritis in the rat.
Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days -1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules (<5%) and absent from glomeruli. During the development of rat anti-GBM disease (days 7 to 21), there was substantial up-regulation of OPN mRNA and protein expression in glomeruli (>5 cells per glomerular cross-section) and tubular epithelial cells (50-75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (/75-85%, P < 0.001) and tubules (/45-60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury. (+info
Effects of interleukin-1 receptor antagonist overexpression on infection by Listeria monocytogenes.
Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring cytokine whose only known function is the inhibition of interleukin-1 (IL-1). Using a reverse genetic approach in mice, we previously showed that increasing IL-1ra gene dosage leads to reduced survival of a primary listerial infection. In this study, we characterize further the role of endogenously produced IL-1ra and, by inference, IL-1 in murine listeriosis. IL-1ra overexpression inhibits, but does not eliminate, primary immune responses, reducing survival and increasing bacterial loads in the target organs. We demonstrate that IL-1ra functions in the innate immune response to regulate the peak leukocyte levels in the blood, the accumulation of leukocytes at sites of infection, and the activation of macrophages during a primary infection. Reduced macrophage class II major histocompatibility complex expression was observed despite increased gamma interferon (IFN-gamma) levels, suggesting that IL-1 activity is essential along with IFN-gamma for macrophage activation in vivo. We also show that IL-1ra plays a more limited role during secondary listeriosis, blunting the strength of the delayed-type hypersensitivity response to listerial antigen while not significantly altering cellular immunity to a second infectious challenge. When these results are compared to those for other mutant mice, IL-1ra appears to be unique among the cytokines studied to date in its regulation of leukocyte migration during primary listeriosis. (+info
Efficacy of sustained blood levels of interleukin-1 receptor antagonist in animal models of arthritis: comparison of efficacy in animal models with human clinical data.
OBJECTIVE: To determine the role of interleukin-1 receptor antagonist (IL-1Ra) in rat adjuvant arthritis and rat type II collagen-induced arthritis, and to compare the efficacy in rat models with that seen in human clinical trials of IL-1Ra. METHODS: Rats with developing adjuvant arthritis or established collagen-induced arthritis were treated with IL-1Ra by continuous infusion in order to determine and maintain efficacious blood levels of this IL-1 inhibitory protein in the rats for comparison with human clinical data. The effects of treatment in the rats were monitored by sequential caliper measurement of the ankle joints, determination of final paw weights, and histologic evaluation with particular emphasis on bone and cartilage lesions. The effects of IL-1Ra on joint swelling and radiographic bone damage in patients with rheumatoid arthritis (RA) in a 6-month trial were compared with the findings in rats. RESULTS: Dramatic differences in the profile of IL-1Ra activity were seen between the 2 groups of rats. Modest antiinflammatory effects were observed in the adjuvant arthritis rats treated with IL-1Ra. However, marked inhibition of bone resorption occurred, even at doses with which antiinflammatory activity was not seen. In contrast, IL-1Ra treatment of rats with established collagen-induced arthritis resulted in nearly complete suppression of all aspects of the disease when adequate blood levels of IL-1Ra were maintained. Treatment of RA patients with IL-1Ra (150 mg daily) resulted in modest inhibition of joint swelling and inhibition of radiographic progression of bone lesions. CONCLUSION: IL-1 appears to be of major importance in mediating the bone resorption that occurs in rat adjuvant arthritis, but is less important in the pathogenesis of periarticular inflammation in this disease. In contrast, IL-1 is of major importance in mediating all aspects of disease progression in rat collagen-induced arthritis. Similar to the response in adjuvant arthritic rats, RA patients treated with IL-1Ra showed only modest antiinflammatory activity, but had evidence of inhibition of progression of bone resorption. However, a comparison of the plasma levels of IL-1Ra in humans and rats suggests that the optimal level of dosing for continuous saturation of IL-1 receptors may not have been achieved in humans, although this was achieved in the rat studies. (+info
Imbalance of IL-1 beta and IL-1 receptor antagonist mRNA in liver tissue from hepatitis C virus (HCV)-related chronic hepatitis.
Increased levels of IL-1 beta and IL-1 receptor antagonist (IL-1Ra) have been found in serum of patients with chronic liver diseases, although their expression in liver tissue has not been extensively investigated. The aim of this study was therefore to examine the relationship between IL-1 beta and IL-1Ra at tissue level in patients with HCV-related chronic active hepatitis (CAH) of varying degrees of severity. IL-1 beta and IL-1Ra mRNA expression was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 31 patients with CAH of varying severity (classified as minimal/mild in 13 cases and moderate/severe activity in 18 cases) and in 12 control subjects. Quantitative evaluation of IL-1 beta and IL-1Ra corresponding bands was performed by densitometric image analysis, and expressed in arbitrary units. The 12 controls expressed a similar pattern with a mean IL-1 beta/IL-1Ra ratio of 1.03 (1.03 +/- 0.15 (mean +/- s.e.m.), median 0.92, range 0.71-1.45). Minimal/mild activity CAH showed a prevalence of IL-1Ra mRNA expression (1.14 +/- 0.64, median 0.43, range 0-8.75) when compared with controls (0.27 +/- 0.04, median 0.23, range 0.11-0.45) and with moderate/severe activity CAH (0.20 +/- 0.04, median 0.12, range 0-0.67; P = 0.01). Since IL-1 beta expression was similar in the three groups, a significantly different IL-1 beta/IL-1Ra ratio emerged between controls, patients with moderate/severe CAH (2.22 +/- 0.48, median 2.76, range 0-6.12) and those with minimal/mild activity CAH (0.62 +/- 0.15, median 0.5, range 0-1.58, P = 0.005). Patients with higher grades of fibrosis showed a higher IL-1 beta/IL-1Ra ratio (2.49 +/- 0.56, median 2.15, range 0.35-6.12) in comparison with lower grade fibrosis (1.06 +/- 0.30, median 0.59, range 0.03-4.50) and control patients (P = 0.01). These results suggest that an imbalance between IL-1 beta and IL-1Ra, at the tissue level, may contribute to the pathogenesis and the activity of chronic active hepatitis C. (+info