Genome-wide associated loci influencing interleukin (IL)-10, IL-1Ra, and IL-6 levels in African Americans

Interleukins (ILs) are key mediators of the immune response and inflammatory process. Plasma levels of IL-10, IL-1Ra, and IL-6 are associated with metabolic conditions, show large inter-individual variations, and are under strong genetic control. Therefore, elucidation of the genetic variants that influence levels of these ILs provides useful insights into mechanisms of immune response and pathogenesis of diseases. We conducted a genome-wide association study (GWAS) of IL-10, IL-1Ra, and IL-6 levels in 707 non-diabetic African Americans using 5,396,780 imputed and directly genotyped single nucleotide polymorphisms (SNPs) with adjustment for gender, age, and body mass index. IL-10 levels showed genome-wide significant associations (p < 5 × 10(-8)) with eight SNPs, the most significant of which was rs5743185 in the PMS1 gene (p = 2.30 × 10(-10)). We tested replication of SNPs that showed genome-wide significance in 425 non-diabetic individuals from West Africa, and successfully replicated rs17365948 in the YWHAZ gene (p = 0.02). IL-1Ra levels showed suggestive associations with two SNPs in the ASB3 gene (p = 2.55 × 10(-7)), ten SNPs in the IL-1 gene family (IL1F5, IL1F8, IL1F10, and IL1Ra, p = 1.04 × 10(-6) to 1.75 × 10(-6)), and 23 SNPs near the IL1A gene (p = 1.22 × 10(-6) to 1.63 × 10(-6)). We also successfully replicated rs4251961 (p = 0.009); this SNP was reported to be associated with IL-1Ra levels in a candidate gene study of Europeans. IL-6 levels showed genome-wide significant association with one SNP (RP11-314E23.1; chr6:133397598; p = 8.63 × 10(-9)). To our knowledge, this is the first GWAS on IL-10, IL-1Ra, and IL-6 levels. Follow-up of these findings may provide valuable insight into the pathobiology of IL actions and dysregulations in inflammation and human diseases.     

Imbalance production between interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1Ra) in bronchial asthma

Genes of the IL-1 family encode three different peptides, IL-1alpha, IL-1beta, and IL-1Ra, respectively. IL-1 operates through IL-1RI, and is involved in airway inflammation in asthmatic subjects, whereas IL-1Ra appears to be a specific competitive inhibitor of IL-1. All genes are on chromosome 2q12-21 where genomewide searches have identified linkage for asthma. To test whether variants of IL-1 relate to asthma, we conducted a genetic association study in a Japanese population. We show that the A2 allele of IL1RN (encoding IL-1Ra) associates with nonatopic asthma [OR = 5.71, 95% CI: 1.63-19. 8, Pc = 0.007]. Both atopic and nonatopic asthmatics with the A2 allele had significantly lower serum IL-1Ra levels in both types of asthmatics. Peripheral blood cells from asthmatics with A2 alleles, however, produced as much IL-1 as did those with A1 homozygotes. Since Th1 and Th2 cytokines differentially regulate the ratio between IL-1beta and IL-1Ra, these findings suggest that dysregulation of IL-1beta/IL-1Ra, probably due to interaction between epithelium and immuno-competent cells in the airway, is important in asthma inflammation.     

In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes

Background  

Chorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.     

ATP-stimulated release of interleukin (IL)-1beta and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage

Interleukin (IL)-1beta and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1beta and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1beta-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X(7)) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1beta and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1beta nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X(7) receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1beta/IL-18. Taken together, these results suggest that, although synthesis of IL-1beta and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1.     

IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells

Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.     

A single amino acid difference between human and monkey interleukin (IL)-1beta dictates effective binding to soluble type II IL-1 receptor

Soluble type II interleukin (IL)-1 receptor (sIL1R-II) binds human IL-1beta with high affinity and neutralizes its activity. Recombinant sIL1R-II is considered a potentially useful anti-IL-1 therapeutic, and preclinical studies have been undertaken with this molecule in primates. To better understand the cytokine-receptor interactions occurring in this nonhuman context, monkey IL-1 and IL1R-II were cloned, and their binding abilities were examined in vitro. IL-1beta from cynomolgus monkey was capable of binding and activating the human type I IL-1 receptor. However, unlike human IL-1beta, it was unable to effectively bind and become neutralized by sIL1R-II. Human and cynomolgus IL-1beta proteins are 96% identical, differing by only six amino acids. Structural and mutational analysis revealed that the unique sIL1R-II binding ability of human IL-1beta is due to a single amino acid difference compared with monkey IL-1beta.     

Hypoxia induces the expression and release of interleukin 1 receptor antagonist in mitogen-activated mononuclear cells

Hypoxia modulates the expression of inflammatory mediators in a variety of cell types. Since interleukin (IL-)1 receptor antagonist (Ra) is a cytokine widely associated with an inflammatory state and is expressed by activated mononuclear cells, we investigated whether hypoxia induces IL-1Ra expression in human peripheral blood mononuclear cells (PBMC) activated by phytohaemagglutinin (PHA). RNase protection assay, conducted on PHA-activated PBMC cultured under hypoxic conditions (2% O(2)) for 16-40 h, revealed that hypoxia enhances IL-1Ra mRNA expression. Further, IL-1Ra release was significantly affected by hypoxia, as determined by ELISA. Concomitantly, hypoxia enhanced, even though at a lesser extent, both IL-1alpha and IL-1beta mRNA expression and release, as determined by RPA and ELISA. However, at 40 h of treatment, hypoxia did not affect cell viability and DNA fragmentation, but caused an inhibition of the proliferation index after PHA stimulation, obtained by MTT assay. These results suggest that activated mononuclear cells tend to respond to hypoxic stress by modulating the expression of IL-1Ra and IL-1-related molecules and their release in the surrounding microenvironment.     

Role of interleukin (IL)-1 type 1 receptor in mycobacterial infection.

It is important to gain a better understanding of IL-1-mediated signaling events in mycobacterial infection. In order to clarify the role of IL-1 receptor type 1 (IL-1 R1) in IL-1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL-1 R1 KO mice developed significantly larger (P< 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild-type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post-infection. Interferon-y production by spleen cells was low in IL-1 R1 KO mice. It is concluded that the IL-1 R1 is essential for IL-1-mediated signaling events in mycobacterial infection.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

The anti-inflammatory cytokine, interleukin (IL)-10, blocks the inhibitory effect of IL-1 beta on long term potentiation. A role for JNK

Several effects of the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1 beta-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1 beta-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1 beta stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1 beta-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1 beta on superoxide dismutase activity and reactive oxygen species production, whereas the H(2)O(2)-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor.     

Short-term treatment of relapsing remitting multiple sclerosis patients with interferon (IFN)-beta1B transiently increases the blood levels of interleukin (IL)-6, IL-10 and IFN-gamma without significantly modifying those of IL-1beta, IL-2, IL-4 and tumour necrosis factor-alpha

We have studied the impact of short-term treatment with interferon (IFN)-beta1b of relapsing remitting (RR) multiple sclerosis (MS) patients' blood levels of type 1 and type 2 cytokines such as IFN-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10 and tumour necrosis factor (TNF)-alpha. These cytokines were measured by solid-phase ELISA. Serum samples were obtained prior to, and 2 and 12 hours after beginning of the treatment and 48 h after the last of 5 s.c. injections with 8 million IU IFN-beta1b given on alternate days for 10 days. The treatment was found to increase the circulating levels of IL-2, IL-6, IL-10 and IFN-gamma at some of the time points considered, with the effect acquiring statistical significance for IL-6, IL-10 and IFN-gamma. The blood levels of IL-1beta, IL-4 and TNF-alpha remained below the limit of sensitivity of the assays at any of the time points considered. If this in vivo study mirrors the impact of IFN-beta1b on MS patients' immune cells, these data demonstrate an activation of the immune system upon early treatment with the drug that does not lead to either type 1 or type 2 cytokine prevalence.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of biologically active chicken interleukin (IL)-12 and IL-18 synthesized by the recombinant fowlpox virus

The single chain chicken interleukin-12 (chIL-12)- or mature chIL-18-encoding region was cloned into the nonessential gene F11L of fowlpox virus (FPV) to generate the recombinant (r) FPV/chIL-12 (rFPV/chIL-12) or rFPV/chIL-18 for rchIL-12 or rchIL-18 production. Splenocytes of chickens were cultured with various dilutions of binary-ethylenimine (BEI)-inactivated rFPV/chIL-12 or rFPV/chIL-18-infected cell lysate for 48 h for interferon-γ (IFN-γ) determination. It was found that 1:10,000 chIL-12 or 1:10 chIL-18 cell lysate stimulated the highest levels of IFN-γ production. When chickens given BEI-treated 1:10 rchIL-12 or rchIL-18 cell lysate, intraperitoneally, or rFPV (0.2 × 10⁵ TCID₅₀) by wing-web puncture, the highest level of IFN-γ was detected in sera on day 3 postinoculation (dpi). In the splenocyte culture supernatants, the highest level of IFN-γ was detected at 14 dpi or 21 dpi in responses to rchIL-12 or rchIL-18, respectively. The results indicated that rchIL-12 or rchIL-18 could induce IFN-γ production both in vitro and in vivo assays, suggesting that both are biologically active and may allow them to be used in the future as the biological adjuvant in the poultry vaccine development, particularly co-administering with vaccine antigens.     

Up-regulation of interleukin (IL)-6 receptor gene expression in vitro and in vivo in IL-6 deprived myeloma cells.

Myeloma cells absolutely require interleukin-6 (IL-6) for growing in vivo in patients with multiple myeloma and exogenous IL-6-dependent myeloma cell lines have been reproducibly obtained. In this study we show a dramatic up-regulation of the IL-6 receptor (gp80 chain) gene expression in myeloma cell lines following the removal of exogenous IL-6. Such a regulation was also known to occur in IL-6-deprived myeloma cells in vivo in three patients who were treated with optimal doses of anti-IL-6 monoclonal antibodies. The direct effect of IL-6 on IL-6 receptor gene expression in myeloma cells was further confirmed by adding IL-6 to an autonomously growing myeloma cell line.     

Anthrax lethal toxin rapidly activates caspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18

Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5). Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7). We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1beta and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1. Studies of the processing of IL-1beta reveal that the levels of activated/cleaved IL-1beta in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT. Enhanced processing of IL-1beta directly correlates with increased levels in the activation of its upstream regulator, IL-1beta-converting enzyme/Caspase-1 (ICE). The extracellular release of IL-1beta and IL-18 in response to anthrax LT is ICE-dependent, as an ICE-specific inhibitor blocks this process. These data indicate that ICE, IL-1beta, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.     

Protein kinase C activation by interleukin (IL)-1 limits IL-1-induced IL-6 synthesis in osteoblast-like cells: Involvement of phosphatidylcholine-specific phospholipase C

We investigated the regulatory mechanism of interleukin-6 (IL-6) synthesis induced by interleukin-1 (IL-1) in osteoblast-like MC3T3-E1 cells. IL-1 stimulated the secretion of IL-6 in a dose-dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL-1-induced secretion of IL-6. The stimulative effect of IL-1 was markedly amplified in PKC down-regulated MC3T3-E1 cells. IL-1 produced diacylglycerol in MC3T3-E1 cells. IL-1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL-1 significantly stimulated the formation of phosphocholine dose-dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the IL-1-induced diacylglycerol production. The IL-1-induced IL-6 secretion was significantly enhanced by D-609. These results indicate that IL-1 activates PKC via phosphatidylcholine-specific phospholipase C in osteoblast-like cells, and the PKC activation then limits IL-6 synthesis induced by IL-1 itself. J. Cell. Biochem. 67:103–111, 1997. © 1997 Wiley-Liss, Inc.     

Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor beta2 chain expression in patients with allergic asthma

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.