Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway

The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.     

Differential utilization of cyclic ADP-ribose pathway by chemokines to induce the mobilization of intracellular calcium in NK cells.

We show here that cyclic adenosine diphosphate-ribose (cADPR) may be a second messenger for chemokines. Extracts collected from NK cells stimulated with IL-8 for 2 min were incubated with beta-NAD for an additional 2 min (designated as IL-8 extracts). This mixture elevated the mobilization of (Ca(2+))(i) in alpha-toxin permeabilized NK cells. This activity was inhibited upon prior incubation of these cells with ruthenium red but not with heparin. Purified cADPR and not Ins 1,4,5 P(3) desensitized NK cells to the calcium mobilization effect of IL-8 extracts. Further analysis showed that ruthenium red and heparin differentially inhibit RANTES-, SDF-1alpha-, or MDC-induced calcium mobilization in IL-2-activated NK cells. Also, introduction of anti-ryanodine receptor antibody inside streptolysin O-permeabilized NK cells resulted in complete inhibition of MDC, and only partial inhibition of RANTES and SDF-1alpha-induced calcium fluxes in NK cells. Collectively, these results suggest that chemokines may utilize the cADPR/ryanodine receptor pathway as well as the Ins 1,4,5 P(3)/Ins 1,4,5 P(3) receptor signaling pathway to induce the accumulation of calcium in NK cells.     

Cytokine changes in patients with heatstroke during pilgrimage to Makkah

Circulating levels and role of IL-6, IL-1ra, TNFsr-II and CRP in patients with heatstroke is not fully known. This study correlated levels of these mediators with outcome in 26 patients. In survivors (n=20), IL-6 concentration declined on cooling, whereas in non-survivors levels continued to increase at 6 h following admission before declining. Admission TNFsr-II concentrations in survivors were significantly lower than non-survivors and levels continued to rise in both groups. IL-1ra levels were markedly elevated in both groups. Changes in cytokine levels were not influenced by renal function. Elevated C-reactive protein levels were observed for both groups and remained so despite cooling, furthermore, there was no correlation with alanine aminotransferase levels. The study demonstrated the elevation of the above mediators and suggested a role in the pathogenesis of heatstroke. Markedly elevated levels or those that remained elevated despite cooling were associated with mortality.     

纳洛酮和电针改善创伤应激大鼠的免疫功能

本工作研究了中枢阿片肽系统在手术创伤介导大鼠免疫功能低下效应中的作用以及电针对创伤介导的免疫功能低下效应的影响。     

The role of natural killer (NK) cells and NK cell receptor polymorphisms in the assessment of HIV-1 neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses.     

Interleukin-1 receptor antagonist transiently impairs antibacterial defense but not survival in murine pneumococcal pneumonia

The inhibition of the biological activity of IL-1 by recombinant human IL-1 receptor antagonist (IL-1ra) has been investigated in several, controlled clinical trials. Encouraging results have been reported, in particular in patients with rheumatoid arthritis. In the present study, we investigated the influence of treatment of wild type mice with IL-1ra, which resulted in an incomplete and transient inhibition of IL-1 activity. Treatment with recombinant human IL-1ra resulted in an enhanced bacterial outgrowth in the lungs of BALB/c and C57BL/6 mice early after induction of pneumococcal pneumonia, without influencing survival or the pulmonary inflammatory response. The effect of IL-1ra on the host response to S. pneumoniae pneumonia is modest and transient. The present data, together with the findings in IL-1R*/* mice in earlier work, suggest that IL-1 occupies a role in the pulmonary immune response to S. pneumoniae that is substantially less prominent than that of TNF-alpha.     

Histone deacetylase inhibitors suppress natural killer cell cytolytic activity

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation.     

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-alpha, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DC(GM/IL-4)) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DC(GM/IL-4) capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.     

阿司匹林对中暑休克大鼠的保护及抗疲劳作用

本研究旨在探讨阿刮匹林是否可以通过降低中暑休克大鼠的白介素-β（interleukin-1β,IL-1β）水平从而发挥抗中暑休克作用。研究包括：（1）预先给产阿刊匹林对人鼠中暑休克的影响;（2）特异性一氧化氮合酶（inducible nitric oxide synthase,iNOS）抑制剂氨基胍（aminoguanidine,AG）对人鼠中暑休克的影响;（3）预先给予阿司匹林对清醒大鼠抗高温疲劳的影响。通过将大鼠置于仿真模拟高温气候舱接受环境离温（环境温度41℃,相对湿度65％）热暴露以诱导中暑休克,建立中暑休克动物模型。实验（1）和（2）分别将大鼠随机分为对照组和阿司匹林处理组,或对照组和AG组,记录热暴露过程中平均动脉压（mean arterial blood pressure,MAP）,结肠温度（colonic temperature,Tco）,心电图（electrocardiograph,ECG）,检测血浆IL-1β或NO浓度。实验（3）将对照组和阿司匹林处理组清醒大鼠置于水温41℃的水箱中,自由游泳,记录生存时间。结果显示,预先给予阿司匹林对大鼠中暑休克形成后血压下降有显著的抑制作用并延长生存时间,抑制血浆IL-1β升高的程度,但对体温变化没有显著影响。预先给予阿司匹林显著延长清醒大鼠在高温疲劳条件下生存时间。AG可以抑制中暑休克形成后大鼠MAP下降并显著延长大鼠生存时间,而且可以显著抑制热暴露后大鼠血浆NO浓度上升,但对大鼠热暴露后体温变化没有显著影响。结果提示,IL-1β可能通过诱导iNOS降低外剧血管张力从而参与中暑休克形成,预防性给予抗炎剂量阿司匹林可能对中暑休克出现的血压降低有一定的保护,同时增强对高温及疲劳耐受性,这种影响可能是通过对IL-1β以及局部iNOS的抑制而实现的。     

Reversal of lovastatin-mediated inhibition of natural killer cell cytotoxicity by interleukin 2

The activation of human natural killer (NK) cell cytotoxicity by interleukin 2 (IL-2) is well established, although the biochemical mechanisms of this stimulation have not yet been fully delineated. Earlier, we reported that treatment of NK cells with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase such as compactin or lovastatin significantly abrogates the in vitro killing of a susceptible human erythroleukemic cell line and that this inhibition can be completely reversed by 2 hr of exposure to mevalonate (J. Cell. Physiology 139:550-557, 1989). We report here that 24 hr of treatment with IL-2 also reverses lovastatin inhibition of NK cell function. In addition to natural cytotoxicity, IL-2 also restores chemotactic and antibody dependent cellular cytotoxicity functions to lovastatin-treated cells. IL-2 does not stimulate proliferation of these cells during this time period, nor does it affect the phenotypic composition of the NK cell preparations. Although IL-2 was able to reverse the lovastatin-mediated inhibition of every cell function we examined, it had no effect on the inhibition of cholesterol biosynthesis as measured by [3H]acetate incorporation into non-saponifiable lipids, nor did it stimulate HMG CoA reductase activity. These findings support the hypothesis that there is a non-sterol isoprenoid product which is required for NK cell cytotoxicity and chemotaxis. In addition, the data suggest that IL-2 stimulation of NK cells proceeds by an isoprenoid-independent pathway.     

Interleukin-2-stimulated natural killer cells are less susceptible to mycophenolate mofetil than non-activated NK cells: possible consequences for immunotherapy

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.     

CD1d1 displayed on cell size beads identifies and enriches an NK cell population negatively regulated by CD1d1

NK cells destroy microbe-infected cells while sparing healthy cells, and are controlled, in part, by inhibitory receptors specific for class I Ag-presenting molecules. CD1d1, a beta(2)-microglobulin-associated class I-like molecule, binds glycolipids and stimulates NKT cells. We previously demonstrated that target cell lysis by IL-2-activated mouse NK cells is inhibited by target cell expression of CD1d1, suggesting that IL-2-activated NK cells may express a CD1d1-specific inhibitory receptor. We now report that a significant subset of mouse IL-2-activated NK cells specifically binds cell size beads displaying either naturally expressed or recombinant CD1d1. In contrast, although tetramers of soluble recombinant CD1d1 loaded with alpha-galactosylceramide identify NKT cells, binding of this reagent to resting or IL-2-activated NK cells was undetectable, even with activated NK cells sorted with CD1d1 beads. Cytotoxicity by the CD1d1 bead-separated NK subset was strongly inhibited by CD1d1, compared with the NK cell subset not bound to CD1d1 beads. An Ab that blocks NKT cell recognition of CD1d1 also reverses CD1d1 inhibition of NK lysis, suggesting that TCRs of NKT cells and NK inhibitory receptor(s) may interact with a similar site on CD1d1. These results provide direct evidence for a physical interaction of NK cells with CD1d1, mediated by a functional, CD1d1-specific low-affinity inhibitory NK receptor. Display of ligands on cell size beads to maximize multivalent interaction may offer an alternative approach to examine NK cell receptor-ligand interactions, particularly those of lower expression and/or lower affinity/avidity that may go undetected using tetrameric reagents.     

Attenuation of seizure in P77PMC rats with an HSV-vector expressing IL-1ra in brain

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High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Interleukin-1 receptor antagonist (IL-1ra) modulates endothelial cell proliferation

Endothelial cell (EC) lifespan controlled by the IL-1 family of cytokines is an important determinant of susceptibility to artery wall disease. Here we show that EC lacking intracellular interleukin-1 receptor antagonist (IL-1ra) have a reduced lifespan compared to controls. Over expression of IL-1ra enhanced proliferation via cyclin dependent kinase 2 activity and retinoblastoma protein phosphorylation. This was not seen in EC lacking IL-1 receptor 1 (IL-1 signalling ability), nor apparent using other stimuli e.g. TNF alpha. These data suggest that IL-1ra has a specific and receptor-dependent function to control the growth and lifespan of EC.     

The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium

We used human tuberculosis as a model to investigate the role of NK cytotoxic mechanisms in the immune response to intracellular infection. Freshly isolated NK cells and NK cell lines from healthy donors lysed Mycobacterium tuberculosis-infected monocytes to a greater extent than uninfected monocytes. Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor, but not the NKp44 receptor. Antisera to NKp46 markedly inhibited lysis of infected monocytes. NK cell-mediated lysis was not due to reduced expression of MHC class I molecules on the surface of infected monocytes or to enhanced production of IL-18 or IFN-gamma. NK cell lytic activity against M. tuberculosis-infected monocytes and NKp46 mRNA expression were reduced in tuberculosis patients with ineffective immunity to M. tuberculosis compared with findings in healthy donors. These observations suggest that 1) the NKp46 receptor participates in NK cell-mediated lysis of cells infected with an intracellular pathogen, and 2) the reduced functional capacity of NK cells is associated with severe manifestations of infectious disease.     

Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and Th1 cell response. Recently IL-1R-related protein (IL-1Rrp) has been cloned as the receptor for IL-18. However, the functional role of IL-1Rrp is still controversial due to its low affinity to IL-18 as well as the possibility of the presence of another high-affinity binding receptor. In the present study, we have generated and characterized IL-1Rrp-deficient mice. The binding of murine rIL-18 was not detected in Th1-developing splenic CD4+ T cells isolated from IL-1Rrp-deficient mice. The activation of NF-kappa B or c-Jun N-terminal kinase were also not observed in the Th1 cells. NK cells from IL-1Rrp-deficient mice had defects in cytolytic activity and IFN-gamma production in response to IL-18. Th1 cell development was also impaired in IL-1Rrp-deficient mice. These data demonstrate that IL-1Rrp is a ligand-binding receptor that is essential for IL-18-mediated signaling events.