Ultrastructural and functional studies of the interaction between IL-12 and IL-2 for the generation of lymphokine-activated killer cells

IL-12 promotes generation of LAK activity in short-term-cultured NK cells, but information on the structure and function of IL-12-induced LAK cells is not yet available. The latter issues have been here investigated with emphasis on interactions between IL-12 and IL-2. Peripheral blood mononuclear cells (MNC) exposed to IL-12 for 5-7 days displayed a decrease in the amount and density of the matrix of large granular lymphocyte (LGL)-associated granules. In cells cultured with IL-12 and IL-2 for 5-7 days, empty vacuoles were predominant and the electron-dense matrix was scanty. In MNC incubated with IL-2 for 5-7 days, most granules were loaded with electron-dense matrix. IL-12 and IL-2 displayed an additive effect on LAK cell cytotoxicity until approximately 48 h in culture which was followed by a sharp decline. Immunocytochemical and biochemical studies demonstrated that MNC cultured for 5-7 days with IL-12 and IL-2 displayed downregulated perforin expression and upregulated granzyme B expression. Fas ligand expression was virtually undetectable in MNC cultured for 5-7 days with or without cytokines. It appears that perforin downregulation plays a major role in the reduced cytotoxicity of MNC cultured with IL-12 and IL-2 for 5-7 days.     

IL-10 fails to inhibit the production of IL-18 in response to inflammatory stimuli

IL-10 is an inhibitor of the production of pro-inflammatory cytokines such as IL-12 and IL-1beta, but it is not known whether it can inhibit the production of IL-18. Therefore, a variety of in vivo and in vitro models were used to determine whether IL-10 is an inhibitor of IL-18 production. Infection of IL-10-/- mice with Toxoplasma gondii results in increased levels of IL-12 in the serum and in recall responses compared to wild type (WT) mice. Surprisingly, although infection resulted in increased levels of IL-18 in serum, there were no differences between WT and IL-10-/- mice. Moreover, splenocytes from infected WT and IL-10-/- mice produced similar levels of IL-18 and addition of exogenous IL-10 did not inhibit their production of IL-18. To address whether endogenous IL-18 inhibitors were masking increased IL-18 production in the IL-10-/- mice, expression of IL-18 binding protein was examined using RT-PCR. Although infection leads to increased expression of IL-18BP mRNA, no difference was seen between WT and IL-10-/- mice. In addition, splenocytes from IL-10-/- mice produced elevated levels of nitric oxide (NO) compared to WT mice, and NO has been shown to inhibit activity of interleukin-1 converting enzyme (ICE), which is required for IL-18 production. However, the addition of an inhibitor of NO production did not alter the levels of IL-18 produced. Finally, analysis of the levels of cytokine mRNA of macrophages stimulated with LPS and IFN-gamma revealed that although IL-10 is a potent inhibitor of IL-12 mRNA accumulation, it did not inhibit IL-1beta or IL-18. Together, these data indicate that IL-10 is not an inhibitor of the production of IL-18.     

Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.     

Vasoactive intestinal peptide synergizes with TNF-alpha in inducing human dendritic cell maturation.

We investigated the effects of different neuropeptides on human dendritic cells (DC) maturation. Immature DC, derived from monocytes cultured for 6 days with IL-4 plus GM-CSF, have been exposed to somatostatin, substance P, or vasoactive intestinal peptide (VIP). Among these neuropeptides, only VIP induces the production of bioactive IL-12 and the neoexpression of CD83 on a fraction of the DC population, with an effect significant at 100 and 10 nM, respectively. These effects of VIP are dose-dependent, unaffected by polymixin B, and partly prevented by a VIP receptor antagonist. Although the effects of VIP alone remain modest, it synergizes with TNF-alpha to induce DC maturation. In the presence of a suboptimal concentration of TNF-alpha, which has no detectable effect on DC by itself, VIP induces the production of high levels of bioactive IL-12, the neoexpression of CD83 on almost all the DC population (with an effect significant at 10 and 0.1 nM, respectively), and the up-regulation of various adhesion and costimulatory molecule expression. Moreover, DC exposed to VIP plus a suboptimal concentration of TNF-alpha are as potent as mature DC obtained by treatment with an optimal concentration of TNF-alpha in stimulating allogenic T cell proliferation. Our data suggest that, in inflammatory sites, VIP may cooperate with proinflammatory mediators, such as TNF-alpha, to induce DC maturation.     

Effect of the tyrosine kinase inhibitor, genistein, on interleukin-1 stimulated PGE2 production in mesangial cells

Prostaglandin production and cAMP formation are two signaling pathways identified for IL-1, though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 micrograms/ml, genistein blocked IL-1 stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.     

Is there an interleukin 2 inhibitor in human serum?

Normal mouse serum has been shown to contain an inhibitor of interleukin 2 (IL-2). Here we report that a molecule with similar activity cannot be found in normal human serum (NHS). Although NHS inhibited the IL-2-dependent proliferation of mouse CTLL cells, as expected of an IL-2 inhibitor, it also had inhibitory activity on IL-3-dependent cells and was cytolytic to IL-2-independent mouse cells as measured by a ⁵¹Cr release assay, indicating a nonspecific effect. In addition, NHS had no effect on the IL-2-dependent proliferation of human peripheral blood T-cell blasts. Fractionation of NHS by size exclusion HPLC failed to separate cytolytic activity from any putative true IL-2 inhibitor activity. The cytolytic component was not related to immunoglobulin since it had a molecular weight of 50,000 to 60,000 and was not bound by protein-A-Sepharose. However, its molecular weight, heat lability, and trypsin sensitivity suggest it to be a protein.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis

Interleukin 9 (IL-9) has been implicated in mast cell-related inflammatory diseases, such as asthma, where vascular endothelial growth factor (VEGF) is involved. Here we report that IL-9 (10-20 ng/ml) induces gene expression and secretion of VEGF from human LAD2. IL-9 does not induce mast cell degranulation or the release of other mediators (IL-1, IL-8, or TNF). VEGF production in response to IL-9 involves STAT-3 activation. The effect is inhibited (about 80%) by the STAT-3 inhibitor, Stattic. Gene-expression of IL-9 and IL-9 receptor is significantly increased in lesional skin areas of atopic dermatitis (AD) patients as compared to normal control skin, while serum IL-9 is not different from controls. These results imply that functional interactions between IL-9 and mast cells leading to VEGF release contribute to the initiation/propagation of the pathogenesis of AD, a skin inflammatory disease.     

Immunoregulatory effects of freeze injured whole tumour cells on human dendritic cells using an in vitro cryotherapy model

Tumour cryotherapy has been described as both immunostimulatory and immunoinhibitory in previous studies. However, previous studies have not accurately reproduced the precise conditions of current clinical cryotherapy. The objective of this study is to assess the immunological effects of cryotreated whole tumour cells on dendritic cells (DC) maturation and function using an in vitro model. Prostate cancer cells were cooled using Endocare cryo-system to mimic temperatures achieved during clinical cryotherapy. Human DC were prepared from cluster of differentiation (CD) 14 monocytes and matured with lipopolysaccharide (LPS). Cryotreated cancer cells were added to DC on day 3. On day 7, DC were harvested and phenotyped. Cytokine gene expression was assessed using real time quantitative polymerase chain reaction (RT-PCR). Functional activity of DC was assessed in allogenic mixed lymphocyte reaction (MLR) and the molecular changes using gene microarray technology. There was statistically significant upregulation of costimulatory molecules and maturation markers (CD86, CD83, CD80 and CL II) in DC loaded with cryotreated whole tumour cells compared to both control DC and DC matured with LPS (P < 0.001). There was a significant increase in stimulatory cytokines gene expression (IL-2, IL-12, IL-15, IL-18 and IFN-γ). However, IL-10 and TGF-β expression reduced significantly. The effect of different freezing temperature was equal. cDNA microarray analysis showed upregulation of interleukin 1 (IL-1) and cycline dependent kinase inhibitor 1A (CDKN1A (p21) and downregulation of Caspase 8 and BCL2. Overall, our findings suggest that the effect of cryotherapy is generally stimulatory to DC which may enhance anti-tumour effects. Therefore, the combination of cryotherapy and DC vaccine may represent a novel method to increase the efficacy of cryotherapy especially at the peripheral zones of the prostate where cells are exposed to sub-lethal temperature.     

In Vivo Production of Various Cytokines in Splenocytes of Sheep Erythrocyte-Immunized Mice after Intravenous Administration of Bacterial Lipid A

The effects of an intravenous administration of lipid A from Salmonella minnesota R595 lipopolysaccharide on the in vivo production of interleukin-2 (IL-2), IL-4, IL-5 and IL-6 in the spleens of mice intravenously immunized with sheep red blood cell (SRBC) antigen were investigated. The increased number of antigen-specific IgM antibody-producing cells and the titer of the IgM serum antibody were measured using the plaque-forming cell (PFC) assay and an enzyme-linked immunosorbent assay (ELISA). Simultaneous injections of SRBC antigen and lipid A adjuvant enhanced IgM-PFC number on days 3 and 4 and the serum IgM titer on days 4 and 5 after the immunization. We found that the enhanced IL-4 and IL-5 levels correlated with the PFC number and IgM titer. When lipid A was injected intravenously 2 days after immunization with SRBC, the PFC number in lipid A-treated groups were similar to those in controls 3 and 4 days after the immunization. However, it was found that a twofold increase in the IgM titer in serum was induced by lipid A 5 days after immunization. In relation to this increase, lipid A stimulated the production of only IL-5 among the cytokines tested.     

Free IL-18 and IL-33 cytokines in chronic spontaneous urticaria

Overproduction of IL-18 has been described in chronic urticaria. To evaluate free IL-18 and IL-33 in chronic spontaneous urticaria (CSU). IL-18, its inhibitor IL-18BP, IL-33 and its soluble receptor ST2 (sST2) were measured (ELISA) in the sera of 73 CSU patients. Free IL-18 was calculated (law of mass action). Autologous serum skin test (ASST) was performed in all patients. Total IL-18, IL-18BP and free IL-18 serum levels were significantly higher in CSU than in controls. IL-18 and IL-18BP increased significantly in both ASST-positive and negative subgroups. Free IL-18 resulted significantly higher in the ASST-negative, but not in the ASST-positive subgroup. No differences in IL-33/sST2 levels were detected between CSU and controls. Increased levels of free IL-18 and IL-18BP, but not IL-33, was detected in CSU. Whether IL-18 up-regulation is a consequence of inflammation or one of the causes of the pathology needs to be addressed.     

Efficacy of an inhibitor of adhesion molecule expression (GI270384X) in the treatment of experimental colitis

Modulation of adhesion molecule expression or function is regarded as a promising therapy for inflammatory conditions. This study evaluates the effects of an inhibitor of adhesion molecule expression (GI270384X) in two experimental models of colitis. Colitis of different severity was induced in C57BL/6J mice by administering 1, 2, or 3% dextran sulfate sodium (DSS). GI270384X (3, 10, or 25 mg.kg(-1).day(-1)) was administered as pretreatment or started 3 days after colitis induction. In IL-10-deficient mice, the highest dose was given for 2 wk. The clinical course of colitis, pathological changes, serum inflammatory biomarkers, expression of adhesion molecules, and leukocyte-endothelial cell interactions in colonic venules were measured in mice treated with vehicle or with active drug. In the most severe forms of colitis (2% and 3% DSS and IL-10-deficient mice), the magnitude of colonic inflammation was not modified by treatment with GI270384X. In a less severe form of colitis (1% DSS), GI270384X treatment dose dependently ameliorated the clinical signs of colitis, colonic pathological changes, and serum levels of biomarkers (IL-6 and serum amyloid A). Administration of 25 mg.kg(-1).day(-1) GI270384X abrogated upregulation of ICAM-1 in the inflamed colon but had no effect on VCAM-1 or E-selectin expression. This was associated with a significant reduction in number of rolling and firmly adherent leukocytes in colonic venules. These results indicate that GI270384X is effective in the treatment of experimental colitis of moderate severity. Reduced adhesion molecule expression and leukocyte recruitment to the inflamed intestine contribute to this beneficial effect.     

Cellular localization and functional role of phosphatidylcholine-specific phospholipase C in NK cells

Although several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (M(r) 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity.