Cytokine regulation of human monocyte interleukin-1 (IL-1) production in vitro. Enhancement of IL-1 production by interferon (IFN) gamma, tumour necrosis factor-alpha, IL-2 and IL-1, and inhibition by IFN-alpha.

IL-1 production (secreted and cell-associated) was measured in monocyte cultures stimulated by a variety of agents in vitro. Monocytes either adherent to conventional plastic culture plates in serum-free conditions, or in suspension in culture medium containing serum were stimulated to produce IL-1 during culture. In non-adherent, serum-free conditions, monocytes produced very low or undetectable amounts of IL-1 during 20 h of culture. Lipopolysaccharide (LPS) induced equivalent amounts of secreted and cell-associated IL-1, although at very low concentrations more cell-associated IL-1 was produced. IL-1 production in response to LPS could be augmented by crude lymphokine, IFN-gamma, or tumour necrosis factor (TNF) alpha. TNF-alpha preferentially augmented the production of cell-associated IL-1 in LPS-stimulated cultures. TNF-alpha induced a significant amount of IL-1 (mainly cell-associated) directly but could also induce IL-1 secretion when combined with IL-2 or IFN-gamma, or when in the presence of serum. IL-2 acted synergistically with low concentrations of IFN-gamma or IL-1 to induce significant levels of IL-1 production. IFN-alpha did not induce any IL-1 production, but was a potent inhibitor of IL-1 production induced by a variety of stimuli. These results suggest that IL-1 production may be enhanced or reduced by different cytokines at concentrations likely to be found in chronic inflammatory lesions.     

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1beta-induced GM-CSF production, but a marked inhibitory effect on TNF-alpha-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1beta or TNF-alpha, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-alpha-induced GM-CSF production, whereas IL-4 and IFN-gamma profoundly attenuated IL-1beta-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1beta or TNF-alpha was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is sensitive to Th1 and Th2 cytokine modulation.     

Regulatory roles of IL-17 and IL-17F in G-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha

The role of the interleukin (IL)-17 family members in the regulation of G-CSF production by lung microvasculature has not been elucidated yet. We therefore investigated the effects of IL-17 and IL-17F on the regulation of G-CSF production by lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not up-regulate G-CSF production from primary human LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced G-CSF production, whereas IL-17F had an enhancing effect on IL-1beta-induced production, but an inhibitory effect on TNF-alpha-induced secretion. G-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17. In contrast, three cytokines IL-1beta, TNF-alpha and IL-17F were combined together, G-CSF production was less than that induced by IL-1beta or IL-1beta plus TNF-alpha or IL-17F. Moreover, IL-17 plus Th1 or Th2 cytokine had a modest stimulatory effect on TNF-alpha-induced G-CSF production, whereas IL-17 plus IFN-gamma had an inhibitory effect on IL-1beta-induced release. Similarly, IL-17F plus IL-10, IL-13 or IFN-gamma had an inhibitory effect on IL-1beta-induced production. Our findings indicate that CD4 T cell cytokines IL-17 and IL-17F play a differential regulatory role in G-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is also sensitive to Th1 and Th2 cytokine modulation.     

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-alpha and nitric oxide from macrophages via IFN-gamma induction

IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-alpha and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-alpha and NO, whereas live mycobacteria alone released TNF-alpha markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-alpha and NO release synergistically. Because IL-12 and mycobacteria also released IFN-gamma from macrophages synergistically, and exogenous IFN-gamma with mycobacteria enhanced TNF-alpha and NO release synergistically, we examined the role of endogenous IFN-gamma in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-gamma, we found that IL-12/mycobacteria-enhanced macrophage TNF-alpha and NO release was mediated through endogenous IFN-gamma. We further demonstrated that IFN-gamma and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-alpha synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-gamma release.     

Modulation of IL-2, IFN-gamma, TNF-alpha and IL-4 production in mice of different ages by thymopentin.

The effect of an immunomodulator drug thymopentin (TP5) on the production of various cytokines (IFN-gamma, IL-2, IL-4, TNF-alpha) in mice of different ages has been studied. TP5 enhanced IL-2, TNF-alpha and IFN-gamma production but reduced the IL-4 secretion by splenocytes from aged mice (greater than 120 week old) in vitro. However, it had no effect on the IL-2, IFN-gamma, TNF-alpha or IL-4 production by splenocytes from young and adult mice. TP5 injected subcutaneously was able to induce high levels of IL-2 production by splenocytes from all groups of mice. The TP5 effect on TNF-alpha and IFN-gamma was similar, even though it was significant only in old mice. Furthermore, TP5 was able to significantly reduce IL-4 production in old mice, which normally produced high levels of this cytokine after mitogen stimulation. Since it has been observed in the mouse that the Th1 cells secrete IFN-gamma and IL-2, whereas the Th2 cells preferentially produce IL-3, IL-4 and IL-5, these results indicate that the immunopotentiatory activity of TP5 is due to the preferential up-regulation of Th1 cells.     

Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1.

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-gamma (IFN-gamma) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CD1a+, HLA-DR+, CD4+, CD40+, CD80+ but CD14- cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-gamma selectively induced HLA-DR and CD86 but did not up-regulate CD1a expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-gamma could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-gamma to GM-CSF plus TNF-alpha during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-alpha mutants, we investigated the relative involvement of TNF-alpha receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-R1 triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-alpha and IL-4; second, that the effect of TNF-alpha in DC generation involves signalling via the TNF-R1 receptor; and third, that IFN-gamma counteracts some of the effects of IL-4 in DC generation.     

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor-alpha (TNF-alpha) activate human alveolar macrophages to inhibit growth of Mycobacterium avium complex.

We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inhibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-alpha, recombinant interferon-gamma (IFN-gamma), and recombinant IL-2. We found that phagocytosis by the various cytokine-stimulated AM was similar to that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-alpha, while no growth inhibition of MAC was observed in the macrophages treated with IFN-gamma or IL-2. Pretreatment with all cytokines tested enhanced the O2- release from AM, but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC. Thus, we concluded that GM-CSF or TNF-alpha could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Effects of associated cytokines (IL-1, TNF-alpha, IFN-gamma and TGF-beta) on collagen and glycosaminoglycan production by cultured human synovial cells

The production of collagen and glycosaminoglycans (GAG) was studied in cultured human synovial cells exposed to four cytokines, alone or in dual combination, namely interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Among these cytokines, only TGF-beta (0.1-10 ng/ml) induced a significant and dose-dependent increase of collagen synthesis in a 24-h incubation. This effect was reversed when the factor was associated with either IL-1 beta (100-500 pg/ml), TNF-alpha (1-100 ng/ml) or IFN-gamma (100 U/ml). Except IFN-gamma which clearly inhibits the collagen production, the other cytokines IL-1 and TNF-alpha were not very effective when tested separately, although they generally induced a small reduction in collagen amount. IL-1 beta and TNF-alpha were found to be more efficient than TGF-beta in stimulating the production of GAG by the synovial cells. IFN-gamma exerted an antagonistic effect on the TGF-beta-induced stimulation of GAG synthesis. TNF-alpha and IL-1 beta were shown to have an additive effect on that production. The results indicate that interactions between cytokines present in the inflamed synovial tissue may modulate their respective actions and thus introduce differentials in their effect on collagen and GAG metabolism which are responsible for the alterations of synovial extracellular matrix in rheumatoid arthritis.     

Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.     

Effects of tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and monocytes on lymphokine-activated killer (LAK) induction from natural killer (NK) cells and T lymphocytes.

Roles of monocytes and cytokines were investigated on LAK induction from T and NK cells. Monocytes augmented more T-LAK induction than did NK-LAK. Expression of IL-1 beta, TNF-alpha and interferon-gamma (IFN-gamma)-mRNA and their cytokine production were superior in NK cells compared with T cells in parallel with their LAK activities. An increase of TNF-alpha, IL-1 beta and IFN-gamma production was induced by co-culturing NK or T cells with autologous monocytes. The augmentation of T cell cytokine production and T-LAK activity by monocytes was more prominent than that of NK cells. TNF-alpha and IL-1 beta were generated 24 h after IL-2 stimulation, and these cytokines were able to almost substitute for monocytes in LAK induction. Conversely, LAK induction was almost completely suppressed by both anti-IL-1 beta and anti-TNF-alpha antibodies, if they were added within 24 h after the start of the LAK induction. IFN-gamma, which was produced at a later stage, scarcely affected LAK induction in spite of the cooperation with TNF-alpha. The results obtained indicate conclusively that the superiority of NK-LAK depends on their superior productivity of both IL-1 beta and TNF-alpha, and that the up-regulation of LAK induction by monocytes is largely due to the enhanced generation of both cytokines.     

Engagement of human monocyte-derived dendritic cells into interleukin (IL)-12 producers by IL-1beta + interferon (IFN)-gamma

Dendritic cells (DCs) are potent antigen-presenting cells and can induce tumour- or pathogen-specific T cell responses. For adoptive immunotherapy purposes, immature DCs can be generated from adherent monocytes using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, and further maturation is usually achieved by incubation with tumour necrosis factor (TNF)-alpha. However, TNF-alpha-stimulated DCs produce low levels of IL-12. In this study, we compared the effects of TNF-alpha, interferon (IFN)-gamma, IL-1beta or IFN-gamma + IL-1beta on the phenotypic and functional maturation of DCs. Our results show that IFN-gamma, but not IL-1beta, augmented the surface expression of CD80, CD83 and CD86 molecules without inducing IL-12 production from DCs. However, IL-1beta, but not IFN-gamma, induced IL-12 p40 production by DCs without enhancing phenotypic maturation. When combined, IFN-gamma + IL-1beta treatment profoundly up-regulated the expression of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II antigens. Furthermore, IFN-gamma + IL-1beta-treated DCs produced larger amounts of IL-12 and induced stronger T cell proliferation and IFN-gamma secretion in primary allogeneic mixed lymphocyte reaction (MLR) than did TNF-alpha-treated DCs. Our results show that IFN-gamma + IL-1beta induced human monocyte-derived DCs to differentiate into Th1-prone mature DCs.     

Secretion of cytokines by natural killer cells primed with interleukin-2 and stimulated with different lipoproteins.

Natural killer (NK) cells were shown to secrete differentially interleukins (IL), IL-1 alpha, IL-1 beta, IL-2, IL-8, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukaemia inhibitory factor (LIF) upon stimulation with optimal concentrations of chylomicrons (CM), very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or acetyl-modified low-density lipoprotein (AcLDL). CM, VLDL, LDL and AcLDL induced LIF secretion which was absent in nonstimulated cells. CM, VLDL, and LDL did not affect IL-1 alpha secretion. CM stimulated IL-8 > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma, and decreased seventeen-fold GM-CSF secretion. VLDL stimulated IL-8 secretion > IL-1 alpha = IL-2 > IFN-gamma > TNF-alpha and decreased fivefold GM-CSF secretion. LDL stimulated IL-8 secretion > IL-1 alpha > IL-2 = IFN-gamma, it did not modify TNF-alpha and inhibited five hundred-fold GM-CSF secretion. HDL stimulated IL-2 secretion = IFN-gamma > IL-8, it decreased GM-CSF secretion > IL-1 alpha > IL-1 beta > TNF-alpha without affecting LIF. AcLDL stimulated IL-8 secretion > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma = IL-1 beta, and decreased GM-CSF secretion eightfold. When NK cells were primed with 10, 100 or 500 IU/ml of IL-2 before the addition of lipoproteins, a decrease in the secretion of cytokines was observed as compared with cells primed with IL-2 only. Differences in cytokine secretion were observed among the diverse type of lipoproteins used for cell stimulus. Thus, lipoproteins may condition NK cytokine secretion and cell activation.     

Benzylpenicillin differentially conjugates to IFN-gamma,TNF-alpha,IL-1beta,IL-4 and IL-13 but selectively reduces IFN-gamma activity

It is known that beta-lactam antibiotics can conjugate to lysine and histidine residues on proteins via the carbonyl group of the opened beta-lactam ring. However, it is not known which proteins these drugs target and there is little work addressing whether conjugation is preferential for some proteins over others or if conjugation has functional consequences for the protein. We have previously shown that the beta-lactam antibiotic benzylpenicillin (BP) conjugates to IFN-gamma and reduces its activity. This interaction demonstrates selectivity, as BP does not bind to IL-4. Here, we extend our study to include other Th1 and Th2 cell-associated cytokines and two cytokines associated with inflammatory responses. We demonstrate by Western blotting that BP also conjugates to IL-1beta, IL-2, IL-5, IL-13 and TNF-alpha but not to IL-10. Densitometric analysis of leading cytokine bands on blots revealed that IFN-gamma always gave more intense BP-positive bands than any other cytokine analysed. Cytokines pre-incubated with BP at 37 degrees C in a protein-containing, serum-free medium were assayed for their biological activity. By in vitro bioassay, BP inhibited the ability of IFN-gamma but not IL-1beta or TNF-alpha to induce CD54 expression on epithelial cells. In addition, BP did not affect IL-4 or IL-13 inhibition of mast cell proliferation. When the pre-incubation temperature was reduced to 4 degrees C, BP did not conjugate to IFN-gamma or modulate its activity. BP retained its inhibitory effect on IFN-gamma activity when 20% FCS was added to the pre-incubation medium. In conclusion, BP conjugates to some cytokines but not others and this does not appear to be related to primary protein structure. Furthermore, of the cytokines studied, conjugation only to IFN-gamma is accompanied by inhibition of activity. This phenomenon is temperature dependent and occurs in the presence of serum. These findings provide further evidence for differential, direct drug-cytokine interactions. Such interactions may have therapeutic implications in terms of targeting cytokines to regulate their activity.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates cytokine induction by 1,3-beta-D-glucan SCG in DBA/2 mice in vitro.

Sparassis crispa Fr. is an edible/medicinal mushroom that recently became cultivable in Japan. SCG is a major 6-branched 1,3-beta-D-glucan in S. crispa showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice strongly react with SCG to produce interferon-gamma (IFN-gamma). In this study, cytokines induced by SCG were screened and found to be IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL-12p70). The addition of recombinant murine GM-CSF (rMuGM-CSF) to spleen cell cultures from various strains of mice synergistically enhanced IFN-gamma, TNF-alpha and IL-12p70 in the presence of SCG. In contrast, neutralizing GM-CSF using anti-GM-CSF monoclonal antibody (mAb) significantly inhibited IFN-gamma, TNF-alpha, and IL-12p70 elicited by SCG. We conclude that GM-CSF is a key molecule for cytokine induction by beta-glucan, and GM-CSF induction by SCG is the specific step in DBA/2 mice in vitro.     

The survival effect of TNF-alpha in human neutrophils is mediated via NF-kappa B-dependent IL-8 release

The capacity of cytokines to modulate neutrophil apoptosis is thought to be a major factor influencing the resolution of granulocytic inflammation. We have previously shown that the late survival effect of TNF-alpha in human neutrophils involves activation of both NF-kappa B and phosphoinositide 3-kinase (PI3-kinase) pathways. In this study, we address how these pathways integrate to prevent cell death. In human neutrophils, TNF-alpha (200 U/ml) induced rapid I kappa B-alpha degradation, NF-kappa B activation and IL-8 release (31.8+/-5.4 pg/10(5) cells/2 h), whereas GM-CSF (10 ng/ml) stimulated an equivalent IL-8 release (26.5+/-4.5 pg/10(5) cells/2 h) without enhanced I kappa B-alpha degradation or NF-kappa B activation compared to control. Importantly, inhibition of PI3-kinase did not modify TNF-alpha -induced I kappa B-alpha degradation, yet fully inhibited the survival effect of both cytokines. Inhibition of I kappa B-alpha phosphorylation, PI3-kinase or ERK1/2 activation blocked IL-8 release by both cytokines. Blocking IL-8 activity by inhibiting its synthesis or by using a neutralizing antibody enhanced the early pro-apoptotic effect of TNF-alpha and inhibited its late survival effect without affecting GM-CSF-induced survival. These data suggest that cross-talk between NF-kappa B and PI3-kinase pathways in TNF-alpha -stimulated neutrophils results from NF-kappa B/ERK1/2-dependent IL-8 production which acts in an autocrine manner to drive PI3-kinase-dependent survival. In contrast, GM-CSF-mediated survival does not involve NF-kappa B activation or IL-8 release.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts

BACKGROUND: Although cigarette smoking is known to have detrimental effects on the immune system, the nature of the immunosuppressive agent or agents is poorly understood. OBJECTIVE: The purpose of the current study was to evaluate the effects of cigarette smoke extracts from high-tar (unfiltered Camel), medium-tar (Marlboro), and low-tar (Carlton) cigarettes on the in vitro production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha. METHODS: The concentrations of hydroquinone and catechol in cigarette smoke extracts were determined by using HPLC. Human PBMCs were treated with cigarette smoke extracts, hydroquinone, or catechol, and stimulated with anti-CD3 and phorbol-12-myristate-13-acetate. Cytokine levels in the supernatants were quantified by ELISA. RESULTS: Pretreatment of PBMCs with cigarette smoke extracts derived from a single high- or low-tar cigarette suppressed the production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha by greater than 90% without significant loss of cell viability. Nicotine, at a concentration comparable with that found in the highest-tar cigarettes (200 microg/mL), suppressed the production of IL-2, IFN-gamma, and TNF-alpha by only 21% to 38%. Catechol (50 micromol/L) inhibited production of IL-2 and IL-1beta by 62% to 73% but had little effect on TNF-alpha or IFN-gamma production. In contrast, hydroquinone inhibited the production of all 4 cytokines with IC(50) values ranging from 3 micromol/L(IL-1beta) to 29 micromol/L (IFN-gamma). However, HPLC determination of the hydroquinone concentrations in cigarette smoke extracts from single Camel (33+/-4 micromol/L), Marlboro (13+/-2 micromol/L), and Carlton (<1 micromol/L) cigarettes clearly demonstrated that the potent inhibitory effects of the low-tar cigarettes could not be accounted for by either hydroquinone or catechol. CONCLUSION: These studies indicate that cigarette smoke contains potent inhibitors of cytokine production, at least one of which is present even in low-tar cigarettes.