Human Peripheral Blood-Derived Dendritic Cells do not Produce Interleukin 1α, Interleukin 1β, or Interleukin 6

Little is known about the non-antigen-specific signals delivered to T cells by dendritic cells (DC). Because several monocyte-derived factors like interleukins 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6) enhance the T-cell proliferative responses, we studied the production of the above-mentioned cytokines by DC separated from human peripheral blood. The intracellular expression of the proteins (IL-1 alpha and IL-6) was studied at a single-cell level using an immunolabelling technique. The supernatants and cell lysates were studied with ELISA (IL-1 alpha and IL-1 beta). Northern blotting analysis was used to quantitate the mRNA levels. Several approaches were taken to stimulate the production of IL-1 alpha, IL-1 beta, and IL-6 by DC. These included the incubation of the DC in the presence of either LPS, rIL-1, or monoclonal anti-HLA-DR antibody, or the stimulation of cells with resting allogeneic T cells. None of the stimuli was able to induce the production of IL-1 alpha, IL-1 beta, or IL-6 by DC, whereas LPS-stimulated monocytes were strong producers of these mentioned cytokines and expressed the respective mRNA. Thus we concluded that IL-1 alpha, IL-1 beta, and IL-6 are primarily monocyte-derived factors and that these factors are not needed or produced during the activation of resting T cells by DC.     

Induction of Interleukin 1α (IL-1α) and IL-1β mRNA Expression and Cellular IL-1 Production by Anti-HLA-DR Antibodies in Human Monocytes

We studied the role of HLA class II antigens in the regulation of interleukin 1 (IL-1) production in human monocytes. Monocytes were cultured with monoclonal anti-HLA-DR antibodies for 24 h after which cellular (i.e. intracellular and membrane-associated) IL-1 production, IL-1 secretion, and the expression of IL-1 alpha and IL-1 beta mRNA were determined. One of the anti-HLA-DR antibodies tested (anti-HLA-DR, Becton Dickinson) clearly induced IL-1 alpha and IL-1 beta mRNA expression and cellular IL-1 production. The other anti-HLA-DR antibody tested (OKIa1, Ortho) had no effect on IL-1 production. The stimulatory effect of anti-HLA-DR was enhanced by IFN-gamma in both fresh and aged monocytes. A synergistic effect by anti-HLA-DR and suboptimal doses of LPS (1 ng/ml) on both cellular IL-1 production and secretion was also demonstrated. The possibility of contaminating LPS causing the IL-1-inducing effect of anti-HLA-DR was excluded by the inability of polymyxin B to abolish the anti-HLA-DR-induced IL-1 production.     

Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production.     

Primary role of interleukin-1 alpha and interleukin-1 beta in lipopolysaccharide-induced hypoglycemia in mice

Within a few hours of its injection into mice, lipopolysaccharide (LPS) induces hypoglycemia and the production of various cytokines. We previously found that interleukin-1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) induce hypoglycemia and that the minimum effective dose of IL-1 alpha or IL-1 beta is about 1/1000 that of TNF-alpha. In the present study, we examined the contribution made by IL-1 to the hypoglycemic action of LPS. Nine other cytokines tested were all inactive at inducing hypoglycemia. LPS produced hypoglycemia in mice deficient in either IL-1 alpha or IL-1 beta but not in mice deficient in both cytokines (IL-1 alpha and -1 beta knockout [IL-1 alpha/beta KO] mice). IL-1 alpha, IL-1 beta, and TNF-alpha induced hypoglycemia in IL-1 alpha/beta KO mice, as they did in normal control mice. The LPS-induced elevation of serum cortisol was weaker in IL-1 alpha/beta KO mice than in control mice, and, in the latter, serum cortisol was markedly raised while blood glucose was declining. IL-1 alpha decreased blood glucose both in NOD mice (which have impaired insulin production) and in KK-Ay mice (insulin resistant). These results suggest that (i). cortisol may not be involved in mediating the resistance of IL-1 alpha/beta KO mice to the hypoglycemic action of LPS, (ii). as a mediator, IL-1 is a prerequisite for the hypoglycemic action of LPS, (iii). IL-1 alpha and IL-1 beta perform mutual compensation, and (iv). IL-1 plays a role as the primary stimulator of the many anabolic reactions required for the elaboration of immune responses against infection.     

Interleukin-1 gene expression in rabbit vascular tissue in vivo.

Cultured human vascular endothelial and smooth muscle cells express interleukin-1 (IL-1) genes when exposed to bacterial lipopolysaccharides (LPS) and a variety of inflammatory mediators. Local production of IL-1 may contribute to the pathogenesis of various vascular diseases. Therefore the ability of intact vascular tissue to accumulate IL-1 mRNA and synthesize de novo biologically active IL-1 protein was examined. Escherichia coli LPS (10 micrograms/kg) was administered intravenously to adult rabbits and total RNA was isolated from aortic tissue at various times after LPS injection. In saline-injected rabbits, RNA extracted from the thoracic aorta contained little or no IL-1 message detected by Northern analysis using IL-1 alpha and beta cDNA probes cloned from an LPS-stimulated rabbit splenic macrophage library. Lipopolysaccharide treatment promptly induced transient accumulation of mRNA for IL-1 alpha and IL-1 beta within the aorta (maximal 1-hour after injection). Short-term organoid cultures of rabbit aorta exposed to LPS in vitro synthesized immunoprecipitable IL-1 alpha protein. Extracts of aortic tissue excised 1.5 to 3.0 hours after intravenous LPS administration contained immunoreactive and biologically active IL-1 alpha. Anti-rabbit IL-1 alpha antibody neutralized the biologic activity (more than 90%). Microscopic and immunohistochemical studies did not disclose adherent or infiltrating macrophages in rabbit aorta at the time of maximal IL-1 mRNA accumulation after LPS administration (1.5 hours), indicating that intrinsic vascular wall cells rather than mononuclear phagocytes probably account for the IL-1 activity induced by LPS. In addition, aortic tissue from rabbits fed an atherogenic diet showed an enhanced ability to accumulate IL-1 alpha and beta mRNA and produce immunodetectable protein in response to LPS administration. These studies demonstrate inducible IL-1 gene expression in rabbit vascular tissue in vivo and support a local role for this cytokine in vascular pathophysiology.     

Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide.

Cytokines produced by intestinal epithelial cells may function as signals to neighbouring immune and inflammatory cells. We investigated production of the neutrophil and T-lymphocyte chemotactic cytokine interleukin-8 (IL-8) by intestinal epithelial cells using four colonic adenocarcinoma cell lines, T84, CaCo-2, HT29 and SW620, as a model system. These cell lines secreted substantial amounts of IL-8 if stimulated with IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), except CaCo-2 cells, which responded only to IL-1 beta. Bacterial lipopolysaccharide (LPS) was also an efficient stimulus of IL-8 release in SW620 and HT29 cells, whereas T84 and CaCo-2 cells were completely unresponsive to LPS, IL-8 secretion was greater at 4 hr after stimulation and was accompanied by induction of IL-8 messenger RNA. In T84 cells IFN-gamma and epidermal growth factor (EGF) stimulated IL-8 secretion synergistically with TNF-alpha, whereas in SW620 cells this synergism occurred only between IFN-gamma and TNF-alpha. IL-4, IL-10 and transforming growth factor-beta (TGF-beta), which can down-regulate IL-8 production in macrophages, had no effect on IL-8 generation by our cell lines. Adenocarcinoma cell culture supernatants also induced rapid transients of intracellular calcium in neutrophils. Depending on cell line and stimulus, supernatant bioactivity was completely or partially abrogated by neutralizing antibodies to IL-8, indicating that the cell lines investigated also generate other neutrophil-activating factors. IL-8 and possibly other chemokines generated by colonic adenocarcinomas may help to attract tumour-infiltrating leucocytes. Possibly, normal intestinal epithelial cells also have the potential to secrete this potent chemoattractant and thus might contribute to inflammatory responses of the intestinal mucosa, for example in inflammatory bowel disease.     

Role of IFN-gamma on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide

Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Significant amounts of interferon-gamma (IFN-gamma) were detected in the PEC cultures on LPS stimulation, and anti-IFN-gamma antibody suppressed the LPS-induced NO, but not TNF-alpha and IL-6, production. Addition of anti-IFN-gamma antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-gamma production, also suppressed the augmented NO production, as did anti-IFN-gamma antibody. Taken together, we propose the following mechanisms: (1) T and NK cells in PEC produce IFN-gamma by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-gamma activates macrophages to augment NO production on LPS restimulation.     

Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide.

The differences between toxic or septic shocks in humans during infections by streptococci and gram-negative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokine-inducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and TNF-beta induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PMBC elicited large amounts of these cytokines, as well as IL-8 and TNF-beta, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-beta in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-alpha was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.     

Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.     

Interleukin-2 augmentation of interleukin-1 and prostaglandin E2 production

Some of the major side effects of interleukin-2 (IL-2) therapy in the treatment of malignancies may be related to increased interleukin-1 (IL-1) and/or prostaglandin E2 (PGE2) production. We examined the effect of recombinant (rIL-2) on the in vitro production of IL-1 beta and PGE2 by unstimulated and LPS-activated human blood mononuclear cells (PBMC). We also compared the effect of rIL-2 on IL-1 beta production by adherent and nonadherent blood mononuclear cell populations. Cultures of PBMC (5 x 10(6)/ml) were incubated for 24 hr in media only (control, 1,000 U/ml rIL-2, 2 micrograms/ml LPS, or both LPS and rIL-2. Supernatants obtained from these cultures were analyzed for levels of IL-1 beta and PGE2 by radioimmunoassays. The addition of rIL-2 caused an increase in IL-1 beta production in 13 of 13 control PBMC cultures and in 11 of 13 LPS-stimulated cultures, which were significant increases as determined by paired t tests. When PBMC were fractionated into plastic adherent and nonadherent populations, the rIL-2 induced increases in IL-1 beta production were more consistent in control (six of seven cases) and LPS (seven of seven cases) cultures of plastic nonadherent cells than in control (three of seven cases) and LPS (four of seven cases) cultures of plastic adherent cells. Recombinant IL-2 did not increase PGE2 production in control PBMC cultures (none of four cases), but did so in LPS-stimulated PBMC cultures (three of four cases]. These results suggest that rIL-2 may increase IL-1 production in vivo and thus possibly account for some of the side effects of this therapy.     

Modulation of IL-1 alpha, IL-1 beta, and 25K Mr non-IL-1 activity released by human mononuclear cells

To determine if the release of IL-1 alpha and IL-1 beta by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFN gamma, latex beads, and indomethacin on the release of IL-1 alpha and IL-1 beta. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1 alpha or anti IL-1 beta monoclonal antibody (mAb) and for the presence of IL-1 alpha or IL-1 beta protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1 alpha or anti-IL-1 beta mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3-to 7-fold by lopopolysaccharide (LPS) (25-50 micrograms/ml). All of this increased activity was due to increased release of IL-1 beta and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1 alpha released. Indomethacin (0.1 microgram/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1 alpha and non-IL-1 activity with a concomitant decrease in IL-1 beta release. Interferon gamma (40-100 U/ml) increased the amount of IL-1 alpha and decreased IL-1 beta and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity. Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1 alpha or IL-1 beta. With certain culture conditions, thymocyte mitogenic activity was detected in the 30-40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30-40K Mr IL-1 alpha, as well as 17K Mr IL-1 alpha and 17K Mr IL-1 beta. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30-40K Mr thymocyte mitogenic activity. Both IL-1 alpha and IL-1 beta protein was detected by ELISA in this Mr range but only the IL-1 alpha was bioactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6. Effect of IL-1 receptor antagonist.

The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells. Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor. In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity. In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition. We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC). We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells. The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity. We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta. The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood. Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing. In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures. The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production.     

Analysis and Characterization of the Interleukin 2 Receptor α and β Chain Expression inCD4−CD8− Cells

The differential effects that the binding of interleukin 2 (IL-2) to its beta or alpha beta receptors might induce in two different CD4-CD8- T-cell lines were analysed. While LD1.T3b, a double-negative T cell derived from MRL/lpr mice, constitutively expressed high levels of the IL-2R beta chain, YAC-1, a Moloney sarcoma virus-transformed CD4-CD8- T cell, expressed (as an activated T cell) the beta and alpha chains. The presence of IL-2 in the culture medium was lethal for LD1.T3b cells, while it had no effect on the growth of YAC-1 cells. IL-2 increased the expression of the beta chain and, to a lesser extent, of the alpha chain in YAC-1 cells. In addition, other markers such as CD4 and CD5 were induced by IL-2 in this cell line.     

Defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens

Neonates do not respond to thymus-independent (TI) antigens (Ag), making them vulnerable to infection with encapsulated bacteria. The antibody (Ab) response of adult and neonatal B cells to TI Ag requires certain cytokines, which are provided by T cells or macrophages (MPhi). Lipopolysaccharide (LPS) failed to induce neonatal MPhi to produce interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) mRNA and to secrete IL-1beta, IL-12, and TNF-alpha. However, LPS induced neonates to secrete some IL-6 and three- to fivefold more IL-10 than adults. Accordingly, adding adult but not neonatal MPhi could restore the response of purified adult B cells to trinitrophenol (TNP)-LPS, a TI Ag. Increased IL-10 is causally related to decreased IL-1beta and IL-6 production, as IL-10(-/-) neonatal MPhi responded to LPS by secreting more IL-1beta and IL-6 than wild-type (WT) neonatal MPhi. When cultures were supplemented with a neutralizing Ab to IL-10, WT neonatal MPhi secreted increased amounts of IL-6 and allowed neonatal MPhi to promote adult B cells to mount an Ab response against TNP-LPS. Thus, neonates do not respond to TI Ag as a result of the inability of neonatal MPhi to secrete cytokines, such as IL-1beta and IL-6, probably as a result of an excess production of IL-10. This dysregulated cytokine secretion by neonatal MPhi may be a result of a reduction in expression of Toll-like receptor-2 (TLR-2) and TLR-4 and CD14.     

Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-gamma

The protopypic long pentraxin 3 (PTX3) is a unique, humoral pattern-recognition receptor, which plays a nonredundant function in innate resistance to pathogens. Dendritic cells (DC) of myelomonocytic origin, but not plasmacytoid DC, are a major source of PTX3 in response to Toll-like receptor (TLR) engagement. The present study was designed to explore the regulation of PTX3 production in DC. PTX3 production was induced by TLR ligands, CD40 ligand, and interleukin (IL)-1beta and was suppressed by dexamethasone, 1alpha, 25-dihydroxivitamin D3, and prostaglandin E2. It was unexpected that lipopolysaccharide (LPS)-stimulated PTX3 production was enhanced by IL-10 and inhibited by IL-4 and interferon-gamma (IFN-gamma). Enhancement of PTX3 production by IL-10 was also evident when Pam3 Cys-Ser-(Lys)4.3HCl, a TLR2-TLR1 agonist, polyionisicpolycytidylic acid, a TLR3 agonist, and IL-1beta were used as stimuli. The effect of IL-10 was blocked by an anti-IL-10 monoclonal antibody (mAb) or an anti-IL-10 receptor alpha mAb, which also reduced the LPS-induced production. Thus, production of PTX3 in DC is subjected to a distinct regulatory network, with inhibition by IFN-gamma and enhancement by IL-10. The amplification by IL-10 of production of a nonredundant component of fluid-phase innate immunity mirrors the IL-10 stimulatory function on B cells in adaptive immunity. As PTX3 is also an extracellular matrix component, IL-10-enhanced PTX3 production may play a role in orchestration of tissue remodeling in chronic inflammation.     

Increased interleukin-1alpha and interleukin-1beta production by macrophages of low-density lipoprotein receptor knock-out mice stimulated with lipopolysaccharide is CD11c/CD18-receptor mediated.

Immune mechanisms, including production of pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF), play an important role in early atherogenesis. The study of the mechanisms responsible for the increased cytokine production capacity of hypercholesterolemic hosts is therefore crucial for finding new strategies aimed to stop the development of atherosclerosis. We assessed the lipopolysaccharide (LPS)-induced cytokine production of macrophages from low-density lipoproteins (LDL)-receptor knock-out (LDLR-/-) mice, which have a seven- to ninefold higher plasma LDL concentration. Macrophages of LDLR-/- mice produced approximately twofold more IL-1alpha and IL-1beta in response to LPS when compared with macrophages of control mice (LDLR+/+). TNF-alpha synthesis was only slightly increased. Removal of CD14 by phospholipase C treatment of cells decreased cytokine production by 50% (IL-1) to 80% (TNF), but the differences between LDLR-/- and LDLR+/+ remained the same. In contrast, treatment of cells with anti-CD11c monoclonal antibody inhibited the IL-1alpha and IL-1beta production in LDLR-/- mice towards normal values, while no effect could be seen on TNF. In conclusion, LDLR-/- macrophages stimulated with LPS synthesize more IL-1alpha and IL-1beta than controls and this phenomenon is mediated by the CD11c/CD18 receptor.     

Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides.

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS.     

Cytokine responses to treponema pectinovorum and treponema denticola in human gingival fibroblasts

Human gingival fibroblasts were challenged with Treponema pectinovorum and Treponema denticola to test three specific hypotheses: (i) these treponemes induce different cytokine profiles from the fibroblasts, (ii) differences in cytokine profiles are observed after challenge with live versus killed treponemes, and (iii) differences in cytokine profiles are noted from different gingival fibroblast cell lines when challenged with these treponemes. Three normal gingival fibroblast cell cultures were challenged with T. pectinovorum and T. denticola strains, and the supernatants were analyzed for cytokine production (i.e., interleukin-1alpha [IL-1alpha], IL-1beta, IL-6, IL-8, IL-10, gamma interferon, macrophage chemotactic protein 1 [MCP-1], platelet-derived growth factor, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor). Unstimulated fibroblast cell lines produced IL-6, IL-8, and MCP-1. T. pectinovorum routinely elicited the greatest production of these cytokines from the fibroblast cell lines, increasing 10- to 50-fold over basal production. While T. denticola also induced IL-6 and IL-8 production, these levels were generally lower than those elicited by challenge with T. pectinovorum. MCP-1 levels were significantly lower after T. denticola challenge, and the kinetics suggested that this microorganism actually inhibited basal production by the fibroblasts. No basal or stimulated production of the other cytokines was observed. Significant differences were noted in the responsiveness of the various cell lines with respect to the two species of treponemes and the individual cytokines produced. Finally, dead T. pectinovorum generally induced a twofold-greater level of IL-6 and IL-8 than the live bacteria. These results supported the idea that different species of oral treponemes can elicit proinflammatory cytokine production by gingival cells and that this stimulation did not require live microorganisms. Importantly, a unique difference was noted in the ability of T. pectinovorum to induce a robust MCP-1 production, while T. denticola appeared to inhibit this activity of the fibroblasts. While the general cytokine profiles of the fibroblast cell cultures were similar, significant differences were noted in the quantity of individual cytokines produced, which could relate to individual patient variation in local inflammatory responses in the periodontium.     

Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne.

Although many cytokines have been implicated in the development and persistence of inflammatory immune responses, it is unknown if any of these are important in inflammatory acne. This study investigated the production of the proinflammatory cytokines interleukin-8 (IL-8), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by human monocytic cell lines, ThP-1 and U937, and by freshly isolated peripheral blood mononuclear cells from acne patients. Both Propionibacterium acnes and supernatants obtained from 72-h P. acnes cultures could induce significant concentrations of IL-1 beta, TNF-alpha, and IL-8 by both cell lines and by peripheral blood mononuclear cells as determined by enzyme-linked immunosorbent assay. There was no significant difference between acne and non-acne subjects. Endotoxin quantification and addition of polymyxin B to assays indicated no lipopolysaccharide (LPS) contamination. P. acnes supernatant was fractionated into components with molecular weights of < 3,000, < 10,000, and < 30,000 and assayed for the ability to induce IL-8 and TNF production in ThP-1 cells. Nearly 90% of the original activity was found in the < 30,000-molecular-weight fraction, 50% was in the < 10,000-molecular-weight fraction, and only 15% remained in the < 3,000-molecular-weight fraction. The effluent from the < 3,000-molecular-weight fraction contained about 70% activity, indicating that the inducing factor was not retained in the membrane. Incubation of P. acnes supernatant with various concentrations of mutanolysin or lysozyme resulted in a loss of 60% of the original activity. The addition of jimson lectin, which binds peptidoglycan, resulted in a loss of 70% of the activity in a dose-response manner, whereas peanut lectin had little or no effect on the activity. Heating of the P. acnes supernatant to 65 degrees C also had no effect on the activity. Blocking of CD14, a receptor for both LPS and peptidoglycan, reduced cytokine production by > 50%, suggesting that the soluble stimulating factor may be a secreted form of peptidoglycan-polysaccharide.