Acute-phase protein synthesis in human hepatoma cells: differential regulation of serum amyloid A (SAA) and haptoglobin by interleukin-1 and interleukin-6.

Interleukin-6 (IL-6, BSF-2 or IFN-beta 2) is thought to be the major regulator of the acute-phase protein response that follows tissue injury and inflammation, with interleukin-1 (IL-1), tumour necrosis factor and more recently, LIF or HSF III, slightly stimulatory on only certain acute phase proteins. The synthesis of the major acute-phase protein SAA, originally described as being synthesized in response to IL-1, has been claimed recently to be mainly under IL-6 regulation. Our results show that in the human hepatoma cell line HuH-7, IL-1 is the major stimulating cytokine increasing SAA synthesis by a factor in excess of 100-fold. We also show that under most conditions interleukin-6 and tumour necrosis factor stimulate additively in combination with IL-1. Isoelectric focusing has demonstrated that SAA1 and SAA2 alpha are expressed but not SAA2 beta. The HuH-7 cell line is IL-6 responsive since haptoglobin is stimulated mainly by IL-6.     

Elevation of serum soluble tumour necrosis factor (TNF) receptor and IL-1 receptor antagonist levels in bronchial asthma

The specific inhibitor for TNF-α activity, soluble form of the 55-kD TNF receptor (sTNF-RI) and soluble form of the 75-kD receptor (sTNF-RII), and the specific inhibitor for IL-1 activity, IL-1 receptor antagonist (IL-1Ra), have been identified. It has been shown that the levels of these inhibitors are elevated in plasma/serum and biological fluids in several diseases, and the protective and inhibitory effect of these inhibitors exist in several inflammatory diseases. In the present study, we measured serum levels of sTNF-RI, STNF-RII and IL-1Ra by ELISA in 36 patients with bronchial asthma (16 atopic and 20 non-atopic) during asthma attacks and in stable conditions in order to assess the state of these inhibitors in allergic inflammation. The levels of sTNF-RI, sTNF-RII and IL-1Ra in sera obtained during bronchial asthma attacks were higher than those in sera obtained in stable conditions. These findings were obtained regardless of atopic status. These results suggest that higher levels of serum sTNF-RI, sTNF-RII and IL-1Ra may reflect up-regulation of TNF-R expression and IL-1Ra production in allergic inflammation, and sTNF-RI, sTNF-RII and IL-1Ra may contribute to regulating TNF-α- and IL-1-mediated production and development of allergic inflammation.     

Human recombinant IL-1 receptor antagonist (IL-1Ra) inhibits leukotriene B4 generation from human monocyte suspensions stimulated by lipopolysaccharide (LPS).

The effect of human recombinant IL-1 receptor antagonist (hrIL-1Ra) on leukotriene B4 (LTB4) release was investigated in activated human monocyte cultures. To stimulate LTB4 generation, LPS was used as an agonist. Detection was performed with the highly sensitive radioimmunoassay method. The cells were treated with scalar concentrations using LPS at 1-1000 ng/ml for different periods of time. The greater LTB4 stimulation was found at LPS 100 ng/ml for 18 h incubation time. Preincubation of monocytes with cytochalasin B (CB) (5 micrograms/ml) for 15 min augmented the release of LTB4 when LPS was used. A dose-dependent inhibition was found when human monocytes were pretreated for 10 min with hrIL-1Ra at different concentrations (0.25-250 ng/ml) and then treated with LPS 100 ng/ml for 18 h. Maximum inhibition was observed at the highest concentration of hrIL-1Ra (250 ng/ml). Macrophages treated with a non-selective 5-lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), used at 10 microM, added 15 min before LPS 100 ng/ml, produce a dose-dependent inhibition of LTB4. Cells pretreated with arachidonic acid, at various concentrations (10(-9)-10(-5) M) for 10 min and then treated with LPS 100 ng/ml for 18 h, were also inhibited in a dose-dependent manner by hrIL-1Ra in their production of LTB4. The inhibition of LTB4 release by hrIL-1Ra, in LPS-stimulated human monocytes, may suggest an important modulatory role for this new cytokine (monokine) in inflammation and immunity and may hold future therapeutic implications for diseases involving LTB4 as a mediator.     

Circulating proinflammatory cytokines (IL-1 beta, TNF-alpha, and IL-6) and IL-1 receptor antagonist (IL-1Ra) in fulminant hepatic failure and acute hepatitis.

Fulminant hepatic failure (FHF) is characterized by massive necroinflammation of the liver tissue and is associated with high mortality. Serum concentrations of IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 receptor antagonist (IL-1Ra) were measured in 30 patients with FHF and in 23 patients with acute hepatitis (AH) before start of treatment and in 23 healthy controls. Levels of all four molecules were increased significantly in FHF compared with AH, in which values were higher than in the healthy controls. High serum levels of IL-1 beta and a significantly reduced ratio of IL-1Ra to IL-1 beta (IL-1Ra/IL-1 beta) were observed in FHF patients who subsequently died compared with subjects who survived. TNF-alpha and IL-6 concentrations were correlated with levels of human hepatocyte growth factor (hHGF), an index of hepatocyte regeneration. Although serum cytokine levels varied considerably between patients within each group studied, it is suggested that the striking elevation in proinflammatory cytokine levels in FHF may reflect both the insufficiency of hepatitis virus elimination and a failure to control a vicious cytokine cascade leading to overwhelming hepatocyte destruction rather than regeneration. The high cytokine levels observed in these patients and the significantly elevated IL-1Ra/IL-1 beta ratio in FHF patients who survived compared with those who did not suggest the possible therapeutic use of cytokine antagonists for the control of this life-threatening disease.     

Expression of multiple forms of IL-1 receptor antagonist (IL-1ra) by human retinal pigment epithelial cells: identification of a new IL-1ra exon

The eye is considered an immunologically privileged organ and is separated from the rest of the body by blood-ocular barriers. Part of the blood-retina barrier consists of the retinal pigment epithelium (RPE). In addition to the physical barrier which the monolayer of RPE cells forms, these cells contribute to ocular immune privilege by producing anti-inflammatory molecules that down-regulate potential damaging immune reactions. In this study the mRNA expression of IL-1 receptor antagonist (IL-1ra) by RPE cells was studied in 15 donor-derived cell lines. Expression of both the intracellular and secreted IL-1ra was detected in unstimulated and IL-1β- or phorbol 12-myristate 13-acetate-exposed RPE. Analysis of IL-1ra protein in RPE cell lysates and cell culture supernatants indicated that these cells produce mainly intracellular IL-1ra. No correlation between IL-1ra expression levels and the IL-1ra gene polymorphism could be detected. In addition to the two known intracellular IL-1ra variants (intracellular IL-1ra type I and type II) evidence is provided for the expression of a hitherto unknown splice variant of the IL-1ra mRNA by RPE cells. Expression was not confined to RPE cells and could also be detected in cultured human fibroblasts and macrophages. This variant, which we have tentatively named intracellular IL-1ra type III, encodes a C-terminally truncated protein of only 27 amino acids.     

Single nucleotide polymorphisms in intron 2 of the human interleukin-1 receptor antagonist (IL-1Ra) gene: further definition of the IL-1 beta and IL-1Ra polymorphisms in North American Caucasians and Taiwanese Chinese

Previous studies have suggested that a variable number tandem repeat (VNTR) polymorphism in the second intron of the interleukin-1 receptor antagonist (IL-1Ra) gene and the single nucleotide polymorphisms at positions -511 and +3954 of the IL-1beta gene might be associated with increased risks of chronic inflammatory diseases, autoimmune diseases and gastric cancer. In the present study, IL-1beta and IL-1Ra genotypes were analyzed among Asians in Taiwan and Caucasians in North America. We identified a novel polymorphism with 3 nucleotide substitutions in the IL-1Ra VNTR 2-repeat allele. One of the substitutions corresponds with the fourth 3' end nucleotide of the reverse primer that is often used for analysis of the IL-1Ra-associated VNTR locus. Mismatching between this primer and the 2-repeat allele can cause misleading amplification results when stringent conditions are used for annealing. The estimated haplotype frequencies of the variant IL-1 genes were significantly different between Taiwanese and Caucasians. The frequency of the pro-inflammatory IL-1Ra 2-repeat allele was significantly lower in Taiwanese than in Caucasians. In contrast, the frequencies of the pro-inflammatory IL-1beta -511T allele and +3954C allele were significantly higher among Taiwanese compared with Caucasians.     

Production of serum amyloid A and C-reactive protein by HepG2 cells stimulated with combinations of cytokines or monocyte conditioned media: the effects of prednisolone.

The hepatic production of the acute phase proteins in response to inflammatory cytokines, and the interaction of corticosteroids within this response, has been the subject of considerable recent research. In this study we have examined the effects of the corticosteroid prednisolone on the production of IL-1 alpha and IL-1 beta by lipopolysaccharide (LPS)-stimulated monocytes, and the ability of the monocyte conditioned media (MOCM) obtained under these conditions to induce human hepatoma HepG2 cells to produce serum amyloid A (SAA) and C-reactive protein (CRP). We also examined the production of SAA and CRP by HepG2 cells exposed to different combinations and concentrations of recombinant human (rh) IL-1 alpha, rhIL-1 beta, rhIL-6, recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and prednisolone. The findings indicate: (i) prednisolone substantially inhibits the production of both IL-1 alpha and IL-1 beta by LPS-stimulated monocytes. The MOCM from prednisolone-treated monocytes induced less SAA and CRP production by HepG2 cells; (ii) IL-1 alpha and IL-1 beta both induced CRP and SAA synthesis by HepG2 cells, but only in the presence of IL-6. IL-1 beta was the more potent inducer for SAA production, but for CRP production IL-1 alpha and IL-1 beta were equivalent; (iii) prednisolone enhances the production of SAA by HepG2 cells, but does not enhance the production of CRP; (iv) TNF-alpha in the presence or absence of IL-6 and/or prednisolone did not induce the production of SAA or CRP by HepG2 cells. These findings offer a tenable solution to a disparate production of SAA compared with CRP in corticosteroid-treated cystic fibrosis (CF) patients.     

Significance of IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12·9 ± 9·5 months (mean ±s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1β and IL-8 were significantly higher in DPB compared with healthy volunteers (P < 0·05, P < 0·05, respectively). IL-1 Ra in patients is considered to have a weak inhibitory activity for IL-1β, with approximately five-fold concentration of IL-1β compared with that in healthy volunteers (approx. nine-fold concentration of IL-1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1β correlated significantly with the concentration of neutrophils in BALF (r = 0·72, P < 0·01), as well as with the level of IL-1Ra (r = 0·688, P < 0·05) and IL-8 (r = 0·653, P < 0·05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r = 0·526, P = 0·053 or r = 0·776, P < 0·01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r = 0·524, P < 0·05). Our results suggest that the relative amounts of IL-1β and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.     

Differential binding of human interleukin-1 (IL-1) receptor antagonist to natural and recombinant soluble and cellular IL-1 type I receptors

A recently described factor, interleukin-1 receptor antagonist binding factor (IL-IraBF), in serum of normal individuals is immunologically related to the interleukin-1 receptor type I (IL-1RI). It is presumably a soluble form of the receptor that binds exclusively to interleukin-1 receptor antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5 fibroblasts and the interference afforded by the soluble receptors. The results show that the protein backbones of IL-1raBF and sIL-1RI are of similar size (≈ 35–40 kDa) and that there are differences in the glycosylation of the two molecules. These carbohydrates were necessary for optimal binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble IL-1RI and that IL-1ra binds differently to these molecules and to cellular IL-1RI.     

Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats

Abstract

To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1β, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5–75?ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2–103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.     

Soluble interleukin-6 receptor strongly increases the production of acute-phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes

Interleukin-6 (IL-6) interacts with a system of receptors, which include a 80-kDa IL-6-binding subunit (IL-6R) and a transducing element (gp130). The soluble form of IL-6R (sIL-6R) can bind its ligand and induce cellular responses by association with gp130, thus acting as an IL-6 agonist. We and others have previously shown that the responsiveness to IL-6 is different in hepatoma and human primary hepatocytes. We therefore compared the effects of sIL-6R on the two types of cells, and on the B9 hybridoma, another IL-6-sensitive cell line. Human primary hepatocytes, hepatoma cells PLC/PRF/5, and B9 cells were incubated with different concentrations of IL-6, sIL-6-R, or both. The hepatocyte culture supernatants were tested for their content of acute-phase proteins (APP). The proliferation of B9 cells was assessed by a colorimetric method. Results showed that sIL-6R alone markedly increases the production of APP by hepatoma cells in a dose-dependent manner, but affects only minimally primary hepatocytes and the proliferation of B9 cells. The combinations of IL-6R and its ligand enhanced the effects of IL-6 alone in both PLC/PRF/5 and B9 cells, but had no effect on primary hepatocytes. An immunohistochemical study indicated that the cell-surface expression of IL-6R was dramatically lower in hepatoma cells than in primary hepatocytes. In conclusion, our results show that the expression of IL-6R is low in the hepatoma cell PLC/PRF/5 when compared with primary hepatocytes and that this difference can, at least partly, explain their deficient responsiveness to IL-6. On the other hand, it appears that IL-6R expression by primary hepatocytes is sufficient and that circulating sIL-6R is unlikely to play a significant role in the modulation of IL-6 effects.     

IL-1 receptor antagonist in saliva; characterization in normal saliva and reduced concentration in Sjo¨gren's syndrome (SS)

The characterization of a salivary factor cross-reacting with IL-1 receptor antagonist (IL-1Ra) is described. The apparent molecular weights of two species were 23 kD, consistent with the secreted peptide (sIL-1Ra), and 20 kD, consistent with the intracellular peptide (icIL-1Ra). It had an inhibitory activity on IL-1-stimulated fibroblasts, which is characteristic of IL-1Ra. Its source was the oral mucosa and not the salivary glands. Saliva from patients with SS contained significantly less IL-1Ra than saliva from controls. The decrease was marked in patients with early dental loss but whose xerostomia was still partial. In SS, the salivary IL-1/IL-1Ra imbalance may promote inflammatory lesions in the mouth and impede mucosal cell differentiation.     

Anti-inflammatory properties of human serum IgA: induction of IL-1 receptor antagonist and FcαR (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-α) and IL-6 in human monocytes

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL-1 and TNF-α accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL-1 receptor antagonist (IL-1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL-1 activity. The present study shows that human serum IgA induces significant IL-1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL-1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL-1β release, and even inhibited LPS-induced IL-1β release. Induction of IL-1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of FcαR with MoAb MY-43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA-induced IL-1Ra production, suggesting that FcαR-mediated induction of tyrosine phosphorylation is required for the IgA-induced stimulation of IL-1Ra release. In addition, triggering of FcαR with MoAb specifically down-regulated TNF-α and IL-6 release in human monocytes activated with Hib. By the induction of IL-1Ra and down-regulation of the release of inflammatory cytokines such as IL-1β, TNF-α and IL-6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.     

Induction of transferrin secretion in murine Sertoli cells by FSH and IL-1: the possibility of different mechanism(s) of regulation

In the present study we examined the capacity of interleukin-1 (IL-1) alpha, beta, interleukin-1 receptor antagonist (IL-1ra) and follicle stimulating hormone (FSH) to induce transferrin secretion by Sertoli cells under in vitro conditions. Primary Sertoli cell (SC) cultures from immature mice secreted constitutively transferrin. Stimulation of these cultures with IL-1alpha, IL-1beta significantly increas\d their capacity to secrete transferrin. Addition of IL-1ra to unstimulated SC cultures did not affect their capacity to secrete transferrin. Stimulation of SC cultures with a combination of both IL-1alpha and FSH or IL-1beta and FSH showed additive effect between IL-1 and FSH in their capacity to induce transferrin secretion by these cells. However, stimulation of Sertoli cells with a combination of both IL-1ra and FSH did not affect their capacity to secrete transferrin compared with FSH-stimulated cultures. Our results may suggest the involvement of testicular paracrine/autocrine factors (IL-1) and endocrine (FSH) factors in the regulation of transferrin secretion by SC. This capacity seems to be differently regulated by these factors. Thus, IL-1alpha and beta may directly affect physiological functions of the testis; which may suggest their involvement in the regulation of spermatogenesis and spermiogenesis processes and male fertility.     

Adhesion to fibronectin via alpha4 integrin (CD49d) protects B cells from apoptosis induced by serum deprivation but not via IgM or Fas/Apo-1 receptors

Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via alpha4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for alpha4beta1 integrin, resulted in statistically significant (P < 0.005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antialpha4 MoAb reverted the protecting effect, thus confirming that it was due specifically to alpha4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated alpha4 integrin, also showed statistically significant higher viability than poly lysine cultures. Alpha4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that alpha4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.     

Stimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1, VCAM-1 or ELAM-1 expression by human vascular endothelial cells

A previous study reported that intercellular adhesion molecule-1 (ICAM-1) expression by human vascular endothelial cells (HUVEC) is augmented by intracellular signal transmission mainly through the protein kinase C (PKC) system stimulated by TXA₂ receptors. In the present study, we show that a TXA₂ receptor agonist, U46619, augments the expression of not only ICAM-1, but also vascular cell adhesion molecule-1 (VCAM-1) or endothelial leucocyte adhesion molecule-1 (ELAM-1) in HUVEC both at protein and mRNA levels. Pretreatment with SQ29,548 (a TXA₂ receptor antagonist) or PKC inhibitors greatly diminished the extent of U46619-induced mRNA accumulation and surface expression of the adhesion molecules. An inhibitor of nuclear factor κB (NF-κB) activation, PDTC, diminishes U46619-induced VCAM-1 mRNA accumulation. NAC, which inhibits NF-κB and activation protein 1 (AP-1) binding activity, inhibits the expression of ICAM-1 or ELAM-1 at protein and mRNA levels. These findings suggest that ICAM-1 or ELAM-1 expression of HUVEC stimulated via TXA₂ receptors is augmented by induction of NF-κB and AP-1 binding activity through the PKC system, and that VCAM-1 expression is augmented by induction of NF-κB binding activity.