Expression and secretion of alpha1-proteinase inhibitor are regulated by proinflammatory cytokines in human pancreatic islet cells

Aims/hypothesis Alpha1-proteinase inhibitor (1-PI) has been considered a key player in inflammatory processes. In humans, the main production site of 1-PI is the liver, but other tissues, including pancreatic islets, also synthesise this molecule. The aims of this study were to assess the islet cell types that produce 1-PI, to determine whether 1-PI is actually secreted by islet cells, and to assess how its production and/or secretion are regulated.Methods Expression of 1-PI in human islet cells was assessed by immunofluorescence, electron microscopy and western blotting. Release of 1-PI was analysed by reverse haemolytic plaque assay and ELISA. The effects of cytokines on 1-PI synthesis and secretion were tested.Results Immunofluorescence showed that alpha and delta cells do express 1-PI, whereas beta cells do not. By electron microscopy, we demonstrated a colocalisation of 1-PI with glucagon and somatostatin within secretory granules. Immunolabelling also revealed localisation of 1-PI within the Golgi apparatus, related vesicles and lysosomal structures. The expression of 1-PI in islet cells was also demonstrated by western blotting and ELISA of protein extracts. ELISA and reverse haemolytic plaque assay showed that 1-PI is secreted into the culture medium. Treatment of islet cells with IL-1 and oncostatin M for 4 days increased the production and release of 1-PI.Conclusions/interpretation Our results demonstrate that 1-PI is expressed by the alpha and delta cells of human islets, and that proinflammatory cytokines enhance the production and release of this inhibitor.     

Acute hematologic effects of interferon alpha,interferon gamma,tumor necrosis factor alpha and Interleukin 2

Summary This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFN-gamma Interleukin 2 and tumor necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFN-gamma also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.     

Presence of interferon and anti-interferon in patients with systemic lupus erythematosus

Summary Sera from 61 patients with systemic lupus erythematosus (SLE) were serially screened over a period of at least 2 years for IFN and anti-IFN antibodies. IFN concentrations were measured both with a cytopathic effect assay and a more sensitive radioimmunoassay. Of the patients 15% (9/61) had IFN in their serum at one or more occasions as measured in the bioassay (6 IU/ml); employing a RIA (1 IU/ml) 28% (17/61) of the patients studied were positive for IFN-. Fifteen patients had a measurable interferonemia over 2–16 months; only two patients had detectable IFN in their serum at only one occasion. In five patients, hourly and daily variations of the IFN titer as measured by RIA were found to amount to less than 80%. The IFN activity found in these sera was characterized as IFN- by means of acid stability, cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous and heterologous antisera. IFN antibodies were quantified with a neutralization bioassay, an ELISA, and a radioimmunoassay. Of the 61 patients 5% (3) possessed high titers of anti-IFN antibodies which persisted over 2 years. The IFN- antibody positive patients had an inactive form of the disease over years without visceral involvement but decreased serum complement levels (C4, C3, CH50) and repeated episodes of Quincke-like edema.     

Short-term inhibition of peroxisome proliferator-activated receptor-γ coactivator-1α expression reverses diet-induced diabetes mellitus and hepatic steatosis in mice

Aims/hypothesis The coactivator of nuclear receptors, peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) has been implicated in a series of events that contribute to the control of glucose metabolism. We have recently reported the use of a PGC-1 antisense oligonucleotide (PGC-1AS) that inhibits up to 60% of PGC-1 expression in pancreatic islets, leading to increased insulin secretion. This oligonucleotide was used in this study to try to ameliorate diet-induced type 2 diabetes in a genetically predisposed mouse strain (Swiss mice).Materials and methods Glucose and insulin tolerance tests, euglycaemic–hyperinsulinaemic clamp, immunoprecipitation assays, immunoblotting assays and immunohistochemistry were used in this investigation.Results Swiss mice became obese and overtly diabetic after 8 weeks of feeding with chow containing 24% saturated fat. One daily dose (1.0 nmol) of PGC-1AS significantly reduced glucose and increased insulin blood levels without affecting food intake and body weight. These effects were accompanied by a reduced area under the glucose curve during an intraperitoneal glucose tolerance test, an increased constant of glucose decay (K_itt) during an insulin tolerance test, and an increased glucose consumption rate during a euglycaemic–hyperinsulinaemic clamp. Moreover, mice treated with PGC-1AS presented an outstanding reduction of macroscopic and microscopic features of hepatic steatosis. These effects were accompanied by reduced expression or function of a series of proteins involved in lipogenesis.Conclusions/interpretation PGC-1 is an attractive target for pharmacological therapeutics in type 2 diabetes mellitus and diet-induced hepatic steatosis.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Expression of EGF-receptor related protein (ERRP) decreases in gastric mucosa during aging and carcinogenesis

Aging and gastrointestinal malignancies, including that of the stomach are associated with increased activation of EGF-receptor (EGFR). Although the intracellular events that regulate this process are poorly understood, we hypothesize that loss of ERRP (EGFR-related protein; GenBank accession number AF187818), a recently identified negative regulator of EGFR, that possesses a substantial homology to the ligand binding extracellular domain of EGFR, may contribute to this event. In support of our hypothesis, we have observed that in Fischer-344 rats, whereas aging is associated with increased activation of EGFR in the gastric mucosa, expression of ERRP decreases in this tissue during this period. The latter is accompanied by a concomitant reduction in the amount of TGF- bound to ERRP. In contrast, the amount of TGF- bound to EGFR is found to be higher in the gastric mucosa of aged than in young rats. This is accompanied by a concomitant rise in EGFR levels. In the gastric mucosa, EGFR and ERRP are found to be colocalized. Gastric adenocarcinoma in humans, which has been shown to be associated with increased activation of EGFR, shows a substantial reduction in ERRP expression, when compared with benign tissues. We conclude that increased activation of EGFR in the gastric mucosa during aging and carcinogenesis may partly be due to the loss of ERRP.     

Plasma leptin and TNF-alpha levels in chronic hepatitis C patients and their relationship to hepatic fibrosis

The aim of this study was to examine the possible relationship between the plasma levels of leptin and tumor necrosis factor (TNF)- and the stage of hepatic fibrosis in a cohort of patients with chronic hepatitis C. Leptin and TNF levels were measured by RIA in 135 patients and in 75 age- and sex-matched controls. Liver disease was evaluated by the stage of fibrosis and the extent of inflammatory infiltrate in the liver biopsy. Leptin levels correlated with BMI values in healthy controls and in patients with chronic hepatitis C (men, r = 0.61, P = 0.0001; women, r = 0.68, P = 0.003). Leptin levels increased as hepatic fibrosis stage progressed both in male and in female patients (P < 0.001); also, TNF levels were higher in patients with an advanced stage of fibrosis (P = 0.006). In these patients, levels of leptin increased according to the progression of the stage of fibrosis; these data suggest that leptin may play a role in the regulation of hepatic fibrosis.     

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon- (IFN-), tumornecrosis factor- (TNF-), andinterleukin-1 (IL-1) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN- and TNF-, but not afterexposure to LPS, SEB or IL-1. The adhesion inducedby either IFN- or TNF- was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN- or TNF-,and peaked after 12 hr of incubation with TNF-and after 24 hr with IFN-. These results indicatethat IFN- and TNF- induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.     

Biliary Interleukin-6 and Tumor Necrosis Factor-α in Patients Undergoing Endoscopic Retrograde Cholangiopancreatography

Cytokines are low-molecular-weight proteinmediators that possess a wide spectrum of inflammatory,metabolic, and immunomodulatory properties. Cytokineshave been shown to be produced by monocytes/macrophages, lymphocytes, fibroblasts, endothelial cells,and more recently, hepatocytes and biliary epithelium.The aim of this study was to define biliary levels ofinterleukin-6 (IL-6) and tumor necrosis factor- (TNF-) in patients undergoing endoscopicretrograde cholangiopancreatography (ERCP) in variousdisease states. Fifty-four patients undergoing ERCPcomprised the study group. IL-6 and TNF- were measured in aspirated bile using an ELISAtechnique. Levels of both TNF- and IL-6 weresignificantly higher in patients with cholangitis (P< 0.00001). Moreover, IL-6 was 100% specific forcholangitis since none of the patients without bacterialcholangitis — including patients with biliaryobstruction secondary to cholangiocarcinoma orpancreatic carcinoma — had measurable IL-6 intheir bile. Low levels of biliary TNF- were detectable in fivepatients without cholangitis; the sensitivity andspecificity of TNF- for cholangitis were 100% and82%, respectively. There was a strong statisticalcorrelation between biliary IL-6 and TNF- levels (r= 0.819, P < 0.0001). In contrast, the correlationsbetween biliary cytokines and serum biochemicalparameters were weak. These results suggest that IL-6and TNF- are sensitive markers for cholangitis and maydifferentiate it from other types of biliary tractdisease.     

Influence of TNF gene polymorphism in patients with acute and fulminant hepatitis

Background Tumor necrosis factor (TNF) is involved in liver damage, especially in fulminant hepatitis (FH). Our previous data showed that the serum level of TNF- was markedly increased in FH. To investigate the mechanism of the overproduction of TNF in FH patients, polymorphism of the TNF gene was studied.Methods We analyzed 120 healthy subjects (controls), 63 patients with acute hepatitis (AH), and 32 patients with FH. Of the 32 FH patients, 21 died or received liver transplantation (FH-D), and 11 survived with intensive therapy (FH-S). The TNF- promoter region at –1031, –863, –857, –308, and –238, and TNF- Nco1 polymorphism sites were studied.Results (1) The four groups showed no differences in polymorphisms of positions –857, –308, and –238. The allelic frequencies of positions –1031C and –863A in the FH-D patients were significantly higher compared to findings in control subjects. (2) The allelic frequency of B2 in the TNF- gene was significantly higher in FH patients, and particularly in the FH-D patients, compared to control subjects. (3) When the patients were divided into four groups by etiology, hepatitis A virus (HAV), HBV, HCV, and non-A non-B non-C, the allelic frequencies of positions –863A and TNF- B2 in FH patients were increased in the non-A non-B non-C group compared to controls.Conclusions FH patients with a poor prognosis had higher frequencies of positions –1031C and –863A in the TNF- promoter region, and higher frequencies of the B2 allele of the TNF- gene. These data suggest that the genomic background may be associated with the prognosis of acute liver failure.     

Nuclear factor-kappaB and TNF-alpha mediate gastric ulceration induced by phorbol myristate acetate

Phorbol esters induce inflammation in rodents by activating protein kinase C. We determined whether nuclear factor-B (NF-B) and tumor necrosis factor- (TNF-) play role in the formation of gastric ulcer induced by phorbol-12-myristate-13-acetate (PMA) in rats. Subserosally injected PMA dose-dependently induced gastric mucosal ulcer. Activation of NF-B in the gastric mucosa corresponding to the PMA injection sites was observed before the ulcers became obvious as assessed by an in situ fluorescence DNA binding assay and electrophoretic mobility shift assay. The NF-B activation and subsequent ulcer formation were significantly inhibited by injection of pyrrolidine dithiocarbamate, proteasome inhibitor (MG132), or NF-B decoy. Antibody against TNF- significantly inhibited ulcer formation without attenuating NF-B activation. These results suggest that both NF-B activation followed by TNF- release contribute to tissue damage in PMA-induced gastric ulcer formation.     

TNFα in ischemia/reperfusion injury and heart failure

Abstract. During myocardial ischemia, both the myocardial and serum TNF concentrations are rapidly increased within the area at risk. With prolongation of ischemia and development of cardiomyocyte necrosis, the TNF concentration increases also in the surrounding viable portions of the myocardium. Indeed, in the scenario of myocardial ischemia/reperfusion, treatment with TNF antibodies reduced the extent of myocardial infarction in rabbits and attenuated the contractile dysfunction following microembolization in dogs. In the latter studies, the serum TNF concentration remained unaltered thereby supporting the notion of a direct action of TNF at the level of cardiomyocytes during ischemia/reperfusion.In heart failure, the serum TNF concentration is also increased, and in patients with advanced heart failure the serum TNF concentration is an independent predictor of mortality. The origin of the increased serum TNF concentration is not clearly identied yet, but TNF derived from the heart and peripheral organs contributes to the increased serum TNF concentration. Treatment with TNF antibodies in the clinical scenario, however, did not improve the prognosis of heart failure patients.     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

TNFα in patients with congestive heart failure

Abstract. It is now generally accepted that chronically extensive stimulation of the cytokine system—and of TNF in particular—is detrimental to the heart and to peripheral tissue and that such stimulation may contribute to the pathogenesis of congestive heart failure of various causes. During the past decade, basic and clinical research has provided growing evidence for the role of systemic and local inammatory responses that, however, have so far failed to translate into new treatments for patients. The present paper represents an attempt to critically review the general concepts that lie behind the dichotomy existing between an impressive bulk of biologic research showing the role of TNF as a pathogen in congestive heart failure and the difculties in translating this evidence into patients treatment.     

Tumor necrosis factor-α and tumor necrosis factor receptors in human heart failure

The basic mechanisms that are responsible for the development and progression of congestive heart failure are not known. Although clinicians have traditionally viewed heart failure as a hemodynamic disorder related to left ventricular pump dysfunction, one of the more recent concepts that has emerged is that the development and progression of heart failure is attributable, at least in part, to the overexpression of biologically active molecules that can contribute both to patient symptomatology as well as to disease progression. In this regard, one of the more recent interesting and intriguing observations in clinical heart failure research is the finding that a proinflammatory cytokine, termed tumor necrosis factor- (TNF-), is expressed in patients with heart failure. Accordingly, the focus of the present brief review is to summarize recent clinical and experimental observations that implicate the elaboration of TNF- and TNF receptors in the progression of human heart failure.     

Effect of recombinant human transforming growth factor beta and tumor necrosis factor alpha on bone marrow progenitor cells of HIV-infected persons

Summary With progressive disease, the majority of patients with human immunodeficiency virus (HIV) infection develop bone marrow failure with anemia, leukopenia, and thrombocytopenia, the cause of which has not yet been clarified. Besides direct infection of bone marrow progenitor cells and immune-mediated cytolysis, the action of inhibitory cytokines, like transforming growth factor beta (TGF-) and tumor necrosis factor alpha (TNF-), has to be discussed with regard to their pathophysiological role in HIV-induced bone marrow failure. Therefore, the influence of recombinant human TGF- and TNF- on colony growth of pluripotent (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells from the bone marrow of HIV-1-infected persons and normal controls was assessed in methylcellulose cultures. Both cytokines inhibited the colony formation of hematopoietic progenitor cells from HIV-positive persons. When added to unseparated bone marrow cells from HIV-infected persons and normal controls, the 50% inhibition (ID₅₀) of BFU-E by TGF- occurred at 1.3 ng/ml and 3.7 ng/ml, respectively, while the ID₅₀ of CFU-GM occurred at 15.5 ng/ml and 142.7 ng/ml. Concentrations of TNF-, causing 50% inhibition of colony formation by bone marrow cells from HIV-infected or noninfected individuals were 6.3 U/ml and 17.0 U/ml for BFU-E, and 24.4 U/ml and >3,000 U/ml for CFU-GM, respectively. The ID₅₀ of the CFU-GEMM growth was below the lowest concentration of both cytokines tested. The suppressive effects were specifically abolished by antibodies against TGF- and TNF-, thus confirming that the inhibitory activities were due to the cytokine preparation used.The study was supported by a grant from theBundesgesundheitsamt     

Systemic sarcoidosis presenting as a granulomatous tattoo reaction secondary to interferon-alpha treatment for chronic hepatitis C and review of the literature

Sarcoidosis is a multisystem granulomatous disease of unknown etiology. Immune alterations involving heightened T-helper-1 responses have been proposed to play a major role in the pathogenesis of sarcoidosis. Interferon-alpha therapy and hepatitis C infection have been implicated in the development of a variety of autoimmune diseases. However, despite the wide use of IFN-alpha therapy for hepatitis C, only a few cases of sarcoidosis have been reported in this context. We report the case of a 42-year-old white female with hepatitis C, who developed systemic sarcoidosis shortly after therapy with IFN-alpha2b. The disease was heralded by the appearance of a cutaneous sarcoid/ foreign body granulomatous reaction at the site of an old tattoo. The sarcoidosis responded to a short course of oral prednisone therapy. We also reviewed the other reported cases and discussed the possible immunological mechanisms involved.     

Inhibition by 16,16-Dimethyl Prostaglandin E2 of Tumor Necrosis Factor-α and Interleukin-1β Production and Messenger RNA Expression in Human Monocytes Stimulated by Helicobacter pylori

We investigated the effects of 16,16-dimethylprostaglandin E₂ on the production of tumornecrosis factor- and interleukin-1 in humanmonocytes stimulated with Helicobacter pylori. Monocytes isolated from human peripheral blood wereincubated for 24 hr with the extract of H. pyloridiluted 1:100 to 1:100,000 by volume, a combination ofthe extract and 16,16-dimethyl prostaglandinE₂, or a vehicle alone. The extract stimulated theproduction of tumor necrosis factor- andinterleukin-1 and the expression of theirmessenger RNA in a dose-dependent manner. 16,16-Dimethylprostaglandin E₂ inhibited the production of thesecytokines and their messenger RNA in the presence of H.pylori at doses higher than 10^-6 M,predominantly with tumor necrosis factor-. These data suggest that antiinflammatory effects ofprostaglandins on gastric mucosa are in part related totheir effects on inhibition of production ofproinflammatory cytokines by monocytes.