Educational level and chronic inflammation in the elderly--the role of obesity: results from the population-based CARLA study.

This study aimed to assess the mediating role of anthropometric parameters in the relation of education and inflammation in the elderly. Cross-sectional data from the population-based CARdio-vascular Disease, Living and Ageing in Halle study were used after excluding subjects with a plasma level of high-sensitive C-reactive protein (hsCRP) above 10 mg L(-1) (916 men/760 women remaining). Education was categorized in accordance with International Standard Classification of Education. As inflammation parameters, the soluble tumour necrosis factor type 1 (sTNF-R1), hsCRP and interleukin 6 (IL-6) were taken into account. Anthropometric parameters were the body mass index (BMI), waist-to-hip ratio (WHR) and waist-to-height ratio (WHeR). We used covariate adjusted mixed models to assess associations. Effect measures were the natural indirect effect (NIE), controlled direct effect and total effect (TE). Education was associated with sTNF-R1, hsCRP and IL-6 in men, and sTNF-R1 and hsCRP in women. Anthropometric parameters correlated with all inflammation parameters after covariate adjustment. BMI and WHeR were strong mediators of educational differences in sTNF-R1 (percentage of NIE of TE: 28% in men; 33% in women) and hsCRP (percentage of NIE of TE: 35% in men; 52% in women), while WHR was the weakest mediator. General obesity mediates roughly one-third of the association of education with chronic inflammation in the elderly.

Inflammation and echocardiographic parameters of ventricular hypertrophy in a cohort with preserved cardiac function.

OBJECTIVE: To investigate the association between inflammation and selective echocardiographic parameters (EP) characteristic for ventricular hypertrophy in cross-sectional and longitudinal population-based analyses. METHODS: Baseline (711 men, 659 women: 45-83 years) and 4-year follow-up data (622 men, 540 women) of the prospective, population-based CARdio-vascular disease, Living and Ageing in Halle (CARLA)study after exclusion of participants with cardiacvascular diseases were analysed. Inflammation parameters: soluble tumour necrosis factor receptor 1 (sTNF-R1), high-sensitivity C reactive protein (hsCRP) and interleukin 6 (IL-6). EPs: left ventricular mass (LVM), left atrial systolic dimension (LADS), interventricular septum diameter (IVSD), posterior wall dimension (PWD), left ventricular diastolic diameter (LVDD), ejection fraction according to Teichholz (EF). For the longitudinal analyses baseline to follow-up differences were considered. Effect sizes were determined by using multiple linear regression and mixed models. Missing values were replaced by means of multiple imputations. RESULTS: Men had higher sTNF-R1 levels; means of hsCRP and IL-6 were similar in men and women. In multiple regression models, sTNF-R1 was associated with LADS (1.4 mm/1000 pg/mL sTNF-R1, 95% CI 0.6 to 2.1) in men. Respecting confounder hsCRP was associated with LVM (5.2 g/10 mg/L hsCRP, 95% CI 1.6 to 8.8), IVSD (0.2 mm/10 mg/L hsCRP, 95% CI 0 to 0.3) and PWD (0.2 mm/10 mg/L hsCRP, 95% CI 0.1 to 0.3) in women, while there were no relevant effects in analysis of IL-6 in both sexes. The baseline to follow-up change in EPs was not relevantly associated with sTNF-R1, hsCRP or IL-6. CONCLUSIONS: STNF-R1, hsCRP and IL-6 were inadequate predictors for structural changes of the heart at follow-up, while weak cross-sectional associations are restricted to certain EPs and depend on sex.

Endogenous nitric oxide and soluble tumor necrosis factor receptor levels are enhanced in infants with congenital heart disease.

OBJECTIVE: This study was designed to investigate cytokine and nitric oxide levels in pediatric patients suffering from chronic heart failure and to investigate effects of beta-blocker treatment on these levels. PATIENTS: Fifteen infants with heart failure resulting from left-to-right shunts with pulmonary overcirculation were compared with 11 infants with cyanotic heart defects with reduced pulmonary blood flow. Four of these patients were finally treated with the beta-blocker propranolol. MEASUREMENTS: Endogenous nitric oxide production was determined by measuring total plasma nitrite/nitrate (Griess method), and levels of soluble tumor necrosis factor receptors type 1 and type 2 (TNF-R1 and TNF-R2, respectively) were measured by commercially available enzyme-linked immunosorbent assay. MAIN RESULTS: In infants with left-to-right shunts, soluble tumor necrosis factor receptor levels were significantly elevated as compared with infants with cyanosis (TNF-R1: 1.7 +/- 0.5 vs. 0.8 +/- 0.3 ng/mL; p =.0003; TNF-R2: 8.1 +/- 4.0 vs. 5.1 +/- 3.2 ng/mL; p =.049). In addition, we found a significant correlation between nitrate/nitrite levels and TNF-R1 (r =.70; p =.0001) or TNF-R2 (r =.62; p =.0013), respectively. Furthermore, the tumor necrosis factor receptor levels in four children after beta-blocker treatment were lower as compared with levels before beta-blocker treatment. CONCLUSIONS: Immune mechanisms, such as cytokine or nitric oxide production, may be involved in pathogenesis of heart failure in children, and may contribute to the beneficial effects of beta-blocker treatment observed in these patients.

Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion.

Lipopolysaccharide (LPS) represents a major virulence factor of Gram-negative bacteria ('endotoxin') that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross-section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin-6 (IL-6)-inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL-6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape.

Bacterial induction of beta interferon in mice is a function of the lipopolysaccharide component.

We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lps(d)]) C57BL/10ScCr mice to produce beta interferon (IFN-beta) when stimulated with bacteria. For this purpose, the IFN-beta and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lps(n)]; C57BL/10ScSn and BALB/c) and Lps(d) (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-beta in the Lps(n) mice but not in the Lps(d) mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-beta in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1alpha [IL-1alpha], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-beta and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-beta in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-beta is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide.

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9-13 and 91-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91-94,96)A, and sCD14(97-101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9-13 and 91-101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.

The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity.

The activation of cells by endotoxin (LPS) is one of the early host responses to infections with Gram-negative bacteria. The lipid A part of LPS molecules is known to represent the endotoxic principle; however, the specific requirements for the expression of biologic activity are still not fully understood. We previously found that a specific molecular conformation (endotoxic conformation) is a prerequisite for lipid A to be biologically active. In this study, we have investigated the interdependence of molecular charge and conformation of natural and chemically modified LPS and lipid A and its transport and intercalation into phospholipid membranes mediated by human LPS-binding protein, as well as IL-6 production after stimulation of whole blood or PBMCs. We found that the number, nature, and location of negative charges strongly modulate the molecular conformation of endotoxin. In addition, the LPS-binding protein-mediated transport of LPS into phospholipid membranes depends on the presence of net negative charge, yet charge is only a necessary, but not a sufficient, prerequisite for transport and intercalation. The biologic activity is determined mainly by the molecular conformation: only conical molecules are highly biologically active, whereas cylindrical ones are largely inactive. We could demonstrate that the net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity.

Catecholamines and lipopolysaccharide synergistically induce the release of interleukin-6 from thymic epithelial cells.

The thymus as the major site of T-cell development is exposed to circulating hormones as well as to neurotransmitters released from peripheral nerves. We investigated the influence of catecholamines on the synthesis of interleukin-1 (IL-1) and IL-6 by cultured rat thymic epithelial cells. Basal or lipopolysaccharide (LPS)-stimulated production of IL-1 was not affected by catecholamines. Release of IL-6 was stimulated only scarcely by catecholamines or tumor necrosis factor-alpha (TNF-alpha) and moderately by LPS alone. However, co-stimulation with adrenaline, noradrenaline, or the beta-adrenoceptor agonist isoproterenol (isoprenaline) had an additive (TNF-alpha) or synergistic (LPS) effect on IL-6 release. The synergistic effect was dose-dependent on catecholamine or LPS concentrations. It was mediated by beta-adrenoceptors that are linked to elevation of intracellular cAMP levels, since it was promoted by beta-adrenoceptor agonists and could be blocked by beta-adrenoceptor antagonists. Co-incubation of LPS with agents directly raising cAMP-levels like forskolin or dibutyryl cAMP yielded even stronger IL-6 induction. After co-stimulation IL-6 mRNA was first detected after 3-4 h and a constant increase of IL-6 bioactivity in the culture supernatant was measured for up to 48 h. Since IL-6 is an important factor for thymocyte differentiation and proliferation, the findings demonstrate an influence of neuronal or hormonal catecholamines on the thymic microenvironment that is created by thymic epithelial cells.

Endotoxin and tumor necrosis factor alpha exert a similar proinflammatory effect in neonatal rat cardiomyocytes, but have different cardiodepressant profiles.

Bolus application of endotoxin to healthy volunteers results in reversible hemodynamic alterations, such as observed in septic cardiomyopathy. Currently, endotoxin-induced cardiodepression is mainly attributed to the endotoxin-induced release of proinflammatory cytokines into the circulation, particularly of tumor necrosis factor alpha and interleukin-1, the serum levels of these cytokines being enhanced in sepsis and septic shock, and also in various heart diseases. In this study, we report a proinflammatory effect of endotoxin (1-10 micrograms/ml, 24-h incubation period) on neonatal rat cardiomyocytes in serum-free culture, evidenced by induction of inducible nitric oxide synthase, enhanced release of nitrite (protein synthesis-dependent) and interleukin-6 into the supernatant, as well as an increase in cell-associated interleukin-1 and a specific cardiodepressant profile: endotoxin disrupts beta-adrenoceptor-mediated increase in pulsation amplitude, but alpha-adrenoceptor-induced increase in pulsation amplitude and arrhythmias are not suppressed. In the presence of dexamethasone (0.1 microM), the endotoxin-mediated blockade of beta-adrenergic responsiveness, as well as induction of inducible nitric oxide synthase, enhanced nitrite release and interleukin-1/-6-production are inhibited. In contrast, tumor necrosis factor alpha at a low concentration (10 U/ml) depresses alpha- and beta-adrenergic responsiveness in the presence of dexamethasone in a nitric oxide-independent manner. These data suggest a stimulatory effect of endotoxin on the cardiomyocyte and a specific proinflammatory and nitric oxide-dependent cardiodepressant profile of endotoxin.

CD26 (dipeptidyl peptidase i.v.) on human T lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts.

We have examined the role of CD26 (dipeptidyl peptidase IV) in the adhesion of resting and activated T lymphocytes to endothelial cells and fibroblasts. For this purpose, we ran a short-time adhesion assay under different strategies: Adhesion of T lymphocytes was determined in the presence of different anti-CD26 monoclonal antibodies, or in the presence of synthetic inhibitors of the enzymatic function of CD26. In addition, the expression of CD26 on T lymphocytes, which were adherent to endothelial cells or fibroblasts, was performed by flow cytometric analysis. We found that the anti-CD26 monoclonal antibodies tested here were not able to inhibit T cell adhesion to monolayers of endothelial cells or fibroblasts. Secondly, synthetic inhibitors of the enzymatic function of CD26 had no effect on the adhesion of T lymphocytes to endothelial cells or fibroblasts. Furthermore, CD26-positive T cells were not accumulated in the adherent population. These results suggest that CD26 on T lymphocytes plays no role in T cell adhesion to endothelial cells or fibroblasts.

Platelet-derived interleukin-1 induces cytokine production, but not proliferation of human vascular smooth muscle cells.

During vascular injury, such as observed in atherosclerosis, restenosis, vasculitides, transplantation, or sepsis, vascular smooth muscle cells (SMC) can be exposed to platelets or platelet products. Under these conditions proliferation or cytokine production of SMC stimulated by platelets or platelet products may contribute to regulation of vascular pathogenesis. Thus, we investigated interleukin-6 (IL-6) and IL-8 production as well as proliferation of SMC in response to platelets or platelet lysates. Platelets not already preactivated by thrombin induced IL-6 (10- to 50-fold) or IL-8 production of unstimulated SMC in a cell number dependent fashion. Preactivation of platelets with thrombin potently increased the platelet-mediated IL-6 (50- to 1,000-fold) and IL-8 production of SMC. Hirudin specifically inhibited the activation of platelets with thrombin. Isolated platelets cultured in the absence of SMC did not contain detectable IL-6 or IL-8. Prestimulation (4 hours) of SMC with pathophysiologically relevant substances (lipopolysaccharide [LPS], tumor necrosis factor-alpha [TNF-alpha], or IL-1alpha) further increased the platelet-induced cytokine production. The platelet-derived SMC stimulatory activity was IL-1, since IL-1 receptor antagonist (IL-1-Ra) inhibited the platelet-induced cytokine production of SMC. Anti-platelet-derived growth factor (PDGF)-antibody did not further reduce this activity. Thrombin itself stimulated expression of IL-6 and IL-8 to some degree and induced IL-6 production of SMC synergistically with IL-1. Platelets also induced proliferation of SMC, however, anti-PDGF antibodies, rather than IL-1-Ra blocked this response. These data show that platelet-derived IL-1 stimulates cytokine production of vascular smooth muscle cells, indicating that platelet-derived IL-1 may contribute to regulation of local pathogenesis in the vessel wall by activation of the cytokine regulatory network.

Apoptosis in monocytes.

Apoptosis is a major mechanism for reducing acute inflammation by elimination of unwanted cellular responses causing tissue damage and monocytes (Mo) and macrophages (Mo) play an important role in these processes. Therefore, in the following we summarize how these cells may contribute to regulation of apoptosis of other cells and discuss the factors involved.

An Fc gamma receptor I (CD64)-negative subpopulation of human peripheral blood monocytes is resistant to killing by antigen-activated CD4-positive cytotoxic T cells.

It has been demonstrated that in monocyte/T cell co-cultures activated with recall antigens, cytotoxic T cells were generated which are able to reduce the number of antigen-presenting monocytes. In previous studies we could show that a minor subset of monocytes, the Fc gamma receptor I-negative (CD64-) monocytes, exhibits significantly higher antigen-presenting capacity than the main population of monocytes (> 90%) which are Fc gamma receptor I-positive (CD64+). Therefore, we addressed the question whether they are also differentially susceptible to T cell-mediated killing. In the present study we demonstrate that the CD64- monocyte subset is more resistant to killing by antigen-activated T cells than CD64+ monocytes, as indicated by a higher viability and recovery of CD64- monocytes. This mechanism involves CD95 (Fas) antigen, since monocyte death in co-cultures with antigen-activated T cells could be partially reduced by blocking anti-Fas monoclonal antibodies (mAb). In agreement with this finding, although CD95 antigen was expressed on CD64+ and CD64- monocytes at comparable levels, killing of CD64- monocytes by activating anti-Fas mAb was lower than of CD64+ monocytes.

Ligation of CD40 activates interleukin 1beta-converting enzyme (caspase-1) activity in vascular smooth muscle and endothelial cells and promotes elaboration of active interleukin 1beta.

Inflammation contributes to a variety of arterial diseases including atherosclerosis. Interleukin 1beta (IL-1beta) in its activated mature 17-kDa form may mediate aspects of vascular inflammation. As shown previously, human vascular wall cells, such as smooth muscle cells (SMC), express the IL-1beta precursor upon stimulation and the IL-1beta-converting enzyme (ICE) constitutively but do not produce mature IL-1beta or express ICE activity. How SMC, the most numerous cell type in arteries, may release active IL-1beta has therefore remained a perplexing problem. We report here that stimulation of human vascular SMC and endothelial cells (EC) through CD40 ligand, a mediator recently localized in human atheroma, induced elaboration of the IL-1beta precursor as well as activation of cell-associated ICE. In addition to the constitutively expressed 45- and 30-kDa immunoreactive ICE proteins, vascular cells incubated with recombinant human CD40 ligand (rCD40L) (but not IL-1 or TNF) showed an increase of a 20-kDa immunoreactive ICE protein by Western blot analysis. Furthermore, SMC and EC stimulated through rCD40L processed recombinant human IL-1beta precursor (pIL-1beta), generating a cleavage product of approximately 17 kDa. Appearance of both the 20-kDa immunoreactive ICE protein and pIL-1beta processing activity required at least 6 h of stimulation with 0.3 or 1.0 microg/ml rCD40L, respectively, and was inhibited by pre-incubation of the ligand with an anti-CD40L antibody. Stimulation of vascular SMC and EC through rCD40L resulted in the release of biologically active IL-1beta, indicating processing of the native IL-1beta precursor induced by the ligand. These findings establish a novel mechanism of IL-1beta activation in human vascular cells and, moreover, indicate a new pathway of ICE-activation, which could participate in inflammatory aspects of atherogenesis and other disease states.

Human vascular smooth muscle cells express interleukin-1beta-converting enzyme (ICE), but inhibit processing of the interleukin-1beta precursor by ICE.

Local immunoregulatory processes during normal vascular biology or pathogenesis are mediated in part by the production of and response to cytokines by vessel wall cells. Among these cytokines interleukin (IL)-1 is considered to be of major importance. Although vascular smooth muscle (SMC) and endothelial cells (EC) expressed both IL-1alpha and IL-1beta as cell-associated, 33-kilodalton (kD) precursors, SMC neither contained detectable mature IL-1beta, nor processed recombinant IL-1beta precursor into its mature 17-kD form. Thus, we investigated the expression and function of IL-1beta-converting enzyme (ICE) in vascular cells. We demonstrate in processing experiments with recombinant IL-1 precursor molecules that EC processed IL-1beta, in contrast to SMC. Despite the failure of SMC to process IL-1beta, these cells expressed ICE mRNA, immunoreactive ICE protein, and the expected IL-1beta nucleotide sequence. The lack of processing was explained by our finding that extracts of SMC specifically and concentration dependently blocked processing of IL-1beta precursor by recombinant or native ICE. The initial biochemical characterization of the inhibitory activity showed that it is heat-labile, has a molecular size of 50-100 kD, and is associated to the cell membrane compartment. Inhibition of processing, i.e., activation of IL-1beta precursor by SMC may constitute a novel regulatory mechanism during normal vascular biology or pathogenesis of vascular diseases.

Beta-adrenoceptor-mediated effects in rat cultured thymic epithelial cells.

1. Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid-induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. 2. In rat cultured thymic epithelial cells mRNA for beta 1- and beta 2-adrenoceptors was detected by reverse transcription-polymerase chain reaction by use of sequence-specific primers. Specific, saturable binding to the cultivated cells was observed with the beta-adrenoceptor agonist CGP 12177. 3. Adrenaline, noradrenaline or the beta-adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose-dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the beta 1- and the beta 2-subtypes. The selective beta 3-adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of beta 1-/beta 2-adrenoceptors to the response. 4. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum-stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T-lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Tumor necrosis factor alpha (TNF alpha) is cardiodepressant in pathophysiologically relevant concentrations without inducing inducible nitric oxide-(NO)-synthase (iNOS) or triggering serious cytotoxicity.

Cardiac hypertrophy and heart failure are frequently accompanied by elevated plasma levels of tumor necrosis factor alpha (TNF alpha), the pathogenetic relevance of this finding being a matter of debate. In human acute septic cardiomyopathy, on the other hand, the negative inotropic impact of TNF alpha on the heart is well documented and frequently ascribed to the induction of inducible nitric oxide (NO) synthase (iNOS) and an enhanced production of NO in the heart. Yet the present study presents evidence that in cardiomyocytes TNF alpha in non-toxic concentrations specifically depresses contractile performance independent of NO. In spontaneously beating neonatal rat cardiomyocytes, TNF alpha in a low, pathophysiologically relevant concentration (10 U/ml, 1-3 days) does not alter basal pulsation amplitude, but blocks alpha- and beta-adrenoceptor-stimulated increase in contractility and beating irregularity and impairs the impact of high extracellular calcium on contractile performance. However, this low TNF alpha-concentration does not suffice to induce iNOS - documented by reverse transcriptase polymerase chain reaction - or enhance nitrite concentrations in the cell culture supernatants as a measure of cellular NO production, neither in the presence nor absence of dexamethasone (0.1 micro M). Only in high concentration - the specific proinflammatory action being documented by an enhanced release of interleukin-6 from cardiomyocytes - TNF alpha (1000 U/mol; 6, 24 h) weakly induces the mRNA for iNOS, with a consecutive moderate rise in cellular nitrite production. TNF alpha-incubation (10-1000 U/ml) does not alter the morphological appearance of the cells displayed by phase contrast microscopy or evoke gross cytotoxicity.

Cytokine production by mononuclear cells following stimulation with a peptide-containing, endotoxin-free Escherichia coli extract.

The beneficial effects of the E. coli extract Colibiogen inj. N (Cb) observed in therapy of inflammatory bowel diseases, allergies, or gastrointestinal tumors are possibly mediated by the induction of cytokines in human leukocytes or vascular cells. Thus, the induction of the cytokines interleukin 1 (IL1), IL6 and tumor necrosis factor (TNF) in human mononuclear cells (MNC) and vascular cells was investigated in vitro. Various administration forms of the extract (including Cb-inj. N, Cb-oral, and Cb-infantibus N) induced the release of IL1 and IL6 from MNC. The compounds stimulated TNF production less potently, possibly due to a lower sensitivity of the TNF assay system, as compared to the IL1 and IL6 detection system. The MNC produced the cytokines with a kinetics similar to that observed with other stimuli. Monospecific antibodies abolished the respective cytokine activity in the biological assays. Addition of submaximal amounts of endotoxin potently enhanced the IL1- and IL6-inducing activity of the bacterial extract, indicating synergism of the extract and endotoxin. These results provide evidence that cytokines produced by MNC following administration of the tested bacterial extract may contribute to the regulation of the immune response during therapy of gastrointestinal tumors. At present the in vivo production of cytokines following treatment with the bacterial extract tested is under investigation in a phase III study.

IL-8 specifically binds to endothelial but not to smooth muscle cells.

The proinflammatory cytokine and potent chemoattractant IL-8 is involved in regulation of infectious or inflammatory processes. Human vascular endothelial cells (EC) and smooth muscle cells (SMC) probably contribute to these responses by recognition and/or production of rIL-8. We demonstrate here in competitive binding studies with radiolabeled rIL-8 that EC and fibroblasts, but not SMC, specifically bind IL-8 with low affinity. The binding was not saturated by ligand concentrations up to 80 nM 125I-rIL-8. Unlabeled neutrophil-activating peptide-2 competed the binding of 125I-rIL-8, although less potently than unlabeled rIL-8, as reported previously for polymorphonuclear neutrophils. In contrast, connective tissue-activating peptide III, platelet factor 4, or lysozyme did not reduce binding of 125I-rIL-8 to EC or fibroblasts. In accordance with these binding studies, EC and fibroblasts, but not SMC, expressed human IL-8 receptor type I mRNA. Neither cell type expressed mRNA for IL-8 receptor type II. Stimulation with IL-1 alpha or LPS did not alter the results obtained in PCR or binding studies. Although SMC did not express specific binding sites for IL-8, Western blot experiments showed that IL-1 alpha-, TNF-, or LPS-stimulated SMC released two major immunoreactive isoforms of IL-8 in a time- and dose-dependent manner. The m.w. were similar to IL-8 isoforms released by EC or mononuclear cells. The differential capacity of EC and SMC to produce IL-8 and express IL-8 binding sites indicates that vascular cell-derived IL-8 may contribute to differential regulation of infectious and inflammatory responses in the vessel wall.