Interleukin 10 Inhibits TNF-Alpha Production in Human Monocytes Independently of Interleukin 12 and Interleukin 1 Beta

Previously we demonstrated that endogenously produced Interleukin (IL-)IO suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via downregulation of paracfine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, EL-10 inhibited monocyte TNF-alpha production by about half Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the EL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interleukin 10 Inhibits TNF-Alpha Production in Human Monocytes Independently of Interleukin 12 and Interleukin 1 Beta

Previously we demonstrated that endogenously produced Interleukin (IL-)IO suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via downregulation of paracfine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, EL-10 inhibited monocyte TNF-alpha production by about half Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the EL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Reversible Attachment of Human Peripheral Blood Monocytes During in Vitro Culture

The attachment kinetics of monocytes to polystyrene surfaces from cultures of human peripheral blood mononuclear leukocytes has been determined. Initial experiments showed that the relative monocyte depletion efficiency was much higher after one hour than after twenty hours of incubation. The supernatants from samples incubated for one hour also contained fewer monocytes than did supernatants from samples incubated for twenty hours. Further studies showed that a large percentage of monocytes which initially attach during the first hour later detach. These cells retain their ability to attach to a new culture substrate and to mature into macrophages.     

Reversible Attachment of Human Peripheral Blood Monocytes During in Vitro Culture

The attachment kinetics of monocytes to polystyrene surfaces from cultures of human peripheral blood mononuclear leukocytes has been determined. Initial experiments showed that the relative monocyte depletion efficiency was much higher after one hour than after twenty hours of incubation. The supernatants from samples incubated for one hour also contained fewer monocytes than did supernatants from samples incubated for twenty hours. Further studies showed that a large percentage of monocytes which initially attach during the first hour later detach. These cells retain their ability to attach to a new culture substrate and to mature into macrophages.     

Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P)

P-selectin (CD62P) is an adhesion molecule expressed on the activated endothelium and activated platelets that is involved in the initial attachment of leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodies (mAbs) and P-selectin-deficient mice have shown that P-selectin is a potential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology. The aim of this study was to generate a novel panel of anti-P-selectin mAbs against the conserved epitopes present in several animal species. To produce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recognize human P-selectin were obtained. Individual mAbs that bound to human, rat, mouse, rabbit and pig activated platelets were characterized by flow-cytometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P-sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL60 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MAb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELISA assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure soluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are unique since they recognize common epitopes conserved during mammalian evolution and they may be useful for studying P-selectin function in inflammatory models in various species.     

Activation of lung macrophage subpopulations in experimental acute pancreatitis

Pulmonary macrophages exist in two different anatomical compartments in the lower respiratory tract: alveolar macrophages in the alveoli and interstitial macrophages in the interstitium. Depending on the micro-environmental stimulation, macrophages follow different activation pathways. According to their inflammatory response pattern, activated macrophages have been characterized as pro-inflammatory (M1), wound-healing (M2a) and regulatory (M2b). Since acute pancreatitis occurs in parallel with acute lung injury, the profile of the different macrophage subpopulations could be relevant in the progression of the disease. The activation of lung alveolar and interstitial macrophages was assessed in an experimental model of severe acute pancreatitis induced in rats by intraductal infusion of 3.5% sodium taurocholate. Alveolar and interstitial macrophages were obtained and the expression of markers of different activations was evaluated. Activation of nuclear factors PPARγ and NF-κB, which are involved in the acquisition of different phenoytpes, was also measured. Alveolar macrophages acquired an early M1 phenotype characterized by the expression of inflammatory cytokines and NF-κB activation. In contrast, interstitial macrophages followed the inhibitory M2b pathway. In these macrophages, PPARγ became activated and the anti-inflammatory cytokine IL-10 was expressed. These results suggest that alveolar and interstitial macrophages play different roles in acute lung injury associated with acute pancreatitis. Alveolar macrophages promote an early inflammatory response, whereas interstitial macrophages help resolve inflammation.     

IL-4 induced leucocyte trafficking in cynomolgus monkeys: correlation with expression of adhesion molecules and chemokine generation

Background Interleukin-4 (IL-4) is an immunoregulatory cytokine which has a wide variety of effects on immune cell function. In addition, recent studies suggest that IL-4 may have effects on other cells including endothelial cells in terms of the regulation of adhesion molecule expression and leucocyte extravasation from the vascular space to sites of tissue inflammation. Consequently, IL-4 may have an important role in the pathogenesis of allergic inflammation and disease. Objective The purpose of this study was to learn more about the potential role of IL-4 in inflammatory disease, specifically in regard to the potential of IL-4 to induce the expression of adhesion molecules on vascular endothelial cells and promote the adherence and transmigration of circulating leucocytes to sites of tissue inflammation. Methods Single subcutaneous injections of human IL-4 were administered to cynomolgus monkeys and tissue biopsy samples were obtained and analysed for adhesion molecule expression on vascular endothehum and inflammatory cell infiltrates. In another series of experiments, multiple subcutaneous injections of human IL-4 were administered (bid on four consecutive days) and the effects on peripheral blood leucocytes and plasma levels of various cytokines and chemokines were examined. Results Intradermal injection of IL-4 induced the expression of vascular cell adhesion molecule-1 (VCAM-l) on cutaneous vascular endothelium that was present at 8hr and persisted out to 24h post injection. The expression of VCAM-1 was associated with an inflammatory cell infiltrate comprised of granulocytes and mononuclear cells. Multiple injections of IL-4 resulted in a dose-related decrease in the relative percentage and total number of circulating lymphocytes and an increase in circulating neutrophils (4.6 ± 1–2.1 ± 0.2 ± 10⁶/mL and 1.7 ± 0.3–7.0 ± 1 ± 10⁶/mL, respectively). Analysis of cell surface markers by flow cytometry revealed a transient decrease in the number of CD4⁺ T lymphocytes and a sustained decrease in CD16⁺ cells. In addition. IL-4 administration resulted in a large increase in plasma MCP-1 concentration. Conclusion This is the first study to demonstrate an acute eflect of IL-4 consistent with lymphocyte trafficking out of the vascular space, the induction of VCAM-l expression on vascular endothelium and increases in plasma levels of MCP-1 in vivo. We suggest that IL-4 may be involved in the early recruitment of mononuclear cells to sites of tissue inflammation by the upregulation of VCAM-l expression on vascular endothelium and the generation and release of potent chemoattractants.     

Identification and characterization of ligands for L-selectin in the kidney. I. Versican, a large chondroitin sulfate proteoglycan, is a ligand for L-selectin

Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer's patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin-IgG chimera (LEC-IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.     

Enhanced In Vitro Phagocytosis of Listeria monocytogenes by Human Monocytes in the Presence of Ampicillin, Tetracycline, and Chloramphenicol

An in vitro system was developed to test the phagocytic activity of human macrophages grown from blood monocytes in the presence of the antibiotics ampicillin, tetracycline, and chloramphenicol. (3)H-labeled Listeria monocytogenes served as test organism. Subinhibitory amounts of the antibiotics enhanced the phagocytic activity significantly (P < 0.025). Macrophages pretreated with the drugs in identical concentrations showed the same phagocytic activity as control cells in the absence of the drugs. Because the drug concentrations used were similar to those that may be attained in man at certain places of inflammation, enhanced phagocytosis in the presence of antibiotics may have clinical significance.     

大鼠血管新生内膜形成过程中NF-κB的活化和ICAM-1及COX-2表达的变化

目的：探讨大鼠血管内膜增生过程中NF-κB活化与内膜炎性增生的相互关系。方法:Western blotting分析血管壁中NF-κB p65、IκBα及其下游炎性基因ICAM-1和COX-2的表达；用免疫沉淀分析检测NF-κB p65的苏氨酸磷酸化。结果:内皮剥脱后，血管总蛋白与核蛋白中p65的含量于术后7 d达峰值；术后21 d下降但仍高于0、1 d组；但胞浆p65含量无明显变化。p65苏氨酸磷酸化水平与NF-κB核转位成负相关关系。IκBα于术后1 d表现出一过性降低，术后14 d开始回升，21 d接近正常组水平；与此相反，ICAM-1和COX-2的表达于内皮剥脱后升高，至14 d时达峰值，21 d时表达量下降但仍高于正常组。结论:血管内皮剥脱诱导的内膜增生过程中伴有持续的NF-κB p65活化和炎性因子的表达。     

Participation of macrophages in segmental endocapillary proliferation preceding focal glomerular sclerosis

We studied infiltrating cells in the glomeruli of eight cases with focal segmental endocapillary proliferation (FSEP) using monoclonal antibodies to leukocyte common antigen, T cells, B cells, and monocytes/macrophages (Mo/Mφ). It was demonstrated by sequential biopsies performed in five cases that FSEP preceded focal glomerular sclerosis (FGS). Cell types in FSEP were compared with those in FGS from 17 patients with persistent nephrotic syndrome, ten non-nephrotic patients, and eight patients with nephrotic syndrome which was initially responsive to steroid therapy but relapsed, as well as minimal change specimens from nine nephrotic patients. In the glomeruli, the mean total leukocyte counts increased significantly in the FSEP group (P < 0.01). The serial sections in FSEP revealed that Mo/Mφ were the predominant cells and were localized in areas of endocapillary proliferation. T-cell or B-cell infiltration was less marked. The extensive intracapillary distribution of p150,95 antigen belonging to the integrin family and acting as a C3bi receptor suggested that FSEP may be mediated by adhesion molecules expressed on Mo/Mφ. These findings indicate that Mo/Mφ may play a key role in FGS which shows endocapillary proliferation in the initial stage.     

The Neuropeptide Substance P Stimulates Production of Interleukin 1 in Human Blood Monocytes: Activated Cells are Preferentially Influenced by the Neuropeptide

Substance P (SP) has recently been reported to induce interleukin 1 (IL-1) production by human monocytes. This was confirmed in our experiments with human monocytes cultivated in the presence of SP or SP together with lipopolysaccharide (LPS). In addition, a wide variability of cell response to the neuropeptide was noticed. Three out of twelve cell cultures were directly stimulated by SP to release IL-1, while four additional cultures needed prestimulation with suboptimal doses of LPS, and no effect was seen in the five remaining experiments. The data may suggest that preferentially activated monocytes respond to SP. The production of IL-1 by SP-stimulated monocytes is of great interest considering the broad spectrum of activity of IL-1 and the increasing evidence of sensory neuron involvement in acute and chronic inflammatory responses.     

Expression of leucocyte–endothelial adhesion molecules is limited to intercellular adhesion molecule-1 (ICAM-1) in the lung of pneumoconiotic patients: role of tumour necrosis factor-alpha (TNF-α)

Coal workers' pneumoconiosis (CWP) is characterized by a chronic inflammatory lung reaction associated with macrophage accumulation in alveolar spaces. In this study, we investigated in CWP the implication of adhesion molecules such as E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) and the role of TNF-α which is one of the cytokines inducing their expression. Adhesion molecule expression was analysed by immunohistochemistry on lung biopsies from patients with CWP and from healthy subjects. In parallel, soluble adhesion molecules were detected in bronchoalveolar lavage fluids (BALF) from patients by specific ELISA. The involvement of TNF in the induction of these adhesion molecules was measured (i) by immunohistochemistry on sections from lung fragments, and (ii) by evaluating in vitro the expression of adhesion molecules on endothelial cells and on alveolar epithelial cells in the presence of alveolar macrophage supernatants. In control subjects, a weak staining of ICAM-1 was detected only in alveolar walls, while E-selectin and VCAM-1 were undetectable. In pneumoconiotic patients, ICAM-1 was expressed at a high level by endothelium, by alveolar and bronchial epithelial cells and by alveolar macrophages. E-selectin and VCAM-1 expression remained undetectable. Measurement of soluble adhesion molecule showed that only the concentration of sICAM-1 was significantly increased in BALF from patients with CWP compared with controls. The involvement of TNF in this ICAM-1 expression was shown by the in vitro effect of alveolar macrophage supernatants on adhesion molecule expresssion by endothelial cells and epithelial cells (this effect was neutralized by anti-TNF antibodies) and by the increased production of TNF in the lung of pneumoconiotic patients. These data provide evidence for the involvement of ICAM-1, induced at least in part by alveolar macrophage-derived TNF, in the development of the inflammatory reaction in CWP.     

Mechanisms Involved in Activation of Human Eosinophil Exocytosis by Substance P: An in Vitro Model of Sensory Neuroimmunomodulation

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10^?6M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10^?7M and 10^?8M primed eosinophils to suboptimal dose (10^?8M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4–11) fragment showed a similar activity while SP(1–4) fragment was not active. SP priming of eosinophils was not affected by Ca²⁺ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induce human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca²⁺ independent pathway.     

Effect of Cholesterol Depletion on Interleukin-8 Production in Human Respiratory Epithelial Cells

Purpose

The lipid entities of cell membranes are components of the immune system and important mediators of inflammation. Despite increasing interest in the function of epithelial cells in inflammation, the role of cholesterol in this process has not been described. Here, we investigated the effect of cholesterol depletion on the inflammatory process in airway epithelial cells via the expression of interleukin (IL)-8 as a marker of inflammation.

Methods

A 549 cells were treated with 0.5% methyl-β-cyclodextrin as a selective cholesterol extractor. The IL-8 level was assessed by enzyme-linked immunosorbent assay and reassessed after cholesterol repletion. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the upstream signaling pathway for IL-8 production in cholesterol-depleted cells.

Results

We found a relationship between the amount of cholesterol in A 549 cells and inflammation of the airway. IL-8 production was increased in cholesterol-depleted A 549 cells and restored by cholesterol repletion. IL-8 production was decreased by pretreatment with the extracellular signal-regulated kinase (ERK) inhibitor U0126 but not with JNK inhibitor II or the p38 MAPK inhibitor SB202190.

Conclusions

Our findings suggest that inflammatory responses are increased in cholesterol-depleted epithelial cells via the MAPK signaling system, predominantly by the ERK pathway. We conclude that the lipid components of airwayepithelial cells may play a role in the inflammatory process.     

Differentiation of Human Monocytes in Vitro Following Exposure to Canova in the Absence of Cytokines

Canova is an immunomodulatory, homeopathic preparation that has been shown to activate macrophages in vitro and in vivo, with resultant enhanced spreading of the cells and formation of microvillus extensions from the cell body. Since monocytes are the precursor cells of macrophages and dendritic cells, the objective of the current study was to investigate the effects of Canova on the differentiation of human blood monocytes in vitro. Monocytes were isolated, grown in culture, and exposed to 10 and 20% Canova without the addition of cytokines. After 48?h, monocytes were prepared for analysis by scanning electron microscopy, while cells kept in culture for 7 days and exposed to Canova on days 1, 3, and 4 were analyzed by flow cytometry for alterations in the levels of expression of CD1a, CD11c, CD14, CD80, CD83, CD86, and HLA-DR. SEM revealed that monocytes exposed to 10% Canova had a morphological appearance similar to that of macrophages. Various cytoplasmic projections were observed with pseudopodia formation. Flow cytometric analysis after exposure of monocytes to 10 and 20% Canova indicated high cell viability and upregulation of CD80, compatible with differentiation into either macrophages or dendritic cells. Exposure to Canova per se causes activation of monocytes with resultant differentiation into large macrophage-like cells of indeterminate phenotype that have increased expression of CD80. Like cytokines, Canova induces differentiation of monocytes, an activity that may underpin the immunomodulatory activity of this product.     

Macrophage Requirement for Production of the Human Mitogenic Factor in Vitro

Highly purified T-cell preparations are not induced by phytohaemagglutinin (PHA) to produce mitogenic factors (MF). As few as 0.1% adherent cells restore PHA induction to MF secretion. Non-adherent, highly purified T-cells, on the other hand, perform independently in producing MF once activated, while adherent cells are not capable of detecting MF secretion.     

Activation process of macrophages after in vitro treatment of mouse lymphocytes with dodecylglycerol.

Alkylglycerols, inflammation products of cancerous membrane lipids, efficiently activate macrophages. A brief in vitro treatment (30 min) of peritoneal cells (mixture of non-adherent and adherent cells) with a small amount (50 ng/ml) of synthetic dodecylglycerol (DDG) resulted in greatly enhanced Fc-receptor-mediated ingestion activity of macrophages. However, treatment of adherent cells (macrophages) alone with DDG produced no significant enhancement of macrophage ingestion activity, implying that macrophage activation requires a contribution of non-adherent cells. DDG-treated non-adherent cells were found to generate a macrophage-activating signal factor. Studies with a serum free-0.1% egg albumin-supplemented RPMI 1640 medium revealed that a serum factor is essential for macrophage activation process. Time course analysis of stepwise transfers of conditioned media of DDG-treated or untreated B cells and T cells revealed that DDG-treated B cells rapidly transmit a factor to untreated T cells which yield the ultimate macrophage-activating factor. This signal transmission among these cells for the macrophage activation process is too rapid to allow time for synthesis of inducible gene products. Thus, we hypothesized that a serum factor is modified by the pre-existing function of DDG-treated B cells and further modified by the pre-existing function of untreated T cells to yield macrophage-activating factor. This hypothesis was confirmed by the demonstration that DDG-treated splenic non-adherent cell ghosts modify a serum factor to yield macrophage-activating factor.