Pro-inflammatory Cytokine Cocktail Research Articles

Production and function of IL-12 in islets and beta cells

IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ). The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Integration of pro-inflammatory cytokines, 12-lipoxygenase and NOX-1 in pancreatic islet beta cell dysfunction

Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1β, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.

Stable antigen‐specific T‐cell hyporesponsiveness induced by tolerogenic dendritic cells from multiple sclerosis patients

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease of the central nervous system. Current therapies decrease the frequency of relapses and limit, to some extent, but do not prevent disease progression. Hence, new therapeutic approaches that modify the natural course of MSneed to be identified. Tolerance induction to self-antigens using monocyte-derived dendritic cells (MDDCs) is a promising therapeutic strategy in autoimmunity. In this work, we sought to generate and characterize tolerogenic MDDCs (tolDCs) from relapsing-remitting (RR) MSpatients, loaded with myelin peptides as specific antigen, with the aim of developing immunotherapeutics for MS. MDDCs were generated from both healthy-blood donors and RR-MSpatients, and MDDCmaturation was induced with a proinflammatory cytokine cocktail in the absence or presence of 1α,25-dihydroxyvitamin-D(3) , a tolerogenicity-inducing agent. tolDCs were generated from monocytes of RR-MSpatients as efficiently as from monocytes of healthy subjects. The RR-MStolDCs expressed a stable semimature phenotype and an antiinflammatory profile as compared with untreated MDDCs. Importantly, myelin peptide-loaded tolDCs induced stable antigen-specific hyporesponsiveness in myelin-reactive T cells from RR-MS patients. These results suggest that myelin peptide-loaded tolDCs may be a powerful tool for inducing myelin-specific tolerance in RR-MS patients.

Properties of the Weibel-Palade Body Fusion Pore

Weibel-Palade bodies (WPBs) are regulated secretory organelles found in endothelial cells (ECs). The major WPB constituent is the haemostatic protein Von Willebrand factor (VWF), however, under specific conditions WPBs also contain a cocktail of small pro-inflammatory cytokines. WPB exocytosis is driven by an increase in intracellular free calcium ion concentration ([Ca2+]i); the majority of WPB fusion events result in complete discharge of cargo components, however, in a small fraction of cases cytokines are selectively released indicating that the fusion pore may act as a molecular size filter. Carbon fibre amperometry has been used to characterize fusion pore behavior in a number of cell types, but to date this approach has not been applied to WPBs. Therefore, we used this technique in combination with simultaneous optical imaging of fluorescent WPB exocytosis and changes in [Ca2+]i and can report, for the first time, the kinetic properties of WPB fusion pore formation and expansion in human cultured endothelial cells. A clear delay (mean ∼50 ms) is seen between the onset of the current spike and the increase in intra-WPB EGFP fluorescence indicating that WPB alkalinsation is delayed by the strong proton buffering capacity of the WPB lumen (55 mM/pH unit). Analysis of current spike parameters reveal a mean 25-75% rise time, peak amplitude and decay time of 1.63 ms, 50 pA and 6.63 ms respectively. Approximately 50% of current spikes were preceded by a foot signal of mean duration 6.34 ms. Occasional low amplitude, prolonged current increases, reminiscent of stand alone foot signals, were observed in conjunction with morphological rounding of WPBs, possibly reflecting kiss-and-run or lingering kiss fusion events. Following characterization of the WPB fusion pore under control conditions the impact of changing cellular parameters, including cholesterol levels, was also assessed.

Poly I: C-activated dendritic cells that were generated in CellGro for use in cancer immunotherapy trials

BackgroundFor clinical applications, dendritic cells (DCs) need to be generated using GMP-approved reagents. In this study, we tested the characteristics of DCs generated in two clinical grade culture media and activated by three maturation stimuli, Poly I: C, LPS and the mixture of proinflammatory cytokines in order to identify the optimal combination of culture media and activation stimulus for the clinical use.MethodWe tested DCs generation using two GMP-certified culture media, CellGro and RPMI+5% human AB serum and evaluated DCs morphology, viability and capapability to mature. We tested three maturation stimuli, PolyI:C, LPS and the mixture of proinflammatory cytokines consisting of IL-1, IL-6, TNF and prostaglandin E2. We evaluated the capacity of activated DCs to induce antigen-specific T cells and regulatory T lymphocytes.ResultsCell culture in CellGro resulted in a higher yield of immature DCs resulting from increased number of adherent monocytes. DCs that were generated in CellGro and activated using Poly I:C were the most efficient in expanding antigen-specific T cells compared to the DCs that were generated in other media and activated using LPS or the cocktail of proinflammatory cytokines. A comparison of all tested combinations revealed that DCs that were generated in CellGro and activated using Poly I:C induced low numbers of regulatory T cells.ConclusionIn this study, we identified monocyte-derived DCs that were generated in CellGro and activated using Poly I:C as the most potent clinical-grade DCs for the induction of antigen-specific T cells.

L-Arginine is essential for pancreatic β-cell functional integrity, metabolism and defense from inflammatory challenge

In this work, our aim was to determine whether L-arginine (a known insulinotropic amino acid) can promote a shift of β-cell intermediary metabolism favoring glutathione (GSH) and glutathione disulfide (GSSG) antioxidant responses, stimulus-secretion coupling and functional integrity. Clonal BRIN-BD11 β-cells and mouse islets were cultured for 24 h at various L-arginine concentrations (0-1.15 mmol/l) in the absence or presence of a proinflammatory cytokine cocktail (interleukin 1β, tumour necrosis factor α and interferon γ). Cells were assessed for viability, insulin secretion, GSH, GSSG, glutamate, nitric oxide (NO), superoxide, urea, lactate and for the consumption of glucose and glutamine. Protein levels of NO synthase-2, AMP-activated protein kinase (AMPK) and the heat shock protein 72 (HSP72) were also evaluated. We found that L-arginine at 1.15 mmol/l attenuated the loss of β-cell viability observed in the presence of proinflammatory cytokines. L-arginine increased total cellular GSH and glutamate levels but reduced the GSSG/GSH ratio and glutamate release. The amino acid stimulated glucose consumption in the presence of cytokines while also stimulating AMPK phosphorylation and HSP72 expression. Proinflammatory cytokines reduced, by at least 50%, chronic (24 h) insulin secretion, an effect partially attenuated by L-arginine. Acute insulin secretion was robustly stimulated by L-arginine but this effect was abolished in the presence of cytokines. We conclude that L-arginine can stimulate β-cell insulin secretion, antioxidant and protective responses, enabling increased functional integrity of β-cells and islets in the presence of proinflammatory cytokines. Glucose consumption and intermediary metabolism were increased by L-arginine. These results highlight the importance of L-arginine availability for β-cells during inflammatory challenge.

Low CCR7-Mediated Migration of Human Monocyte Derived Dendritic Cells in Response to Human Respiratory Syncytial Virus and Human Metapneumovirus

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses.

Two-step maturation of immature DCs with proinflammatory cytokine cocktail and poly(I:C) enhances migratory and T cell stimulatory capacity

Effective induction of cell-mediated immune responses strongly depends on the ability of dendritic cells (DCs) to produce Th1-polarizing cytokines, migrate to lymph nodes and stimulate T cells through antigen-presenting complex and costimulatory molecules. While various protocols for optimizing DC maturation with single or multiple stimuli mimicking infections or inflammatory milieu have been proposed for the generation of DCs with features desired for clinical application, stepwise maturation of DCs by these multiple stimuli has not been systemically assessed. Among the combinations of several immune-modulating factors with known effects on DC maturation, we found that stepwise DC maturation with cytokine cocktail (TNF-α + IL-6 + IL-1β + PGE 2) followed by poly(I:C) stimulation enhanced the production of IL-12 with strong allostimulatory capacity. While there were no significant differences between DC matured by simultaneous or sequential activation by cytokine cocktail and poly(I:C) in expression of markers and costimulatory molecules of mature DCs, the delivery of inflammatory signal prior to poly(I:C) results in sustained interleukin-12 expression with reduced IL-10 than DC matured by simultaneous stimulation. This sequential stimulation significantly increased migratory capacity in response to CCL21 and CXCL12 compared to DC matured with cytokine cocktail. Furthermore, these DCs retained their responsiveness to CD40L stimulation in secondary IL-12 production and efficiently generated autologous antigen-specific effector T cells as evidenced by ELISPOT assay. Thus, we propose a novel DC maturation protocol in which stimulation of DCs with cytokine cocktail and subsequently with poly(I:C) generates DCs with a high migratory capacity with a preferential Th1 inducing capacity.

Airway smooth muscle: telling the eosinophil how to behave

Airway smooth muscle: telling the eosinophil how to behave

Pre-clinical testing of T cell responses to influenza vaccine/adjuvant combinations in older adults (132.5)

Abstract Objective: The purpose of this experiment was to determine whether inflammatory cytokines or TLR ligands could be combined with influenza vaccine in older adult PBMC to enhance the response to live influenza virus challenge. Methods: PBMC from five older adults (&amp;gt;65 years old) and six young (20-38 years old) adults were obtained at six months post-vaccination and cultured for five days with split-virus vaccine (SVV) and the TLR3 ligand, poly I:C, or a cocktail of pro-inflammatory cytokines (TNF-α, IL-1, IL-6; T/1/6). Stimulated PBMC were then challenged with live influenza virus for 20 hours and supernatants and lysates prepared for assays of IL-1, IL-6, TNF-α in 5-day supernatants, and the IFN-γ:IL-10 ratio and granzyme B activity in 20-hour virus-challenged PBMC. Results: We observed an increased response to SVV in younger compared to older adults, but a dose-response relationship between poly I:C (added to a fixed dose of SVV) and the IFN-γ:IL-10 ratio and granzyme B levels improved the response in older adult PBMC. This response was associated with a dose-response increase in pro-inflammatory cytokine levels in SVV/poly I:C-stimulated PBMC. However, a cocktail of these inflammatory cytokines (T/1/6) added to the PBMC culture suppressed rather than enhanced the T cell response in older adults. Conclusion: Poly I:C combined with influenza vaccine was shown to enhance the T cell response to influenza in aged PBMC by mechanisms that cannot be replicated by the simple addition of inflammatory cytokines to the vaccine.

Lipocalin-2-induced renal regeneration depends on cytokines

This study investigated whether the renal regeneration occurring in the recovery phase of kidney ischemia-reperfusion (I/R) is mediated by endogenously generated lipocalin-2 (Lcn2). A second objective was to examine whether Lcn2-mediated cell effects could be regulated by the inflammatory cytokines in the environment through their action on Lcn2 receptors (Lcn2R and megalin). Male Swiss mice were subjected to 30 min of renal ischemia with a reperfusion period of 24 h (early reperfusion, expected time for maximum inflammation) and 96 h (late reperfusion, expected time for maximum regeneration). Different experimental groups underwent I/R, I/R with iv anti-mouse Lcn2 monoclonal antibody injected during the early/inflammatory or late/recovery phase, and I/R with proinflammatory cytokine cocktail administration (recombinant mouse IL-1beta, TNF-alpha, and IFN-gamma). Compared with control nonischemic mice, the expression of three proliferation markers (stathmin, PCNA, and Ki-67, analyzed by quantitative RT-PCR) increased significantly in the I/R-treated animals. Blockade of Lcn2 by addition of anti-Lcn2 antibody significantly decreased the expression of these three proliferation markers when administered in the late/reparative phase, but had the opposite effect when administered in the early/inflammatory phase. Proinflammatory cytokine cocktail administration reduced the proliferative effects of Lcn2, and repressed Lcn2R and megalin expression. In conclusion, endogenously generated Lcn2 induces renal cell regeneration depending on the inflammatory cytokines in kidney I/R.

Human immature monocyte-derived dendritic cells produce and secrete α-defensins 1–3

Defensins are effector molecules of the innate immunity with a broad antimicrobial spectrum, including HIV. They also link innate and adaptive immunity, displaying chemotactic activity for monocytes, T cells, and dendritic cells (DCs). alpha-Defensins 1-3 are mainly produced by neutrophils, but their production by other leukocyte subsets has also been reported. Herein, we studied whether monocyte-derived DCs (MDDCs), which are regarded as a model for myeloid DCs, produce alpha-defensins 1-3. We found that immature MDDCs (imMDDCs) produce alpha-defensins 1-3 mRNA, but this production is undetectable or barely detectable following 48 h of maturation with the proinflammatory cytokine cocktail (IL-1beta+IL-6+TNF-alpha) or LPS. It is surprising that alpha-defensins 1-3 production was up-regulated when exposed to each one of the proinflammatory cytokines alone, especially IL-1beta. alpha-Defensins 1-3 produced by imMDDCs were mainly secreted peptides. Production and secretion of alpha-defensins 1-3 by imMDDCs can have biological relevance for the antigen processing of pathogens and can contribute to understanding differences in susceptibility to infections, an issue of special interest in the field of HIV infection.

Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line

Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

The ethyl pyruvate analogues, diethyl oxaloproprionate, 2-acetamidoacrylate, and methyl-2-acetamidoacrylate, exhibit anti-inflammatory properties in vivo and/or in vitro

Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. EP has been shown to decrease the expression of various pro-inflammatory mediators, including nitric oxide (NO ), tumor necrosis factor (TNF), cyclooxygenase-2, and interleukin (IL)-6, in a variety of in vitro and in vivo model systems. In an effort to better understand the chemical features that might explain the anti-inflammatory properties of EP, we screened 15 commercially available compounds for cytoprotective or anti-inflammatory effects using two in vitro assay systems: TNF and NO production by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage-like cells and changes in the permeability of Caco-2 human enterocyte-like monolayers stimulated with a cocktail of pro-inflammatory cytokines called cytomix (1000 U/ml IFN-γ plus 10 ng/ml TNF-α plus 1 ng/ml IL-1β). Two compounds, namely diethyl oxaloproprionate (DEOP) and 2-acetamidoacrylate (2AA), demonstrated consistent anti-inflammatory or cytoprotective pharmacological properties in this screening process. Treatment of mice with either of these compounds ameliorated LPS-induced ileal mucosal hyperpermeability to the fluorescent probe, fluorescein isothiocyanate-labeled dextran (average molecular mass 4 kDa), and bacterial translocation to mesenteric lymph nodes. Treatment with either of these compounds also improved survival in mice challenged with a lethal dose of LPS. Finally, in a study that compared 2AA to its methyl ester, we showed that methyl-2-acetamidoacrylate is at least 100-fold more potent than the parent carboxylate as an inhibitor of LPS-induced NO production by RAW 264.7 cells. Collectively, these data are consistent with the view that anti-inflammatory activity is demonstrable for a number of compounds that either incorporate an olefinic linkage conjugated to a carbonyl moiety or are capable of undergoing tautomeric rearrangement to form such a structure. Moreover, our findings suggest that esters with these general characteristics, perhaps because of their greater lipophilicity or electrophilicity, are more potent anti-inflammatory agents than are the parent carboxylates.

Enhancing Effects of CCL19 on Antigen Uptake and Antigen Presenting Ability of Human Mature Dendritic Cells (DCs).

Recently, it has been demonstrated that CCL19 induces not only chemotaxis but also the extension of dendrites and endocytosis by mature DCs derived from mouse splenocytes. There are some reports presenting CCL19-associated inhibition on the generation of cytotoxic T lymphocytes towards allogeneic DCs, but another reports presenting the inhibition of allogeneic response by the antagonist of CCL19. In the present study, we tried to induce CCR7 expression on human monocyte-derived mature DCs using cytokines and examined CCL19 effects on the functions of these mature DCs. In this present study, we demonstrated that CCL19 could promote the ability of endocytosis and allogeneic antigen presentation of monocyte-derived mature DCs. CD14+ cells were separated from human PB-MNCs using anti-CD14 conjugated magnetic microbeads. The cells were seeded in tissue culture dish and cultured in RPMI medium containing GM-CSF and IL-4. On day 4, a pro-inflammatory cytokine cocktail (TNF-α, IL-1α, IL-6 and INF-γ) and PGE2 ware added to this dish as the DC maturation stimuli and the cultures were continued for more one day. Surface phenotypes such as CD1a, CD14, CD80, CD83, CD86, HLA DR and CCR7 were analyzed by flow cytometry. To analyze endocytosis, immature and mature DCs were incubated with FITC-dextran at 37°C and 4°C in the presence or absence of CCL19 for 5 minutes. Cells were washed with ice-cold PBS then incubated for 10 minutes on ice. After washing, the cells were re-suspended in PBS and the fluorescence intensity of the cells was analyzed by flow cytometry. Incubation of cells with the FITC-dextran on ice was used as a background control. Antigen presenting ability was analyzed by 3H-thymidin incorporation assay in the presence or absence of CCL19, in which autologous and allogeneic lymphocytes were used as responder cells. After the culture using TNF-α, IL-1α, IL-6, INF-γ and PGE2 (TIIIP) as maturation stimuli, DCs highly expressed CD83, CD80, CD86 and HLA-DR and moderately expressed CCR7 in almost the same manner of mature DCs cultured with LPS. About the internalization of FITC-dextran in immature DCs, approximately 50% of the cells rapidly became FITC-dextran positive after 5–10 minutes of incubation without the addition of CCL19. Mature TIIIP-induced mature DCs, however, scarcely internalized FITC-dextran at 5–10 minutes; the proportion of FITC+ cells was less than 5% of the total cells. But the addition of CCL19 increased the uptake by TIIIP-induced mature DCs. In the presence of CCL19, the proportion of mature DCs positive for FITC-dextran was about 30% after 5 minutes of incubation. In allogeneic MLC, 3H-thymidin uptake of lymphocytes stimulated with TIIIP-induced mature DCs was definitely increased by CCL19 addition, while there were no effects of CCL19 in immature DCs. We recognized the role of CCL19 in the positive regulation of endocytosis in human mature DCs. In addition to CCL19-associated enhancement of endocytosis of antigens by mature DCs, CCL19 has also been demonstrated to promote the antigen presenting ability of mature DCs. These findings suggested that the regulation of CCR7-CCL19 interaction could be usefully applied in generating efficient DCs with possessing both active phagocytosis and potent antigen presenting ability for DC-based immunotherapy in various disorders.

Evidence for a sustained increase in clonal beta-cell basal intracellular Ca2+ levels after incubation in the presence of newly diagnosed Type-1 diabetic patient sera. Possible role in serum-induced inhibition of insulin secretion.

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).

Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.