Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10(-8) mol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10(-8) mol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10(-8) mol/l and glucose accumulation at 10(-7) mol/l in HMVEC-Cs. TNF-α dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.

The relative contribution of the pro-inflammatory cytokines tumour necrosis factor (TNF)-alpha and interleukin (IL)-1 beta and the lipopolysaccharide (LPS)-induced pathways that result in endothelial activation during sepsis are not fully understood. We have examined the effects of plasma obtained from LPS-treated human whole blood on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) on human endothelial cells. Stimulation of blood with 10 pg/ml of LPS is sufficient to produce plasma that induces E-selectin and ICAM-1 expression, while direct induction by LPS alone requires a 100-fold higher concentration. Characteristics for the plasma-induced adhesion molecule expression were similar to the LPS-induced production of TNF-alpha and IL-1 beta in blood. A complete inhibition of E-selectin and ICAM-1 expression was observed when antibodies against TNF-alpha and IL-1 beta were added to plasma prior to the incubation to endothelial cultures. Significant inhibition was even observed if antibodies were added to the cultures up until 3 h after LPS-conditioned plasma. The plasma-induced adhesion molecule response could also be prevented with inhibitors of nuclear factor (NF)-kappaB, such as pyrollidine dithiocarbamate. These findings emphasize the central role of TNF-alpha and IL-1 beta in LPS-induced endothelial activation and suggest that simultaneous neutralization of these cytokines or their common pathways may, even after the initial stimulus, prevent endothelial response during sepsis.

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid beta (Abeta) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Abeta in the absence of stimuli or following stimulation with IL-1beta, TNFalpha, IL-6, and TGF-beta1. Neither did combinations of TNFalpha and IL-1beta, IL-6 or TGF-beta1, or the coadministration of IFNgamma and IL-6 or TGF-beta1 induce Abeta production. In contrast, pronounced production of Abeta1-40 and Abeta1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNgamma and TNFalpha or IFNgamma and IL-1beta. Induction of Abeta production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsbeta. Our results suggest that astrocytes may be an important source of Abeta in the presence of certain combinations of inflammatory cytokines. IFNgamma in combination with TNFalpha or IL-1beta seems to trigger Abeta production by supporting beta-secretase cleavage of the immature APP molecule.

Sjögren's syndrome (SS), an idiopathic, autoimmune exocrinopathy, is partly characterized by diminished salivary flow, acinar cell atrophy, and increased expression of several cytokines. Several in vivo characteristics of the sialoadenitis are also evident in a human salivary gland ductal epithelial cell line (HSG) treated with cytokines. HSG cells differentiate to the acinar phenotype when cultured on Matrigel (Becton Dickinson, Bedford, MA), a basement membrane extract. To elucidate mechanisms of salivary gland pathology, the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on cell cycle progression and integrin expression were evaluated in HSG acinarlike cells. Flow cytometry experiments showed that cytokine treatment for 2 days arrested cells in G(1) phase of the cell cycle, and this preceded significant morphologic changes and decreased viability. Whereas only modest cytokine-mediated increases in protein expression for the alpha 3 and beta 1 integrin subunits were seen by immunoprecipitation, a form of alpha 3 integrin displaying enhanced electrophoretic mobility was evident after 6 days of cytokine treatment. To our knowledge, this is the first report demonstrating an IFN-mediated alteration in the electrophoretic mobility of integrin subunits. From this study, it was evident that the combination of IFN-gamma and TNF-alpha resulted in a block in G(1) phase for acinar cells before accumulation of the alpha 3 integrin variant or significant degenerative cellular changes. Information from the present and previous studies suggests that cytokines may alter the pattern of integrin expression and block cell cycle progression in salivary gland cells grown in three-dimensional acinarlike clusters. These experiments may provide a new cell culture model to study the effects of cytokines in normal and diseased salivary glands, including SS.

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.