Interleukin-8 production in human adipose tissue. inhibitory effects of anti-diabetic compounds, the thiazolidinedione ciglitazone and the biguanide metformin.

Obesity and Type 2 diabetes are associated with an increased risk of developing cardiovascular disease. Reports have suggested that the chemokine, interleukin-8, may be involved in the development of diabetic macroangiopathy as well as in the pathogenesis of atherosclerosis. Two classes of drugs, the biguanides and the insulin-sensitizing thiazolidinediones seem to have additional beneficial effects on cardiovascular risk-factors besides their effects on glucose homeostasis. In this study, we investigated the effects of the thiazolidinedione, Ciglitazone, the peroxisome proliferator-activated receptor alpha-agonist 5,8,11,14-eicosatetraynoic acid (ETYA) and the biguanide, Metformin on interleukin-8 gene expression and production in human adipose tissue in vitro. Ciglitazone 10-100 M inhibited interleukin-8 release by 25-33% (p < 0.05) and mRNA expression by 33-60% (p < 0.05). Metformin 0.1-10 mM inhibited interleukin-8 release by 20-50% (p < 0.05) and mRNA expression by 20-90% (p < 0.05). However, ETYA did not effect the production of interleukin-8 in the adipose tissue. In conclusion, we demonstrate the ability of two anti-diabetic compounds to decrease the release of interleukin-8 from human adipose tissue in vitro. These findings open the possibility that the beneficial effects on cardiovascular risk-factors of these anti-diabetic compounds might involve a reduction in the interleukin-8 produced in human adipose tissue.     

Interleukin-6 release from human skeletal muscle during exercise: relation to AMPK activity.

We tested the hypothesis that IL-6 release from muscle during exercise may be related to muscle activity of 5'-AMP-activated protein kinase (AMPK). Eight healthy, well-trained young men completed two 60-min trials on a bicycle ergometer at 70% of their peak oxygen uptake in either a glycogen-depleted or a glycogen-loaded state. IL-6 was released from the leg already after 10 min of exercise in the glycogen-depleted state, whereas no significant release was observed at any time in the loaded state. Nevertheless, plasma IL-6 increased similarly in the two trials from approximately 0.8 pg/ml at rest to approximately 4.5 pg/ml after 60 min of exercise. Activity of alpha1-AMPK (160%) and alpha2-AMPK (145%) was increased at rest in the glycogen-depleted compared with the loaded situation. During exercise, alpha1-AMPK activity did not change from resting levels in both trials, whereas alpha2-AMPK activity increased only in the glycogen-depleted state. After 60 min of exercise in the glycogen-depleted state, individual values of alpha2-AMPK activity correlated significantly (r = 0.87, P < 0.006) with individual values of IL-6 release as well as with average IL-6 release over the entire 60 min (r = 0.86, P < 0.006). The present data are compatible with a role for AMPK in IL-6 release during exercise or a role for IL-6 in activating AMPK. Alternatively, both AMPK and IL-6 are independent sensors of a low muscle glycogen concentration during exercise. In addition, leg release of IL-6 cannot alone explain the increase in plasma IL-6 during exercise.     

IL-8 inhibits histamine release from human basophils induced by histamine-releasing factors, connective tissue activating peptide III, and IL-3.

We have previously purified and partially characterized histamine releasing factors (HRF), which were derived from a mixture of human mononuclear cells and platelets. We now report the effect of IL-8 upon HRF-, connective tissue activating peptide III (CTAP III)-, and IL-3-induced histamine release from human basophils. We determined that IL-8 itself, at concentrations between 10(-7) to 10(-11) M, does not release histamine from basophils, although positive results are observed in two of 26 subjects at 10(-7) M. Unfractionated (crude) HRF released histamine in 25 of 26 donors, in the range of 6.7% to 100% of total basophil histamine stores. When basophils were preincubated with IL-8 (10(-7) to 10(-11) M) for 5 min, followed by a 40-min incubation with HRF, histamine release was significantly inhibited in 20 of 25 donors. Inhibition was observed at as little as 10(-11) M IL-8, with maximal inhibition being attained at 10(-9) M. HRF-containing supernatants contain a mixture of different histamine-releasing moieties. To better define which factor(s) may be inhibited by IL-8, fractionated supernatants, purified CTAP III, and IL-3 were studied. Histamine release produced by two different HRF-containing chromatographic fractions (HRFvoid and HRFpeak 2) and purified CTAP-III (5 micrograms/ml) was inhibited by IL-8 in 10 of 12 donors, three of three donors, and seven of 10 donors, respectively. IL-3 (5000 U/ml)-dependent histamine release was inhibited by IL-8 in all subjects tested. In contrast, histamine release by anti-IgE and FMLP was not affected by IL-8. Thus, IL-8 appears to be an inhibitor of cytokine-like molecules that induce histamine release and may represent the previously described 8-kDa histamine release inhibitory factor present in mononuclear cell supernatants.     

Comparison of PGE2, prostacyclin and leptin release by human adipocytes versus explants of adipose tissue in primary culture

The present studies were designed to investigate the sites of PGE(2), prostacyclin and leptin formation in human adipose tissue. Most of the PGE(2) and prostacyclin formation by adipose tissue explants from obese humans after 48 h in primary culture was due to blood vessels and other tissues not digested by collagenase. However, there was appreciable PGE(2) formation by adipocytes over a 48 h incubation and leptin formation was only seen in adipocytes. An increase in COX-2 immunoreactive protein was also seen after incubation of isolated human adipocytes for 48 h. The release of PGE(2) by adipocytes incubated for 48 h was about 4% that by intact adipose tissue explants while the release of prostacyclin was about 1.5% that by tissue. However, in a different experimental design where PGE(2) formation was measured over 2 h in the presence of 20 microM arachidonic acid the formation of PGE(2) by adipocytes after 48 h prior incubation in primary culture was 38% of that by tissue explants. Dexamethasone enhanced leptin release by adipocytes while inhibiting PGE(2) release and COX-2 up-regulation. The mechanisms involved in up-regulation of COX-2 activity during primary culture of adipocytes and the inhibition of this by dexamethasone do not appear to involve p38 MAPK or p42-44 MAPK. Interleukin I(beta) further enhanced PGE(2) formation by adipocytes but did not affect leptin formation. In conclusion, these data indicate that leptin release is exclusively a function of adipocytes while prostanoids are made by both adipocytes and the other cells present in human adipose tissue     

Interleukin-8 receptor-mediated chemotaxis of normal human epidermal cells.

Normal human keratinocytes show chemotactic behavior towards interleukin-8 (IL-8). Under physiological conditions this cytokine seems to be present in an equilibrium between monomeric and dimeric forms, as indicated by Western blotting data. Radioligand binding studies suggest that keratinocyte chemotaxis is mediated by receptors specific for IL-8 dimers. IL-8 receptor-specific mRNA can be detected in a keratinocyte cell line by polymerase chain reaction.     

Interleukin-8, a chemotactic and inflammatory cytokine.

Interleukin-8 (IL-8) belongs to a family of small, structurally related cytokines similar to platelet factor 4. It is produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin-1 or tumor necrosis factor) and activates neutrophils inducing chemotaxis, exocytosis and the respiratory burst. In vivo, IL-8 elicits a massive neutrophil accumulation at the site of injection. Five neutrophil-activating cytokines similar to IL-8 in structure and function have been identified recently. IL-8 and the related cytokines are produced in several tissues upon infection, inflammation, ischemia, trauma etc., and are thought to be the main cause of local neutrophil accumulation.     

Opioids regulate the release of human chorionic gonadotropin hormone from trophoblast tissue.

Opioid ligands were investigated for their effect on hCG release from trophoblast tissue obtained from term human placenta. Data obtained indicate that opiate agonists stimulate in vitro basal hCG release from trophoblast tissue. The potency of these opioid agonists correspond to their kappa receptor selectivity, i.e., the greater the selectivity the lower is the effective concentration causing maximum stimulation. Opioid antagonists inhibit the release of hCG due to their reversal of the stimulation caused by endogenous opioid peptides. Potency of the antagonists correspond also to their kappa receptor selectivity. Antagonists reverse the stimulation of hCG release caused by agonists indicating that the ligand's action is mediated by the placental kappa opioid receptors. The bell shaped response curves for agonists and antagonists suggest that opioids play a role in the regulation of hCG release from trophoblast tissue, but other mechanism(s) may also exist.     

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue.

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandins E (PGE) and F2alpha (PGF2alpha) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF2alpha and the metabolites when incubated with SRS or antigen. In in vivo experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized quinea pigs. These effects, but not the action of injected PGF2alpha and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly "secondary" mediators (in the temporal sense) of the antigen-antibody reaction.     

Comparison of LPS-stimulated release of cytokines in punch versus transwell tissue culture systems of human gestational membranes

Background  

Cytokine signaling within the amnionic, chorionic and decidual extraplacental gestational membranes plays an important role in membrane rupture and the timing of birth. The predominant in vitro explant culture system for evaluating cytokine induction in human gestational membranes has been the free-floating biopsy punch culture. Punch systems have been used to investigate the impact of various toxicants, pharmaceuticals and genetic variation on expression of pro-inflammatory cytokines. More recently, a dual compartment transwell culture system has been developed that more closely mimics the intrauterine compartment. The current study compares these two systems with respect to release of pro- and anti-inflammatory cytokines in response to lipopolysaccharide (LPS), a model stimulant.     

Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants

Spontaneous and potassium-induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium-induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion-dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.     

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.

Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.     

The regulation of prostaglandin biosynthesis in cells derived from human gestational tissues: effects of fetal calf serum.

We have evaluated the production of prostaglandin E2 (PGE2) and its regulation in amnion, chorion, and decidual cells in the presence and absence of fetal calf serum (FCS), and in the absence of FCS but with supplementation with substrate arachidonic acid (AA). Basal rates of PGE2 biosynthesis in amnion, chorion and decidual cell cultures were significantly reduced in the absence of FCS. The magnitudes of the responses to various stimulatory agents were different between cells from different tissues and the different culture media. We conclude that these different experimental conditions must be taken into account when interpreting the results of such in vitro experiments.     

IV. Immunologic mechanisms for release of chemical mediators of anaphylaxis from human lung tissue

The antigen-induced, IgE-dependent release of chemical mediators from human lung tissue in vitro is modulated by a variety of pharmacologic maneuvers involving alterations in the intracellular levels of cyclic nucleotides. Increase in the level of cyclic AMP inhibits the immunologic release of histamine, SRS-A and ECF-A; β-adrenergic agents, prostaglandins, cholera toxin and methylxanthines all produce accumulations of cAMP in human lung tissue. Depletion of cAMP after α-adrenergic, low-dose prostaglandin and imidazole stimulation is associated with enhancement of the release of mediators. Studies involving purified preparations of rat peritoneal mast cells confirm that alterations in the cAMP levels of a homogeneous population of target cells indeed influence histamine release in a fashion analogous to that of human lung tissue.Furthermore, cholinergic stimuli produce a marked enhancement of the antigen-induced release of mediators from human lung through an apparently independent mechanism, presumably acting through alterations in the tissue concentration of cyclic GMP. This latter observation suggests an important interaction between endogenously released parasympathetic neurohormones and the immunologic release of the chemical mediators of asthma.     

Enhanced proliferation of cells from human tissue explants following irradiation in the presence of environmental carcinogens

Summary A method which allows growth of normal human tissue to be studied in vitro is used to investigate possible interactive effects of radiation and environmentally important carcinogens on oesophageal and urothelial cell growth. Carcinogens chosen were selected for their known or suspected effect on the oesophageal mucosa or urothelium in vivo. The results indicate that with carcinogens alone concentrations can be identified that result in increased proliferation of cells. With radiation alone inhibition of cell proliferation occurs at all dose points examined. However, at precise combinations of radiation and carcinogen, greatly enhanced cell proliferation could be detected, suggesting a synergistic interaction between the two agents. The results may have implications for the design and interpretation of experiments aimed at elucidating early or premalignant changes in epithelial tissues and may indicate hitherto unsuspected interactions between radiation and environmentally important carcinogens.