The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.     

Robust growth hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH)

Aging is associated with a blunted growth hormone (GH) secretory response to GH-releasing hormone (GHRH), in vivo. The objective of the present study was to assess the effects of aging on the GH secretory response to GH-releasing hexapeptide (GHRP-6), a synthetic GH secretagogue. GHRP-6 (30 micrograms/kg) was administered alone or in combination with GHRH (2 micrograms/kg) to anesthetized female Fischer 344 rats, 3 or 19 months of age. The peptides were co-administered to determine the effect of aging upon the potentiating effect of GHRP-6 on GHRH activity. The increase in plasma GH as a function of time following administration of GHRP-6 was lower (p less than 0.001) in old rats than in young rats; whereas the increase in plasma GH secretion as a function of time following co-administration of GHRP-6 and GHRH was higher (p less than 0.001) in old rats than in young rats (mean Cmax = 8539 +/- 790.6 micrograms/l vs. 2970 +/- 866 micrograms/l, respectively; p less than 0.01). Since pituitary GH concentrations in old rats were lower than in young rats (257.0 +/- 59.8 micrograms/mg wet wt. vs. 639.7 +/- 149.2 micrograms/mg wet wt., respectively; p less than 0.03), the results suggested that GH functional reserve in old female rats was not linked to pituitary GH concentration. The differential responses of old rats to individually administered and co-administered GHRP-6 are important because they demonstrate that robust and immediate GH secretion can occur in old rats that are appropriately stimulated. The data further suggest that the cellular processes subserving GH secretion are intact in old rats, and that age-related decrements in GH secretion result from inadequate stimulation, rather than to maladaptive changes in the mechanism of GH release.     

Serum level of soluble interleukin-2 receptor alpha correlates with the clinical course and activity of Wilms' tumour and soft tissue sarcomas in children.

Wilms' tumour (WT) and soft tissue sarcomas (SA) in children lack reliable biochemical markers. This study was carried out to determine the clinical significance of serum soluble interleukin-2 receptor alpha (sIL-2Ralpha) in the diagnostics and treatment monitoring of children with WT and SA. The study included 48 children: ten with WT, eight with SA and 30 healthy controls. The sIL-2Ralpha levels (ELISA) and rates of elevated sIL-2Ralpha values were estimated prospectively at diagnosis and in complete remission during treatment and after therapy. As the dependence on age was determined, the levels of sIL-2Ralpha were expressed as multiplications of the upper value of the normal range for a particular age ( xN). Median pretreatment levels of sIL-2Ralpha in patients exceeded those of healthy controls (1.79 xN for WT and 1.53 for SA vs. 0.61 for controls; p < 0.001) as did the rates of elevated sIL-2Ralpha values (80% of WTand 87.5% of SA patients vs. 0% of controls). Good response to therapy was paralleled by a significant decline of pretreatment sIL-2Ralpha levels and its elevated rates. Thus, sIL-2Ralpha determination may be of some value in the diagnostics and treatment monitoring of childhood WT and SA.     

Differences in expression, affinity, and function of soluble (s)IL-4Ralpha and sIL-13Ralpha2 suggest opposite effects on allergic responses

IL-4 and IL-13 are each bound by soluble receptors (sRs) that block their activity. Both of these sRs (sIL-4Ralpha and sIL-13Ralpha2) are present in low nanogram per milliliter concentrations in the serum from unstimulated mice, but differences in affinity and half-life suggest differences in function. Serum IL-4/sIL-4Ralpha complexes rapidly dissociate, releasing active IL-4, whereas sIL-13Ralpha2 and IL-13 form a stable complex that has a considerably longer half-life than uncomplexed IL-13, sIL-13Ralpha2, IL-4, or sIL-4Ralpha. Approximately 25% of sIL-13Ralpha2 in serum is complexed to IL-13; this percentage and the absolute quantity of sIL-13Ralpha2 in serum increase considerably during a Th2 response. sIL-13Ralpha2 gene expression is up-regulated by both IL-4 and IL-13; the effect of IL-4 is totally IL-4Ralpha-dependent while the effect of IL-13 is partially IL-4Ralpha-independent. Inhalation of an IL-13/sIL-13Ralpha2 complex does not affect the expression of IL-13-inducible genes but increases the expression of two genes, Vnn1 and Pira-1, whose products activate APCs and promote neutrophilic inflammation. These observations suggest that sIL-4Ralpha predominantly sustains, increases, and diffuses the effects of IL-4, whereas sIL-13Ralpha2 limits the direct effects of IL-13 to the site of IL-13 production and forms a stable complex with IL-13 that may modify the quality and intensity of an allergic inflammatory response.     

Effects of acute intravenous injection of two growth hormone-releasing hormones (GHRH 1-40 and 1-29) on serum growth hormone and other pituitary hormones in short children with pulsatile growth hormone secretion

We administered two different growth hormone-releasing hormones (GHRH) to 20 short, prepubertal children who had spontaneous secretion of growth hormone (GH), assessed from 24-hour GH secretion profiles (72 sampling periods of 20 min). We compared one i.v. injection of 1 microgram/kg of GHRH 1-40 with that of GHRH 1-29 regarding serum concentrations of GH, prolactin, luteinizing hormone, follicle-stimulating hormone and IGF-I. The children were allocated to two groups without statistical randomization. Both groups were given both peptides, with at least 1 week in between. The first group started with GHRH 1-40, the other with GHRH 1-29. The peptides both induced an increased serum concentration of GH of the same magnitude: mean maximal peak of 89 +/- 12 mU/l after GHRH 1-40 and 94 +/- 10 mU/l after GHRH 1-29 (n.s.). The mean difference in maximum serum GH concentration in each child after injection was 52 +/- 9 mU/l, range 1-153 mU/l. GHRH 1-29 also induced a short-term, small increase in the concentrations of prolactin (p less than 0.05), luteinizing hormone (p less than 0.01) and follicle-stimulating hormone (p less than 0.05). We conclude that the shorter sequence GHRH 1-29, when given in a dose of 1 microgram/kg, gives a rise in serum concentration of GH similar to that after the native form GHRH 1-40.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

Glucocorticoid sensitivity of interleukin-1 agonist and antagonist secretion: the effects of age and gender

Interleukin-1 (IL-1) is a primary mediator of inflammation that is regulated, in part, by the hypothalamic-pituitary-adrenal axis. The purpose of this study was to determine if gender- or age-related differences exist in the sensitivity of IL-1-producing cells to hydrocortisone. Peripheral blood mononuclear cells (PBMC) isolated from men and women (21-77 yr old) were incubated with hydrocortisone (0, 50, 100, 500, or 1,000 ng/ml) with or without lipopolysaccharide (LPS). Secretion of IL-1beta and IL-1 receptor antagonist was inhibited in a dose-dependent manner (P = 0.001) without age- or gender-related differences. Hydrocortisone decreased soluble IL-1 receptor type II (sIL-1RII) secretion by unstimulated cells (P = 0. 0001), but it increased secretion by LPS-stimulated cells (P = 0. 0001) in all groups. Unstimulated cell supernatants from men contained greater concentrations of sIL-1RII than the supernatants from women (P = 0.011). Compared with men, PBMCs from women were less responsive to hydrocortisone inhibition of sIL-1RII secretion, regardless of age (P = 0.001), and compared with the follicular phase, sIL-1RII secretion was lower in the luteal phase of the menstrual cycle (P < 0.05). These data indicate that basal secretion and glucocorticoid modulation of sIL-1RII secretion by cultured PBMCs are gender dependent. Moreover, glucocorticoid influences on sIL-1RII secretion depend on the presence or absence of gram-negative bacterial toxins.     

Neuropeptide Y cotransmission with norepinephrine in the sympathetic nerve-macrophage interplay

The CNS modulates immune cells by direct synaptic-like contacts in the brain and at peripheral sites, such as lymphoid organs. To study the nerve-macrophage communication, a superfusion method was used to investigate cotransmission of neuropeptide Y (NPY) with norepinephrine (NE), with interleukin (IL)-6 secretion used as the macrophage read-out parameter. Spleen tissue slices spontaneously released NE, NPY, and IL-6 leading to a superfusate concentration at 3-4 h of 1 nM:, 10 pM:, and 120 pg/ml, respectively. Under these conditions, NPY dose-dependently inhibited IL-6 secretion with a maximum effect at 10(-10) M: (p = 0.012) and 10(-9) M: (p < 0.001). Simultaneous addition of NPY at 10(-9) M: and the alpha-2-adrenergic agonist p-aminoclonidine further inhibited IL-6 secretion (p < 0.05). However, simultaneous administration of NPY at 10(-9) M: and the beta-adrenergic agonist isoproterenol at 10(-6) M: or NE at 10(-6) M: significantly increased IL-6 secretion (p < 0.005). To objectify these differential effects of NPY, electrical field stimulation of spleen slices was applied to release endogenous NPY and NE. Electrical field stimulation markedly reduced IL-6 secretion, which was attenuated by the NPY Y1 receptor antagonist BIBP 3226 (10(-7) M, p = 0.039; 10(-8) M, p = 0.035). This indicates that NPY increases the inhibitory effect of endogenous NE, which is mediated at low NE concentrations via alpha-adrenoceptors. Blockade of alpha-adrenoceptors attenuated electrically induced inhibition of IL-6 secretion (p < 0.001), which was dose-dependently abrogated by BIBP 3226. This indicates that under blockade of alpha-adrenoceptors endogenous NPY supports the stimulating effect of endogenous NE via beta-adrenoceptors. These experiments demonstrate the ambiguity of NPY, which functions as a cotransmitter of NE in the nerve-macrophage interplay.     

Soluble Interleukin IL-15Ralpha is generated by alternative splicing or proteolytic cleavage and forms functional complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.     

Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble interleukin-2 receptor (sIL-2R) concentrations in peripheral blood as markers of pituitary tumours

Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and soluble interleukin-2 receptor (sIL-2R) are important cytokines. They are secreted by normal pituitary glands and those with all types of adenomas and may be involved in pituitary tissue growth. The peripheral blood concentrations of VEGF, bFGF and sIL-2R in nineteen patients (17-70 years) with pituitary tumours and ten healthy subjects (23-34 years) were studied. Hypersecretion of prolactin (five cases), human growth hormone (four cases), and thyroid stimulating hormone (one case) was recorded in some patients, and the remaining subjects were diagnosed as having nonfunctional pituitary tumours. Increased peripheral blood plasma levels of VEGF (310.82 +/- 59.17 pg/ml) compared with controls (40.32 +/- 11.80 pg/ml; p < 0.01), as well as bFGF (87.27 +/- 7.58 pg/ml) versus controls (11.14 +/- 2.43 pg/ml; p < 0.001) were recorded. The levels of sIL-2R did not differ between the pituitary tumour patients (4,490.58 +/- 581.50 pg/ml) and control subjects (3,617.01 +/- 1,397.18 pg/ml; p > 0.05). The concentrations of VEGF and bFGF in the peripheral blood are useful additional markers of the presence of tumours.     

Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17)

This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.     

Effects of IL-6 and its soluble receptor on proteoglycan synthesis and NO release by human articular chondrocytes: comparison with IL-1. Modulation by dexamethasone.

Contradictory results have been reported on the effects and role of IL-6 on proteoglycan (PG) synthesis. Having shown recently that in vitro IL-6 depends on the presence of soluble IL-6 receptor alpha (sIL-6Ralpha) to fully exert its effects on chondrocytes, we conducted the present study to analyse the effects of IL-6 on PG synthesis by human articular chondrocytes in the presence of sIL-6Ralpha. PG synthesis was quantified by specific ELISA using a monoclonal antibody (MAB) raised against the keratan sulphate region of PG as a capture antibody, and a MAB to the acid binding region as a detector. It proved specific for PG from primary (differentiated) chondrocytes. In the absence of sIL-6Ralpha, IL-6 had a slight inhibitory effect on PG synthesis by articular chondrocytes. sIL-6Ralpha alone also had slight but consistent inhibitory effects. When adding sIL-6Ralpha at concentrations of 50 ng/ml corresponding to levels found in synovial fluid, the effects of IL-6 increased consistently. However, even at optimal concentrations (30-100 ng/ml of IL-6sR per 100 ng/ml of IL-6), maximal inhibition (48%) did not equal the degree of inhibition achieved by IL-1 at 1 ng/ml (66%). Similar effects, although slightly weaker, were observed on osteoarthritic cells. Dexamethasone, over a wide range of concentrations, markedly enhanced proteoglycan synthesis and completely reversed the downregulatory effects of IL-1 and IL-6 + sIL-6Ralpha. The effects of IL-1 were partially inhibited by an anti-IL-6 antibody. Finally, unlike IL-1, IL-6 + sIL-6Ralpha only weakly stimulated nitric oxide (NO) synthesis. In conclusion, sIL-6Ralpha potentiates the inhibitory effect of IL-6 on PG synthesis by articular chondrocytes, but the overall effect of IL-6 + IL-6sR is moderate compared to the effects of IL-1.     

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.     

Comparative effect of clonidine and growth hormone (GH)-releasing hormone on GH secretion in adult patients on chronic glucocorticoid therapy.

Glucocorticoids are thought to inhibit growth hormone (GH) secretion through an enhancement of endogenous somatostatin tone. The aim of our study was to evaluate the effects of GH-releasing hormone (GHRH) and clonidine, an alpha-2-adrenergic agonist which increases GH secretion acting at the hypothalamic level with an unknown mechanism, on GH secretion in seven adult patients (3M, 4F) with non endocrine diseases and on daily immunosuppressive glucocorticoid therapy. Eleven normal subjects (7M, 4F) served as controls. Steroid-treated patients showed a blunted GH response to GHRH (GH peak 8.3 +/- 3 micrograms/L) with respect to normal subjects (GH peak 19.3 +/- 2.4 micrograms/L). The GH responses to clonidine were also blunted (p less than 0.05) in steroid-treated patients (GH peak 5.8 +/- 2.8 micrograms/L) with respect to normal subjects (GH peak 17.6 +/- 2.3 micrograms/L). No significant differences between the GH responses to GHRH and clonidine were observed either in steroid-treated or in normal subjects. Clonidine is not able to enhance GH secretion similar to GHRH in patients chronically treated with steroids. It can be hypothesized that clonidine does not elicit GH secretion decreasing hypothalamic somatostatin tone.     

Influence of chronic treatment with the growth hormone secretagogue Ipamorelin,in young female rats: somatotroph response in vitro

Growth hormone (GH) is secreted in the anterior pituitary gland by the somatotroph cells. Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the composition of the somatotroph cell population and on somatotroph GH content, an in vitro analysis was performed of the percentage of somatotroph cells (% of total), the ratio of different GH cell types (strongly/weakly-staining) and individual GH content, in pituitary cell cultures obtained from young female rats receiving Ipamorelin over 21 days (Ipamorelin group) and the effects were compared with those of GHRH (GHRH group) or saline (saline group). The ultrastructure of somatotroph cells did not change, but the volume density of secretion granules was increased (P<0.05) by previous in vivo Ipamorelin or GHRH treatment. In 3-day basal pituitary cell monolayer cultures, the percentage of somatotroph cells showed no modifications between groups, nor was there any change in the ratio of strongly/weakly immunostaining GH cells. In the Ipamorelin group alone, in vitro treatment with Ipamorelin (10(-8) M), or GHRP 6 (10(-8) M), or GHRH (10(-8) M) for 4 hours, increased the percentage of somatotroph cells, without modifying the ratio of strongly/weakly immunostained GH cells. Basal intracellular GH content in somatotroph cells over 4 hours was lower in the Ipamorelin group and the GHRH group than in the saline group. Only in the Ipamorelin group did Ipamorelin (10(-8) M), GHRP 6 (10(-8) M) and GHRH (10(-8) M) prompt increased intracellular GH content. These data suggest that, at least in the young female rat, the GHS Ipamorelin is able to exert a dynamic control effect on the somatotroph population and on GH hormone content.     

Effect of soluble interleukin-6 receptor alpha and interleukin-6 secreted by polymorphonuclear leukocytes on tumor necrosis factor-alpha expression and its production by peripheral blood mononuclear cells

BACKGROUND: It has recently been shown that soluble interleukin-6 receptor (sIL-6R) alone or complexed with interleukin (IL)-6, besides their regulatory role in a wide variety of both normal and abnormal biologic reactions mediated by IL-6, could be an effective stimulator of the cell function. AIMS: The key question of the present study is whether the sIL-6Ralpha or sIL-6R with IL-6 released by polymorphonuclear leukocytes (PMN) can influence cytokine secretion such as tumor necrosis factor-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMC), which together with PMN develop the inflammatory and immune response of a host. METHODS: Cells were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 1 h at 37 degrees C in 5% CO(2). After incubation, the culture supernatant of PMN was removed and was added to PBMC. The PBMC were cultured for 1 h at 37 degrees C in the same conditions. In the culture supernatants and lysates of PMN, we examined the concentrations of sIL-6R by enzyme-linked immunosorbent assay (ELISA). TNF-alpha was measured at both protein and mRNA levels. Protein levels were determined by ELISA. To examine TNF-alpha mRNA expression, we isolated mRNA from PBMC after culture, using TRIZOL Reagent. The quantity of mRNA TNF-alpha was determined by the Quantikine mRNA assay. RESULTS AND CONCLUSION: The results obtained revealed that sIL-6R with IL-6 secreted by PMN may play a regulatory role in the immune response by modulating the TNF-alpha expression and its production by PBMC. This may have a significant influence on an early phase of the inflammation and other reactions mediated by TNF-alpha.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

Insulin-like growth factor-I feedback regulation of growth hormone and luteinizing hormone secretion in the pig: evidence for a pituitary site of action

Three experiments (EXP) were conducted to determine the role of insulin-like growth factor-I (IGF-I) in the control of growth hormone (GH) and LH secretion. In EXP I, prepuberal gilts, 65 ± 6 kg body weight and 140 days of age received intracerebroventricular (ICV) injections of saline (n = 4), 25 μg (n = 4) or 75 μg (n = 4) IGF-I and jugular blood samples were collected. In EXP II, anterior pituitary cells in culture collected from 150-day-old prepuberal gilts (n = 6) were challenged with 0.1, 10 or 1000 nM [Ala15]-h growth hormone-releasing hormone-(1-29)NH2 (GHRH), or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 1000 nM GHRH. Secreted GH was measured at 4 and 24 h after treatment. In EXP III, anterior pituitary cells in culture collected from 150-day-old barrows (n = 5) were challenged with 10, 100 or 1000 nM gonadotropin-releasing hormone (GnRH) or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 100 nM GnRH. Secreted LH was measured at 4 h after treatment. In EXP I, serum GH and LH concentrations were unaffected by ICV IGF-I treatment. In EXP II, relative to control all doses of GHRH increased (P < 0.01) GH secretion. Only 1, 10, 30 nM IGF-I enhanced (P < 0.02) basal GH secretion at 4 h, whereas by 24 h all doses except for 30 nM IGF-I suppressed (P < 0.02) basal GH secretion compared to control wells. All doses of IGF-I in combination with 1000 nM GHRH increased (P < 0.04) the GH response to GHRH compared to GHRH alone at 4 h, whereas by 24 h all doses of IGF-I suppressed (P < 0.04) the GH response to GHRH. In EXP III, all doses of IGF-I increased (P < 0.01) basal LH levels while the LH response to GnRH was unaffected by IGF-I (P > 0.1). In conclusion, under these experimental conditions the results suggest that the pituitary is the putative site for IGF-I modulation of GH and LH secretion. Further examination of the role of IGF-I on GH and LH secretion is needed to understand the inhibitory and stimulatory action of IGF-I on GH and LH secretion.     

In vitro effects of genistein and resveratrol on the production of interferon-gamma (IFNgamma) and interleukin-10 (IL-10) by stimulated murine splenocytes.

Phytoestrogens are a group of plant-derived biologically active substances with a chemical structure that resembles that of 17beta-estradiol (E2). As the presence of estrogen receptors (ER) has been identified in several immune cells, phytoestrogens may also have a great impact on the immune system. The aim of our study was to determine the in vitro effects of genistein and resveratrol on the production of interferon-gamma (IFNgamma) and interleukin-10 (IL-10) by stimulated murine splenocytes and compare them with the effects of natural E2. Phorbol 12-miristate 13-acetate (PMA) together with ionomycin was used to stimulate the cells. E2 and genistein did not show any significant effects on the stimulated production of IFNgamma. Resveratrol had a mild inhibitory effect on IFNgamma production at the concentration of 10(-7)M; however, this difference did not reach statistical significance (p>0.05). IL-10 levels in the splenocytes culture supernatants were found to be increased in the presence of E2, genistein and resveratrol; however, these effects were also not statistically significant. To determine whether the exposure to our studied phytoestrogens induced a shift in the T-helper 1/T-helper 2 (Th1/Th2) balance, we calculated the ratio between the production of IFNgamma, the prototypic Th1 cytokine, and the production of IL-10, the prototypic Th2 cytokine, at different concentrations of our tested substances. Genistein at the concentrations of 10(-6) and 10(-7)M and resveratrol at the concentrations of 10(-6)M decreased significantly the IFNgamma/IL-10 ratio. This decrease was comparable to that of E2 at the concentrations of 10(-7)M. From our in vitro experiments we conclude that genistein and resveratrol, similarly to E2, by decreasing the IFNgamma/IL10 ratio may shift the Th1/Th2 balance towards the Th2 response.     

Relationship between circulating interleukin-10 (IL-10) with interleukin-6 (IL-6) type cytokines (IL-6, interleukin-11 (IL-11), oncostatin M (OSM)) and soluble interleukin-6 (IL-6) receptor (sIL-6R) in patients with multiple myeloma

We investigated the serum concentration of the interleukin-10 (IL-10), along with cytokines of interleukin-6 (IL-6) family (IL-6, IL-11 and oncostatin M - OSM), as well as soluble receptor for IL-6 (sIL-6R), in 121 patients with multiple myeloma (MM) and 28 healthy subjects. We studied the interactions between IL-10 and other cytokines, and the receptor. The correlation between IL-10 and some clinical and laboratory parameters associated with the disease activity were also analysed. The IL-10 was detectable in all patients with multiple myeloma and in all controls. The IL-10 concentration was significantly increased in myeloma patients compared with healthy persons (mean - 7.09 and 2.1 pg/ml, respectively) (p = 0.008). The level of IL-10 correlated positively with the advanced stage of disease estimated according to the Salmon and Durie classification (I versus III stage - p = 0.03). Higher values of IL-10 were found in patients with the light chain disease, hypercalcaemia, and correlated with the elevated concentrations of C-reactive protein (CRP). IL-6 was detected in 117 of the 121 patients and in all controls. The concentration of IL-6 was statistically increased in MM patients compared with control group (mean - 16.06 and 4.49 pg/ml, respectively) (p = 0.01). We found a positive correlation between IL-10 and IL-6 serum levels in MM patients. The relationship, expressed as Spearman's rank sum coefficient (rho = 0.249, p = 0.006) was significant. IL-11 was detected in 26 of the 121 MM patients and in 3 of the 28 healthy subjects at the mean concentration of 1.2 and 0.6 pg/ml respectively (p > 0.05). OSM was at detectable levels in 51 of the 121 patients and in only 4 of the 28 controls (mean - 3.84 and 0.1 pg/ml, p = 0. 002). The correlation between IL-10 and IL-11 levels in MM patients was not significant, but there was a strong statistical correlation between IL-10 and OSM concentrations (rho= 0.327, p = 0.0002). The serum concentration of sIL-6R was measurable in all patients and all controls (mean - 66.00 and 39.57 ng/ml respectively), but the difference between these groups was not significant. We found significant, positive correlation between the levels of IL-10 and sIL-6R (rho= 0.233, p = 0.01). In conclusion, we state that the serum concentrations of IL-10, IL-6, OSM and sIL-6R in MM patients may be a useful markers for the evaluation of the disease activity.