Developmental IL-6 Exposure Favors Production of PDGF-Responsive Multipotential Progenitors at the Expense of Neural Stem Cells and Other Progenitors.

Interleukin-6 (IL-6) is increased in maternal serum and amniotic fluid of children subsequently diagnosed with autism spectrum disorders. However, it is not clear how increased IL-6 alters brain development. Here, we show that IL-6 increases the prevalence of a specific platelet-derived growth factor (PDGF)-responsive multipotent progenitor, with opposite effects on neural stem cells and on subsets of bipotential glial progenitors. Acutely, increasing circulating IL-6 levels 2-fold above baseline in neonatal mice specifically stimulated the proliferation of a PDGF-responsive multipotential progenitor accompanied by increased phosphorylated STAT3, increased Fbxo15 expression, and decreased Dnmt1 and Tlx expression. Fate mapping studies using a Nestin-CreERT2 driver revealed decreased astrogliogenesis in the frontal cortex. IL-6-treated mice were hyposmic; however, olfactory bulb neuronogenesis was unaffected. Altogether, these studies provide important insights into how inflammation alters neural stem cells and progenitors and provide new insights into the molecular and cellular underpinnings of neurodevelopmental disorders associated with maternal infections.

An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations.

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.

Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.

BACKGROUND: Genetically modified mice offer the unique opportunity to gain insight into the pathophysiology of pulmonary arterial hypertension. In mice, right heart catheterization is the only available technique to measure right ventricular systolic pressure (RVSP). However, it is a terminal procedure and does not allow for serial measurements. Our objective was to validate a noninvasive technique to assess RVSP in mice. METHODS AND RESULTS: Right ventricle catheterization and echocardiography (30-MHz transducer) were simultaneously performed in mice with pulmonary hypertension induced acutely by infusion of a thromboxane analogue, U-46619, or chronically by lung-specific overexpression of interleukin-6. Pulmonary acceleration time (PAT) and ejection time (ET) were measured in the parasternal short-axis view by pulsed-wave Doppler of pulmonary artery flow. Infusion of U-46619 acutely increased RVSP, shortened PAT, and decreased PAT/ET. The pulmonary flow pattern changed from symmetrical at baseline to asymmetrical at higher RVSPs. In wild-type and interleukin-6-overexpressing mice, the PAT correlated linearly with RVSP (r(2)=-0.67, P<0.0001), as did PAT/ET (r(2)=-0.76, P<0.0001). Sensitivity and specificity for detecting high RVSP (>32 mm Hg) were 100% (7/7) and 86% (6/7), respectively, for both indices (cutoff values: PAT, <21 ms; PAT/ET, <39%). Intraobserver and interobserver variability of PAT and PAT/ET were <6%. CONCLUSIONS: Right ventricular systolic pressure can be estimated noninvasively in mice. Echocardiography is able to detect acute and chronic increases in RVSP with high sensitivity and specificity as well as to assess the effects of treatment on RVSP. This noninvasive technique may permit the characterization of the evolution of pulmonary arterial hypertension in genetically modified mice.

An assessment of the effects of central interleukin-1beta, -2, -6, and tumor necrosis factor-alpha administration on some behavioural, neurochemical, endocrine and immune parameters in the rat.

Despite a vast amount of research into the actions of cytokines within the central nervous system, the pharmacological role and/or physiological function of the various cytokines within the central nervous system is still not fully understood. The present study evaluated the effects of intracerebroventricular administration of interleukin-1beta, -2, -6 (20 ng) and tumour necrosis factor-alpha (40 ng) on elevated plus maze behaviour, monoamine levels in the hypothalamus, hippocampus and amygdala, plasma corticosterone and catecholamine concentrations and Concanavalin A-induced splenic lymphocyte proliferation in the rat. Both interleukin-1beta and tumour necrosis factor-alpha induced "anxiogenic-like" effects on the elevated plus maze, whereas interleukin-2 and interleukin-6 did not. However only interleukin-1beta led to endocrine variations often associated with stress and anxiety. Cytokine specific alterations in monoamine levels were evident in the hypothalamus and hippocampus, while neurotransmitter concentrations in the amygdala were not significantly altered by cytokine treatment. In addition, interleukin-1beta reduced Concanavalin A-induced lymphocyte proliferation, whereas the other cytokine treatments failed to significantly alter this response. These results demonstrate that in some, but not all, respects interleukin-1beta administration produced "stress like" effects on behaviour, monoamine neurotransmitters, hypothalamic pituitary adrenal axis activity and immune function, while the other cytokines produced less consistent effects on these parameters. It is noteworthy that although interleukin-1beta and tumour necrosis factor-alpha provoked an anxiogenic response in the elevated plus maze test of anxiety, neither cytokine significantly altered amygdaloid noradrenergic or serotonergic activity, as many previous studies have implicated increased amygdaloid noradrenergic and/or serotonergic activity in the pathophysiology of anxiety.

Generation of murine monoclonal antibodies in serum-free medium.

Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells. Serum is also required for subcloning of hybridoma cells to support low density cell growth. IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells. We found that the serum requirement in media used for generation of hybridomas can be totally eliminated by substituting with 300 units/ml of IL-6. Stable hybridoma cell lines were generated to peptide and protein antigens using serum-free adapted P3.653 myelomas as the fusion partner and medium containing IL-6. Our results indicate that, in general, the fusion efficiencies of serum-free IL-6 supplemented fusions are lower than the fusions employing serum containing media (40%-60% vs. 80%-100%). However, in spite of the lower fusion efficiency, the number of antigen-specific clones generated using IL-6 was equal to or greater than fusions using serum supplements. The use of IL-6 instead of serum in the generation of monoclonal antibodies (MAbs) has several advantages. We are able to eliminate the costly need for serum in media by using IL-6 that is prepared in house. In addition, we eliminate the need for time-consuming serum-free adaptation of hybridoma cell lines prior to transfer to hollow fiber bioreactors.

Dose effects of recombinant human interleukin-6 on pituitary hormone secretion and energy expenditure.

Interleukin-6 (IL-6), the main circulating cytokine, is putatively a major mediator of the effects of the immune system on several endocrine axes and intermediate metabolism. We performed dose-response studies of recombinant human IL-6 on pituitary hormone secretion in 15 healthy male volunteers, using 5 single, escalating subcutaneous doses of IL-6 (0.1, 0.3, 1.0, 3.0 and 10.0 micrograms/kg body weight), each in 3 volunteers. We measured resting metabolic rate (RMR) with indirect calorimetry and plasma anterior pituitary hormones and vasopressin (AVP) at baseline and half-hourly over 4 h after the injection. All doses examined were tolerated well and produced no significant adverse effects. Dose-dependent RMR increases were observed in response to the 3.0- and 10.0-microgram/kg doses of IL-6, beginning at 60 min and slowly peaking between 180 and 240 min. Plasma adrenocorticotropic-hormone concentrations increased dramatically and dose-dependently in all the patients who received the 3.0- and 10.0-microgram/kg doses of IL-6, respectively, peaking to 150 and 255 pg/ml at 60 min, and slowly returning to normal by 4 h. Corresponding plasma cortisol levels peaked dose-dependently between 90 and 150 min, but remained elevated throughout the sampling period. In contrast, the growth hormone (GH) dose-response was bell-shaped, with maximum (approximately 100-fold) stimulation achieved by 3.0 micrograms/kg IL-6. Prolactin (PRL) showed a similar but less pronounced response pattern. Thyroid-stimulating hormone (TSH) dose-dependently and progressively decreased over the 240 min, while gonadotropins showed no clear-cut changes. In conclusion, subcutaneous IL-6 administration induced synchronized dose-dependent increases in the RMR and hypothalamic-pituitary-adrenal axis activity, suggesting that hypothalamic corticotropin-releasing hormone may mediate both of these functions in humans. IL-6 also acutely stimulated GH and PRL secretion and suppressed TSH secretion. The dose of 3.0 micrograms/kg could be used safely in the study of patients with disturbances of the hypothalamic-pituitary unit or of thermogenesis.

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines.

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1 beta or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5-107.6 ng/10(6) cells/24h) in different MC lines. IL-1 beta up-regulated the synthesis by 2.4-4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1.76 ng/h/10(6) cells under IL-1 stimulation versus basal rate of 0.37 ng/h/10(6) cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.

Biological function of recombinant IL-6 expressed in a baculovirus system.

The cDNA of human interleukin-6 (IL-6) was cloned into baculovirus DNA. Insect cells (Spodoptera frugiperda cells) infected with the recombinant baculovirus secreted a large amount of 22K protein into the culture medium. This culture fluid contained high biological activity of growth stimulation of a mouse myeloid cell line (MH-60). The IL-6 was purified by a one-step procedure employing immunoaffinity chromatography of monoclonal antibody to IL-6. The specific activities (BSF-2 reference units/mg protein) of the original culture medium and the purified material from the one-step purification were 0.3-1 x 10(6) and greater than 2 x 10(7), respectively. The highly purified IL-6 could not induce an antiviral state in human diploid fibroblast (FS-4) cells.