Induction of nitric oxide (NO) synthesis in murine macrophages requires potassium channel activity

The activation of macrophages for antimicrobial responses is a multistage event involving numerous intracellular signalling cascades that makes possible target cell destruction by these effector cells. This study examined the effects of different potassium channel inhibitors and activators on the NO production of murine macrophage-like cell lines P388D.1 and B10-4(S). We found that the potassium channel inhibitors tetraethylammonium, 4-aminopyridine, and quinine caused dose-dependent reductions in the NO production of macrophages, and that the potassium channel activator, minoxidol, caused a dose-dependent enhancement of NO production. The inhibition of NO production was due to involvement of potassium channels in the priming stage of macrophage activation, since pretreatment with the priming agent interferon-gamma partially restored the NO response of the macrophages. The results of this study demonstrate a link between potassium channel activity and the activation of anitimicrobial functions of murine macrophages.     

Inhibition of Polyclonally Induced Immunoglobulin Secretion by Aurothiomalate

The influence of sodium aurothiomalate on the secretion of immunoglobulins by normal human lymphocytes in vitro was investigated by means of a reverse hemolytic plaque forming cell (PFC assay, Aurothiomalate inhibited the FFC response induced by pokeweed mitogen (PWM) and by Epstein-Barr virus (EBV) in a dose dependent manner. The inhibition was irreversible, as pre-incubation for 2 h with the drug followed by extensive washing and further culture in gold salt–free medium still caused an inhibition of the PFC response to PWM and to EBV. Cell proliferation was not significantly affected, suggesting that the inhibition of PFC formation was not due to cytotoxicity. Pre–incubation of monocytes/macrophages (Mø's), T lymphocytes and B lymphocytes with the gold compound prior to culture with PWM showed that M0's and B cells were highly sensitive, whereas T lymphocytes where resistant to the drug. The findings indicate that aurothio-malate inhibits the polyclonally induced PFC response by interfering with accessory Me function and by affecting the B lymphocyte itself.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase–wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus and peri-, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5^? B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human μ chain antibody (a-μ-Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5⁺ B cells and subsequently fractionated on Percoll density gradients. While resting CD5⁺ B cells proliferated and produced IgM molecules in response to a-μ-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5^? B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5⁺ B cells had the typical phenotype of mantle zone B cells (IgM⁺ IgD⁺ CD39+ CD38^? CD10^? CDw75^dim), whereas resting CD5^? B cells were CD38^? CD39^? CD 10^? CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38⁺ CD10⁺ CD39^? CDw75^bright, IgG⁺) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5^? B cells. However, some of the data collected suggest possible relationships between CD5^? B cells and germinal center cells. The CD5^? B cells isolated from the 50 % Percoll fraction proliferated in response to a-μ-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5⁺ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5⁺ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5^? B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5^? B cells did not express CD5. The CD5^? B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro. The present study underlines the phenotypic and functional heterogeneity of CD5^? B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Changes in isometric force- and relaxation-time,electromyographic and muscle fibre characteristics of human skeletal muscle during strength training and detraining

HÄKKINEN, K., ALÉN, M. & KOMI, P.V. 1985. Changes in isometric force- and relaxation-time, electromyographic and muscle fibre characteristics of human skeletal muscle during strength training and detraining. Acta Physiol Scand 125, 573–585. Received 26 January 1985, accepted 9 May 1985. ISSN 0001–6772. Department of Biology of Physical Activity and Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland. Eleven male subjects (20–32 years) accustomed to strength training went through progressive, high-load strength training for 24 weeks with intensities ranging variably between 70 and 120% during each month. This training was also followed by a 12-week detraining period. An increase of 26.8% (P < 0.001) in maximal isometric strength took place during the training. The increase in strength correlated (P < 0.05) with significant (P < 0.05–0.01) increases in the neural activation (IEMG) of the leg extensor muscles during the most intensive training months. During the lower-intensity training, maximum IEMG decreased (P < 0.05). Enlargements of muscle-fibre areas, especially of fast-twitch type (P < 0.001), took place during the first 12 weeks of training. No hypertrophic changes were noted during the latter half of training. After initial improvements (P < 0.05) no changes or even slight worsening were noted in selected force-time parameters during later strength training. During detraining a great (P < 0.01) decrease in maximal strength was correlated (P < 0.05) with the decrease (P < 0.05) in the maximum IEMGs of the leg extensors. This period resulted also in decreases (P < 0.05) of the mean muscle-fibre areas of both fibre types. It was concluded that improvement in strength may be accounted for by neural factors during the course of very intensive strength training. Selective training-induced hypertrophy also contributed to strength development but muscle hypertrophy may have some limitations during long-lasting strength training, especially in highly trained subjects.     

Avoidance conditioning in rats following changes in activity of the noradrenergic and cholinergic systems of the brain

Bilateral coagulation of the ventral noradrenergic pathways of the brain in male rats of the Wistar strain disrupted the conditioned passive avoidance response after unconditioned stimulation with a current of 0.75 mA. Injecting 10 mg/kg galantamine into these rats 20 min before training and increasing the unconditioned stimulus to 3 mA improved subsequent avoidance responses. Injecting galantamine under analogous conditions into normal rats impaired this reaction. Disruption of the conditioned avoidance response following the operation may be due to a change in the intracentral interrelation of the noradrenergic and cholinergic systems of the brain.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 68, No. 3, pp. 297–302, March, 1982.