TNF-α and Temporal Changes in Sleep Architecture in Mice Exposed to Sleep Fragmentation

TNF-α plays critical roles in host-defense, sleep-wake regulation, and the pathogenesis of various disorders. Increases in the concentration of circulating TNF-α after either sleep deprivation or sleep fragmentation (SF) appear to underlie excessive daytime sleepiness in patients with sleep apnea (OSA). Following baseline recordings, mice were subjected to 15 days of SF (daily for 12 h/day from 07.00 h to 19.00 h), and sleep parameters were recorded on days1, 7 and 15. Sleep architecture and sleep propensity were assessed in both C57BL/6J and in TNF-α double receptor KO mice (TNFR KO). To further confirm the role of TNF-α, we also assessed the effect of treatment with a TNF- α neutralizing antibody in C57BL/6J mice. SF was not associated with major changes in global sleep architecture in C57BL/6J and TNFR KO mice. TNFR KO mice showed higher baseline SWS delta power. Further, following 15 days of SF, mice injected with TNF-α neutralizing antibody and TNFR KO mice showed increased EEG SWS activity. However, SWS latency, indicative of increased propensity to sleep, was only decreased in C57BL/6J, and was unaffected in TNFR KO mice as well as in C57BL/6J mice exposed to SF but treated with TNF-α neutralizing antibody. Taken together, our findings show that the excessive sleepiness incurred by recurrent arousals during sleep may be due to activation of TNF-alpha-dependent inflammatory pathways, despite the presence of preserved sleep duration and global sleep architecture.     

TNF regulates thymocyte production by apoptosis and proliferation of the triple negative (CD3-CD4-CD8-) subset

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3(-)CD4(-)CD8(-) triple negative (TN) subset within a broad range of dosages (10(1)-10(5) pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44(-)CD25(+)) and TN4 (CD44(-)CD25(-)) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (10(4)-10(5) pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin alpha and beta triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.     

Diurnal variation of heart rate, locomotor activity, and body temperature in interleukin-1 alpha/beta doubly deficient mice.

This study was investigated the roles of interleukin-1 (IL-1) on diurnal rhythms of heart rate (HR), locomotor activity (LA), and body temperature (BT). For this purpose, HR, LA, and BT were recorded from conscious and unrestrained IL-1 alpha/beta doubly deficient (KO) and normal C57BL/6J mice using a telemetry system. These parameters were continuously recorded from just after to 2 weeks after transmitter implantation, because we thought that the surgical stress-induced IL-1 might affect the biobehavioral activities of the animals. At 1 day after implantation, HR and LA in IL-1 alpha/beta KO mice were higher than those in C57BL/6J mice. While BT in IL-1 alpha/beta KO mice was lower than that in C57BL/6J mice. Moreover, diurnal rhythmicity in these parameters after implantation in IL-1 alpha/beta KO mice appeared earlier than in C57BL/6J mice. At 2 weeks after implantation, there were no significant differences in the light- and dark-phase values of each parameter between IL-1 alpha/beta KO and C57BL/6J mice, however, IL-1 alpha/beta KO mice showed clear ultradian rhythmicity. It is thought that a phenotypical difference in biobehavioral activities between IL-1 alpha/beta KO and C57BL/6J mice may reflect IL-1 induced febrile and behavioral responses. These results suggest that IL-1 may play important physiological and pathophysiological roles on biobehavioral activities.     

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex? multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.     

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.     

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.     

Identification of a locus on distal mouse chromosome 12 that controls resistance to tumor necrosis factor-induced lethal shock

Administration of recombinant murine tumor necrosis factor (TNF) to mice results in lethal shock, characterized by hypotension, hypothermia, and dramatic induction of cytokines released in the circulation, such as interleukin-6 (IL-6). The sensitivity of mice to the effects of murine TNF varies from strain to strain. DBA/2 mice were found to be considerably more resistant to TNF than C57BL/6 mice. The resistance proved to be dominant since (C57BL/6 x DBA/2)F1 mice were also resistant. Using BXD recombinant inbred mice and a dose of TNF lethal for C57BL/6 but not for DBA/2 mice, we found that the resistance to TNF links to loci coding for corticosteroid-binding globulin (Cbg), alpha1-protease inhibitor (Spi1), contrapsin (Spi2) and the contrapsin-regulating gene Spi2r that form a gene cluster on chromosome 12. Quantitative trait-loci analysis of TNF-induced induction of IL-6 and of hypothermia also points to the importance of this locus (P < 0.0002 and P = 0.017, respectively), more particularly the Cbg and Spi2 loci, in the resistance to TNF. We propose to name the locus "TNF protection locus." The data suggest that endogenous protease inhibitors and/or glucocorticoids play a significant role in the attenuation of TNF-induced lethal shock. This study also demonstrates that loci affecting important biological responses can be identified with very high resolution using recombinant inbred mice.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

LPS-induced serum TNF production and lethality in mice: effect of L-carnitine and some acyl-derivatives

The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.     

A quantitative trait loci analysis to map genes involved in lipopolysaccharide-induced inflammatory response: identification of macrophage scavenger receptor 1 as a candidate gene

Septic shock, which is a major complication observed after trauma and other human diseases, is likely the product of a prolonged and poorly controlled systemic inflammatory response. Symptoms of sepsis can be partially reproduced by injection of bacterial LPS in mice. Differences in mortality between C57BL/6J(high) and A/J(low) mice after LPS injection have been previously observed and correlated with differences in the inflammatory response between these two inbred strains. In the present study, we have mapped four loci responsible for differences in levels of LPS-induced IL-10, named modifier of IL-10, between the two strains. A locus within mouse chromosome 8 was confirmed using chromosome 8 consomic mice. This locus was further reduced in size by haplotype analysis and evaluated by the presence of potential candidate genes. The macrophage scavenger receptor 1 (Msr1) within this locus emerged as a candidate gene based on differences at the expression and structural levels between C57BL/6J and A/J mice. In comparison with wild-type (C57BL/6J) mice, Msr1 knockout mice displayed reduced levels of LPS-induced IL-10, but not of TNF-alpha or IL-6, confirming a specific role for this gene in the regulation of IL-10. These results suggest that Msr1 is involved in the regulation of the anti-inflammatory process, thus offering a new perspective on the molecular mechanisms involved in endotoxemia and sepsis.     

Modulation of endotoxin-induced NF-kappa B activation in lung and liver through TNF type 1 and IL-1 receptors

We investigated the requirement for tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 receptors in the pathogenesis of the pulmonary and hepatic responses to Escherichia coli lipopolysaccharide (LPS) by studying wild-type mice and mice deficient in TNF type 1 receptor [TNFR1 knockout (KO)] or both TNF type 1 and IL-1 receptors (TNFR1/IL-1R KO). In lung tissue, NF-kappaB activation was similar among the groups after exposure to aerosolized LPS. After intraperitoneal injection of LPS, NF-kappaB activation in liver was attenuated in TNFR1 KO mice and further diminished in TNFR1/IL-1R KO mice; however, in lung tissue, no impairment in NF-kappaB activation was found in TNFR1 KO mice and only a modest decrease was found in TNFR1/IL-1R KO mice. Lung concentrations of KC and macrophage-inflammatory peptide 2 were lower in TNFR1 KO and TNFR1/IL-1R KO mice after aerosolized and intraperitoneal LPS. We conclude that LPS-induced NF-kappaB activation in liver is mediated through TNF-alpha- and IL-1 receptor-dependent pathways, but, in the lung, LPS-induced NF-kappaB activation is largely independent of these receptors.     

Contact hypersensitivity responses following ribavirin treatment in vivo are influenced by type 1 cytokine polarization, regulation of IL-10 expression, and costimulatory signaling.

We previously described the promotion of type 1 cytokine responses by the nucleoside analogue, ribavirin, in human T cells in vitro. In this study, we examined whether type 1 cytokine polarization by ribavirin in vivo could promote contact hypersensitivity (CHS) responses to dinitrofluorobenzene, a type 1 cytokine-mediated immune response. Unexpectedly, although type 1 cytokine responses were enhanced following ribavirin treatment in vitro and in vivo, the magnitude of CHS responses in BALB/c and C57BL/6 mice was influenced more by a second ribavirin-regulated pathway. The key regulatory molecule in this pathway was IL-10. Ribavirin-mediated suppression of IL-10 in BALB/c mice was associated with increased B7-2 expression and enhanced CHS responses, whereas enhanced IL-10 levels, following ribavirin administration, led to increased B7-1 expression and impaired CHS responses in C57BL/6 mice. The effect of ribavirin on the expression of B7 molecules and on CHS responses was neutralized by IL-10 administration in BALB/c and by anti-IL-10 Ab in C57BL/6. Thus, ribavirin controlled CHS responses directly through the modulation of IL-10 expression, and in vivo outcome was dictated by the preferential expression of either B7-1, an inappropriate costimulatory molecule in CHS, or B7-2, the predominant costimulatory molecule in CHS. Replacing dinitrofluorobenzene priming with IFN-alpha stimulation, we showed that the ribavirin-regulated pathway could function independent of Ag priming. Altogether, these data showed that, although ribavirin treatment induced a type 1 cytokine bias in contact allergen-primed BALB/c and C57BL/6 mice, in vivo CHS responses were dependent on ribavirin-mediated regulation of both IL-10 and preferential costimulatory signaling.     

IL-6 is essential for development of gut barrier dysfunction after hemorrhagic shock and resuscitation in mice

We sought to determine the role of IL-6 as a mediator of the alterations in gut barrier function that occur after hemorrhagic shock and resuscitation (HS/R). C57Bl/6 wild-type (WT) and IL-6 knockout (KO) mice on a C57Bl/6 background were subjected to either a sham procedure or HS/R. Organ and tissue samples were obtained 4 h after resuscitation. In WT mice, HS/R significantly increased ileal mucosal permeability to fluorescein isothiocyanate-labeled dextran (average molecular mass, 4 kDa) and bacterial translocation to mesenteric lymph nodes. These alterations in gut barrier function were not observed in IL-6 KO animals. HS/R increased ileal steady-state mRNA levels for IL-6, TNF, and IL-10 in WT but not in IL-6 KO mice. Ileal mucosal expression of the tight junction protein, ZO-1, decreased after HS/R in WT but not IL-6 KO mice. Collectively, these data support the view that expression of IL-6 is essential for the development of gut barrier dysfunction after HS/R.     

Electroencephalographic changes with age in male mice.

Electroencephalographic (EEG) changes, as measured by the awake state, slow-wave sleep (SWS), rapid-eye movement (REM) patterns and ratio of REM/total sleep, were recorded in aging male mice of DBA/2J and C57BL/6J strains. Results indicate that there is a significant increase in the awake state accompanied by significant decrease in SWS with advancing age for both strains, although these changes appear more pronounced in DBA/2J mice than C57BL/6J mice. Of considerable significance is the finding that REM sleep is absent in mice of DBA/2J strain at 23.5 months of age. Based on these findings, the conclusion was reached that strain DBA/2J ages significantly faster than C57BL/6J. The difference in aging between the two strains emphasizes the need for additional studies dealing with genetic aspects of aging.     

Tumour necrosis factor receptor deficiency alters anxiety-like behavioural and neuroendocrine stress responses of mice

Tumour necrosis factor (TNF)-alpha is known to be involved in anxiety and the regulation of the hypothalamic-pituitary-adrenal axis. To examine the role of its receptors in neuroendocrine immunomodulation, we studied behaviour, corticosterone production and T-cell activation in mice with a C57BL/6J background and deficient for one or both TNF receptors (TNFR1-/-, TNFR2-/-, and TNFR1+2-/-) compared to wildtype C57BL/6J mice with and without psychological stress. Stress was induced by social disruption (SDR), and anxiety-like behaviour was examined using the elevated plus maze (EPM). Anxiety of unstressed TNFR1+2-/- mice was increased compared to C57BL/6J mice as shown by reduced ratios of entries into open arms relatively to total entries. SDR-stressed TNFR1+2-/- mice showed reduced ratios of entries into open arms relatively to total entries, reduced ratios of distances walked in open relatively to distances walked in both arms and reduced time in open arms compared to C57BL/6J mice. Locomotor activity of unstressed and SDR-stressed TNFR1-/- and TNFR2-/- mice was reduced. Serum corticosterone concentrations of control mice do not differ between mouse strains. However, TNFR1+2-/- mice had significantly higher corticosterone concentrations than C57BL/6J mice after SDR. EPM testing significantly increased corticosterone concentrations in all strains. Mitogen-induced activation-marker expression was reduced in TNFR1-/- T-helper cells under control and stress conditions, while activation marker expression of TNFR2-/- and TNFR1+2-/- cells was only slightly affected by stress compared to C57BL/6J T cells. Our study suggests that both TNF receptors contribute to anxiety-like behaviour and corticosterone responses, whereas TNFR1 has a larger impact on T-cell activation.     

Tumor necrosis factor-α receptor type 1, not type 2, mediates its acute responses in the kidney

Acute administration of tumor necrosis factor-α (TNF-α) resulted in decreases in renal blood flow (RBF) and glomerular filtration rate (GFR) but induced diuretic and natriuretic responses in mice. To define the receptor subtypes involved in these renal responses, experiments were conducted to assess the responses to human recombinant TNF-α (0.3 ng·min(-1)·g body wt(-1) iv infusion for 75 min) in gene knockout (KO) mice for TNF-α receptor type 1 (TNFαR1 KO, n = 5) or type 2 (TNFαR2 KO, n = 6), and the results were compared with those obtained in corresponding wild-type [WT (C57BL/6), n = 6] mice. Basal levels of RBF (PAH clearance) and GFR (inulin clearance) were similar in TNFαR1 KO, but were lower in TNFαR2 KO, than WT mice. TNF-α infusion in WT mice decreased RBF and GFR but caused a natriuretic response, as reported previously. In TNFαR1 KO mice, TNF-α infusion failed to cause such vasoconstrictor or natriuretic responses; rather, there was an increase in RBF and a decrease in renal vascular resistance. Similar responses were also observed with infusion of murine recombinant TNF-α in TNFαR1 KO mice (n = 5). However, TNF-α infusion in TNFαR2 KO mice caused changes in renal parameters qualitatively similar to those observed in WT mice. Immunohistochemical analysis in kidney slices from WT mice demonstrated that while both receptor types were generally located in the renal vascular and tubular cells, only TNFαR1 was located in vascular smooth muscle cells. There was an increase in TNFαR1 immunoreactivity in TNFαR2 KO mice, and vice versa, compared with WT mice. Collectively, these functional and immunohistological findings in the present study demonstrate that the activation of TNFαR1, not TNFαR2, is mainly involved in mediating the acute renal vasoconstrictor and natriuretic actions of TNF-α.     

TLR4 signaling attenuates ongoing allergic inflammation

The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Role of IL-6 in Exercise Training- and Cold-Induced UCP1 Expression in Subcutaneous White Adipose Tissue

Expression of brown adipose tissue (BAT) associated proteins like uncoupling protein 1 (UCP1) in inguinal WAT (iWAT) has been suggested to alter iWAT metabolism. The aim of this study was to investigate the role of interleukin-6 (IL-6) in exercise training and cold exposure-induced iWAT UCP1 expression. The effect of daily intraperitoneal injections of IL-6 (3 ng/g) in C57BL/6 mice for 7 days on iWAT UCP1 expression was examined. In addition, the expression of UCP1 in iWAT was determined in response to 3 days of cold exposure (4°C) and 5 weeks of exercise training in wild type (WT) and whole body IL-6 knockout (KO) mice. Repeated injections of IL-6 in C57BL/6 mice increased UCP1 mRNA but not UCP1 protein content in iWAT. Cold exposure increased iWAT UCP1 mRNA content similarly in IL-6 KO and WT mice, while exercise training increased iWAT UCP1 mRNA in WT mice but not in IL-6 KO mice. Additionally, a cold exposure-induced increase in iWAT UCP1 protein content was blunted in IL-6 KO mice, while UCP1 protein content in iWAT was lower in both untrained and exercise trained IL-6 KO mice than in WT mice. In conclusion, repeated daily increases in plasma IL-6 can increase iWAT UCP1 mRNA content and IL-6 is required for an exercise training-induced increase in iWAT UCP1 mRNA content. In addition IL-6 is required for a full induction of UCP1 protein expression in response to cold exposure and influences the UCP1 protein content iWAT of both untrained and exercise trained animals.