B-1 cells temper endotoxemic inflammatory responses

Sepsis syndrome is caused by inappropriate immune activation due to bacteria and bacterial components released during infection. This syndrome is the leading cause of death in intensive care units. Specialized B-lymphocytes located in the peritoneal and pleural cavities are known as B-1 cells. These cells produce IgM and IL-10, both of which are potent regulators of cell-mediated immunity. It has been suggested that B-1 cells modulate the systemic inflammatory response in sepsis. In this study, we conducted in vitro and in vivo experiments in order to investigate a putative role of B-1 cells in a murine model of LPS-induced sepsis. Macrophages and B-1 cells were studied in monocultures and in co-cultures. The B-1 cells produced the anti-inflammatory cytokine IL-10 in response to LPS. In the B-1 cell-macrophage co-cultures, production of proinflammatory mediators (TNF-alpha, IL-6 and nitrite) was lower than in the macrophage monocultures, whereas that of IL-10 was higher in the co-cultures. Co-culture of B-1 IL-10(-/-) cells and macrophages did not reduce the production of the proinflammatory mediators (TNF-alpha, IL-6 and nitrite). After LPS injection, the mortality rate was higher among Balb/Xid mice, which are B-1 cell deficient, than among wild-type mice (65.0% vs. 0.0%). The Balb/Xid mice also presented a proinflammatory profile of TNF-alpha, IL-6 and nitrite, as well as lower levels of IL-10. In the early phase of LPS stimulation, B-1 cells modulate the macrophage inflammatory response, and the main molecular pathway of that modulation is based on IL-10-mediated intracellular signaling. (C) 2010 Elsevier GmbH. All rights reserved.

Gene expression reprogramming protects macrophage from septic-induced cell death

Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance affects inflammatory response. To comprehend potential new mechanisms of immune regulation in endotoxemia, we examined macrophage mRNA expression by macroarray affected by LPS tolerance. LPS tolerance was induced with subcutaneous administration of 1 mg/kg/day of LPS over 5 days. Macrophages were isolated from the spleen and the expression of 1200 genes was quantitatively analyzed by the macroarray technique. The tolerant group displayed relevant changes in the expression of 84 mRNA when compared to naive mice. A functional group of genes related to cell death regulation was identified. PARP-1, caspase 3, FASL and TRAIL genes were confirmed by RT-PCR to present lower expression in tolerant mice. In addition, reduced expression of the pro-inflammatory genes TNF-alpha and IFN-gamma in the tolerant group was demonstrated. Following this, animals were challenged with polymicrobial sepsis. Flow cytometry analysis showed reduced necrosis and apoptosis in macrophages from the tolerant group compared to the naive group. Finally, a survival study showed a significant reduction in mortality in the tolerant group. Thus, in the current study we provide evidence for the selective reprogramming of the gene expression of cell death pathways during LPS tolerance and link these changes to protection from cell death and enhanced survival rates. (C) 2010 Elsevier Ltd. All rights reserved.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Endotoxin tolerance: Selective alterations in gene expression and protection against lymphocyte death

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0 mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6 h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge. (C) 2009 Elsevier GmbH. All rights reserved.

The time course of vasoconstriction and endothelin receptor A expression in pulmonary arterioles of mice continuously exposed to ambient urban levels of air pollution

The present study aimed to verify the time course of the effects of environmental levels of urban air pollution toxicity on lung arterioles. BALB/c mice (n = 56) were continuously exposed to selective chambers equipped with (filtered, F) or without (non-filtered, NF) filter devices for particles and toxic gases for 24 h/day, over 14, 21, 30 or 45 days. After exposure, we evaluated the lumen-wall relationship (an estimator of arteriolar narrowing), endothelial nitric oxide synthase (eNOS) and endothelin type A receptor (ETAr) expression in the vascular wall and inflammatory influx of the peribronchiolar area. Concentrations of fine particulate matter (PM <= 2.5 mu g/m(3)), nitrogen dioxide (NO(2)), black smoke (BS), humidity and temperature in both the environment and inside the chambers were measured daily. Filters cleared 100% of BS and 97% of PM inside the F chamber. The arteriole wall of the lungs of mice from NF chamber had an increased ETAr expression (p <= 0.042) concomitant to a decrease in the lumen/wall ratio (p = 0.02) on the early days of exposure, compared to controls. They also presented a progressive increment of inflammatory influx in the peribronchiolar area during the study (p = 0.04) and decrement of the eNOS expression on the 45th day of exposure in both vascular layers (p <= 0.03). We found that after 14 days of exposure, the ambient levels of air pollutants in Sao Paulo induced vasoconstriction that was associated with an increase in ETAr expression. These vascular results do not appear to be coupled to the progressive inflammatory influx in lung tissue, suggesting a down-regulation of vasoconstrictive mechanisms through an imbalance in the cytokines network. It is likely that these responses are protective measures that decrease tissue damage brought about by continuous exposure to air pollutants. (C) 2010 Elsevier Inc. All rights reserved.

Effects of high-intensity aerobic interval training vs. moderate exercise on hemodynamic, metabolic and neuro-humoral abnormalities of young normotensive women at high familial risk for hypertension

Exercise training has an important role in the prevention and treatment of hypertension, but its effects on the early metabolic and hemodynamic abnormalities observed in normotensive offspring of hypertensive parents (FH+) have not been studied. We compared high-intensity interval (aerobic interval training, AIT) and moderate-intensity continuous exercise training (CMT) with regard to hemodynamic, metabolic and hormonal variables in FH+ subjects. Forty-four healthy FH+ women (25.0+/-4.4 years) randomized to control (ConFH+) or to a three times per week equal-volume AIT (80-90% of VO(2MAX)) or CMT (50-60% of VO(2MAX)) regimen, and 15 healthy women with normotensive parents (ConFH-; 25.3+/-3.1 years) had their hemodynamic, metabolic and hormonal variables analyzed at baseline and after 16 weeks of follow-up. Ambulatorial blood pressure (ABP), glucose and cholesterol levels were similar among all groups, but the FH+ groups showed higher insulin, insulin sensitivity, carotid-femoral pulse wave velocity (PWV), norepinephrine and endothelin-1 (ET-1) levels and lower nitrite/ nitrate (NOx) levels than ConFH- subjects. AIT and CMT were equally effective in improving ABP (P<0.05), insulin and insulin sensitivity (P<0.001); however, AIT was superior in improving cardiorespiratory fitness (15 vs. 8%; P<0.05), PWV (P<0.01), and BP, norepinephrine, ET-1 and NOx response to exercise (P<0.05). Exercise intensity was an important factor in improving cardiorespiratory fitness and reversing hemodynamic, metabolic and hormonal alterations involved in the pathophysiology of hypertension. These findings may have important implications for the exercise training programs used for the prevention of inherited hypertensive disorder. Hypertension Research (2010) 33, 836-843; doi:10.1038/hr.2010.72; published online 7 May 2010

A high-fat meal impairs muscle vasodilatation response to mental stress in humans with Glu27 beta(2)-adrenoceptor polymorphism

Background: Forearm blood flow responses during mental stress are greater in individuals homozygous for the Glu27 allele. A high-fat meal is associated with impaired endothelium-dependent dilatation. We investigated the impact of high-fat ingestion on the muscle vasodilatory responses during mental stress in individuals with the Glu27 allele and those with the Gln27 allele of the beta(2)-adrenoceptor gene. Methods: A total of 162 preselected individuals were genotyped for the Glu27Gln beta(2)-adrenoceptor polymorphism. Twenty-four individuals participated in the study. Fourteen were homozygous for the Gln27 allele (Gln27Gln, 40 +/- 2 years; 64 +/- 2 kg), and 10 were homozygous for the Glu27 allele (Glu27Glu, 40 +/- 3 years; 65 +/- 3 kg). Forearm blood flow was evaluated by venous occlusion plethysmography before and after ingestion of 62 g of fat. Results: The high-fat meal caused no changes in baseline forearm vascular conductance (FVC, 2.2 +/- 0.1 vs. 2.4 +/- 0.2; P = 0.27, respectively), but reduced FVC responses to mental stress (1.5 +/- 0.2 vs. 0.8 +/- 0.2 units; P = 0.04). When volunteers were divided according to their genotypes, baseline FVC was not different between groups (Glu27Glu = 2.4 +/- 0.1 vs. Gln27Gln = 2.1 +/- 0.1 units; P = 0.08), but it was significantly greater in Glu27Glu individuals during mental stress (1.9 +/- 0.4 vs. 1.0 +/- 0.3 units; P = 0.04). High-fat intake eliminated the difference in FVC responses between Glu27Glu and Gln27Gln individuals (FVC, 1.3 +/- 0.4 vs. 1.2 +/- 0.4; P = 0.66, respectively). Conclusion: These findings demonstrate that a high-fat meal impairs muscle vasodilatation responses to mental stress in humans. However, this reduction can be attributed to the presence of the homozygous Glu27 allele of the beta(2)-adrenoceptor gene.