Effects of exposure of mice to hindlimb unloading on leukocyte subsets and sympathetic nervous system activity.

The hindlimb unloading (HU) rodent model was developed to simulate some of the aspects of spaceflight conditions. Our previous studies showed that exposure to HU for 48 h (h) followed by bacterial challenge, reduces the ability of mice to resist infection. The purpose of this study was to investigate the physiological changes in mice during the 48 h of exposure to HU to understand the mechanisms involved in the increased susceptibility to infection observed in mice subjected to these conditions. Female Swiss Webster mice were hindlimb-unloaded during 48 h. Blood samples, spleen and peritoneal cells were removed before and after 18 or 48 h of HU-exposure. Leukocyte subset analysis was performed in spleen and peritoneal cells by flow cytometry, and catecholamine levels were measured in plasma and whole spleen by a catecholamine enzyme immunoassay. Catecholamine levels measured in plasma and spleen were significantly greater in mice exposed to HU compared to control. This increase coincided with significant reductions in spleen size in the HU group. Flow cytometric analyses showed a significant reduction of splenic CD19 + B-cells and NK1.1+ cells in mice exposed to HU with a concomitant increase in T-cells. These results suggest that exposure to HU increases the activity of the sympathetic nervous system (SNS) and induces lymphocyte sub-population changes that may contribute to the deregulation of immunity seen in mice exposed to HU and, more importantly may predispose the otherwise healthy host to the subsequent reduced ability to resist infections.

Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection.

BACKGROUND: Infection is a serious, costly, and common complication of surgery and constitutes the principal cause of late death in patients undergoing surgery. The objective of this study was to clarify the mechanisms by which active hexose correlated compound (AHCC) increases survival in a murine model of intramuscular infection. METHODS: Food-deprived mice receiving either AHCC or excipient were infected with bacteria. Kinetics of bacterial load, white blood cell counts, cytokine levels, and antibody levels were compared between groups. RESULTS: AHCC-treated mice had reduced bacterial load at day 5 and cleared bacteria entirely at day 6. Levels of interleukin-12, tumor necrosis factor-alpha, and interleukin-6 peaked earlier in this group (day 3) compared with controls (day 5). Increased percentages of peripheral lymphocytes and monocytes and decreased numbers of polymorphonuclear cells were detected in the AHCC group. CONCLUSIONS: AHCC appears to induce an early activation of the immune response, leading to an effective clearance of bacteria and rapid recovery.

Kinetics of systemic cytokine and brain chemokine gene expression in murine toxoplasma infection.

Toxoplasma gondii often migrates to the central nervous system in immunocompromised patients, where it induces a severe inflammation referred to as Toxoplasma encephalitis. The mechanisms involved in control of parasite multiplication and prevention of Toxoplasma encephalitis remain unclear. The objective of the present study was to characterize the inflammatory response in the brains of mice during acute T. gondii infection, with emphasis on the expression of chemokine receptors. Susceptible C57BL/6 mice were orally infected with 10 cysts of the low-virulent ME49 strain of T. gondii. Levels of cytokines (TNF-alpha, IFN-gamma, IL-10, IL-6, and IL-12p70) and chemokines (CCL/2MCP-1) were measured in plasma at 5, 10, 15, 20, and 30 days after infection. In addition, the mRNA expression of chemokines (CCL5/RANTES, CCL2/MCP-1, CCL4/MIP-1beta) and chemokine receptors (CCR1, CCR2, CCR5, CCR7, CCR8, CXCR4, and CXR5) were measured in brain tissues at the same time points. Plasma levels of IFN-gamma and CCL2/MCP-1 were highly expressed at day 5, whereas TNF-alpha had a moderate increase at day 5, peaked at day 10, and returned to normal levels by day 30. Plasma levels of IL-10, IL-6, and IL-12p70 were not detected throughout the study. Analyses of mRNA expression of chemokines and chemokine receptors in the brain showed that CCL5/ RANTES, CCR7, CXCR4, and CXCR5 were upregulated, peaking after 10 days of T. gondii infection. IgM-specific antibody levels increased at day 5 and peaked at days 10 and 30, whereas IgG levels increased at day 10 and continued to increase thereafter, reaching maximum levels at day 30 postinfection (PI). Our results suggest that T. gondii infection is controlled at local and systemic levels, and that proinflammatory proteins and their receptors may be acting coordinately to induce stage conversion and prevent parasite multiplication and development of Toxoplasma encephalitis. The early production of IFN-gamma and the delayed expression of CXCR4 and CXCR5 indicate that T. gondii induces an early robust cellular immune response, followed by a strong and sustained antibody-mediated immunity.

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats.

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.

Elastase promotes aortic dilation by inhibiting Ca2+ influx into vascular smooth muscle.

Abdominal aortic aneurysm (AAA) is a common vascular disease with, as of yet, unclear mechanism. Increased elastase activity and elastin degradation in the aorta are consistent findings in human AAA. Also, elastase perfusion of the aorta promotes aortic dilation in animal models of AAA. Although elastase-induced degradation of extracellular matrix proteins and the ensuing inflammation of the aortic wall have been implicated as possible causes of the aortic dilation in AAA, little is known regarding the effects of elastase on the mechanisms of aortic smooth muscle contraction. The purpose of this study was to test the hypothesis that elastase promotes aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats non-treated or treated with elastase. Initial experiments suggested that elastase alone caused matrix degradation. To avoid potential degradation of the extracellular matrix proteins by elastase, the same experiments were repeated in the presence of saturating concentrations of elastin (10 mg/ml). In normal Krebs (2.5 mM Ca2+), phenylephrine (Phe, 10(-5) M) caused contraction of the aortic strips that was significantly inhibited by elastase. The elastase-induced inhibition of Phe contraction was concentration- and time-dependent. At 5 U/ml elastase, the inhibition of Phe contraction was rapid in onset (2.4 +/- 0.3 minutes) and complete in 32 +/- 4 minutes. The inhibitory effects of elastase on Phe contraction were partially reversible. In Ca2+-free (2 mM EGTA) Krebs, Phe caused a small contraction that was not inhibited by elastase, suggesting that elastase does not inhibit Ca2+ release from the intracellular stores. Membrane depolarization by 96 mM KCl, which stimulates Ca2+ entry from the extracellular space, caused a contraction that was inhibited by elastase in a time-dependent and reversible fashion. The reversible inhibitory effects of elastase, particularly in the presence of saturating concentrations of elastin, suggest that they are not due to dissolution of the extracellular matrix or permanent damage to the smooth muscle contractile proteins. Elastase also caused significant inhibition of Phe- and KCl-induced 45Ca2+ influx. These data suggest that elastase promotes aortic relaxation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction, and thus further explain the role of increased elastase activity during the early development of AAA.

Gender, sex hormones, and vascular tone.

The greater incidence of hypertension and coronary artery disease in men and postmenopausal women compared with premenopausal women has been related, in part, to gender differences in vascular tone and possible vascular protective effects of the female sex hormones estrogen and progesterone. However, vascular effects of the male sex hormone testosterone have also been suggested. Estrogen, progesterone, and testosterone receptors have been identified in blood vessels of human and other mammals and have been localized in the plasmalemma, cytosol, and nuclear compartments of various vascular cells, including the endothelium and the smooth muscle. The interaction of sex hormones with cytosolic/nuclear receptors triggers long-term genomic effects that could stimulate endothelial cell growth while inhibiting smooth muscle proliferation. Activation of plasmalemmal sex hormone receptors may trigger acute nongenomic responses that could stimulate endothelium-dependent mechanisms of vascular relaxation such as the nitric oxide-cGMP, prostacyclin-cAMP, and hyperpolarization pathways. Additional endothelium-independent effects of sex hormones may involve inhibition of the signaling mechanisms of vascular smooth muscle contraction such as intracellular Ca2+ concentration and protein kinase C. The sex hormone-induced stimulation of the endothelium-dependent mechanisms of vascular relaxation and inhibition of the mechanisms of vascular smooth muscle contraction may contribute to the gender differences in vascular tone and may represent potential beneficial vascular effects of hormone replacement therapy during natural and surgically induced deficiencies of gonadal hormones.

Reduced endothelial NO-cGMP-mediated vascular relaxation and hypertension in IL-6-infused pregnant rats.

Placental ischemia during pregnancy is associated with increased plasma cytokines such as interleukin-6 (IL-6), which may contribute to increased vascular resistance and hypertension of pregnancy. We tested the hypothesis that an increase in plasma IL-6 during pregnancy is associated with impaired endothelium-dependent relaxation, enhanced vascular contraction, and hypertension. Systolic blood pressure was measured in virgin and pregnant Sprague-Dawley rats non-treated or infused with IL-6 (200 ng/kg per day for 5 days). Isometric contraction was measured in isolated aortic strips, and endothelial nitric oxide (NO) synthase (eNOS) was measured in aortic homogenate using Western blots. Blood pressure was greater in IL-6-infused (146+/-3) than in control pregnant rats (117+/-2 mm Hg). In endothelium-intact vascular strips, phenylephrine (Phe) caused greater increase in active stress in IL-6-infused (maximum: 10.6+/-0.6) than in control pregnant rats (maximum: 4.1+/-0.3x10(4) N/m2). Acetylcholine (ACh)-induced relaxation of Phe contraction and vascular eNOS protein and nitrite/nitrate production were less in IL-6-infused than in control pregnant rats. N(omega)-nitro-L-arginine methyl ester (10(-4) mol/L), inhibitor of NOS, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (10(-5) mol/L), inhibitor of cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in control but not IL-6-infused pregnant rats. Endothelium removal enhanced Phe-induced stress in control but not in IL-6-infused pregnant rats. The blood pressure and vascular Phe-induced contraction, ACh relaxation, and eNOS protein were not different between control and IL-6-infused virgin rats. Thus, an endothelium-dependent NO-cGMP-mediated relaxation pathway is inhibited in systemic vessels of pregnant rats infused with IL-6. The results support a role for IL-6 as a possible mediator of the increased vascular resistance during hypertension of pregnancy.

Elastase-induced suppression of endothelin-mediated Ca2+ entry mechanisms of vascular contraction.

Abdominal aortic aneurysm (AAA) is associated with increased endothelin (ET-1), both systemically and locally in the aorta. Also, elastase activity is increased in human AAA, and elastase perfusion of the aorta induces aneurysm formation in animal models of AAA. However, whether elastase directly affects the ET-1-induced mechanisms of aortic smooth muscle contraction is unclear. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats and treated with elastase (5 U/mL). To avoid degradation of the extracellular matrix proteins by elastase, experiments were performed in the presence of elastin (10 mg/mL). In normal Krebs solution (2.5 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused contraction of aortic strips that was inhibited by elastase (5 U/mL). The elastase-induced inhibition of ET-1 contraction was slow in onset (4.6+/-0.4 minutes), time-dependent, complete in 34+/-3 minutes, and reversible. In Ca2+-free Krebs solution, caffeine (25 mmol/L) caused a small contraction that was not inhibited by elastase, suggesting that elastase does not inhibit Ca2+ release from the intracellular stores. Membrane depolarization by 96 mmol/L KCl, which stimulates Ca2+ entry from the extracellular space, caused a contraction that was inhibited by elastase in a concentration-dependent, time-dependent, and reversible fashion. The reversible inhibitory effects of elastase, particularly in the presence of elastin, suggest that they are not due to dissolution of the extracellular matrix or smooth muscle contractile proteins. Elastase also inhibited ET-1 and KCl-induced 45Ca2+ influx. Thus, elastase directly inhibits ET-1-induced Ca2+ entry mechanisms of vascular smooth muscle contraction, which may explain the role of elastase and ET-1 during the development of AAA.