Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-alpha and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca(2+), both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca(2+) increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-alpha, but not arachidonate, released Ca(2+) from endoplasmic stores and increased mitochondrial Ca(2+). Furthermore, Ca(2+) depletion abrogated TNF-alpha responses partially, but arachidonate responses completely. These differences in Ca(2+) mobilization by TNF-alpha and arachidonate were reflected in spatial patterning in the capillary in that the TNF-alpha effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-alpha- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca(2+) mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-alpha revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.     

Effect of lactate on FFA and glycerol turnover in resting and exercising dogs.

The rates of appearance of FFA (RaFFA) and that of glycerol (RaGLY) were measured simultaneously with [1-14 C]palmitate and [2-3H]-glycerol, in dogs with indwelling arterial and venous catheters. Lipolysis was stimulated by exercise (treadmill run on 10% slope) or by the infusion of norepinephrine (0.5 mug/kg-min). Na-L(+)-lactate (L), Na-pyruvate (Py), or Na-nicotinate (N) were infused. All three components decreased RaFFA. RaGLY was increased by L, unaltered by Py, and decreased by N. There was an inverse correlation (P less than 0.001) between the logarithms of RaFFA and plasma lactate. A linear correlation was obtained between RaGLY and plasma lactate when this latter was increased by the infusion of L. It is suggested that a) lactate in physiological concentrations inhibits the release of FFA stimulated by exercise and b) the increase of the NADH/NAD ratio leads to the formation of alpha-glycerophosphate which in turn yields glycerol. Therefore changes in plasma glycerol do not reflect lipolysis when blood lactate increases. c) The effect of lactate on RaFFA can be explained by an enhanced reesterification, although a direct inhibition on lipase could not be excluded.     

Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat.

Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen-stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF-alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS-treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4.     

Stimulation of lymphocyte migration by endotoxin, tumor necrosis factor, and interferon

Since several studies have demonstrated that lipopolysaccharide (LPS), tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhanced lymphocyte binding to endothelial cells in vitro, we examined the effects of these agents on lymphocyte migration in vivo. Small peritoneal exudate lymphocytes (sPEL), which perferentially migrate into inflammatory sites, were radiolabeled with 111In and injected iv into rats. The id injection of LPS was a strong stimulus for the migration of these cells into the skin. TNF alpha was also a good stimulator of lymphocyte migration, while TNF beta and IL-1 alpha were weak or nearly inactive. Kinetic analysis demonstrated that migration to TNF was rapid, with a peak at 6 hr, followed by a steady decline, while migration to LPS was sustained for 24 hr. TNF alpha, TNF beta, and IL-1 alpha, when combined with interferon-gamma (IFN-gamma) or IFN-alpha/beta produced striking synergistic increases in lymphocyte migration. Combinations of the TNFs and IL-1 had less than additive effects, as did combinations of the IFNs. Qualitatively similar migration responses were found when spleen T cells instead of sPEL were studied.