Assay of soluble tumor necrosis factor receptors.

Over the last decade, numerous basic biological as well as experimental and clinical studies have firmly established the significance of tumor necrosis factor (TNF) as a principal proximal mediator of sepsis (1-4). One of the major insights that has emerged during recent years has been that under physiological circumstances, TNF activity is tightly controlled and locally restricted. In this respect, the soluble TNF receptors (sTNF-Rs) have been recognized to exert an important regulatory control on the biological actions of TNF, not only in the normal host defense against infection, but also in systemic inflammatory disorders that are related to infectious as well as noninfectious etiologies. Moreover, elevated systemic levels of sTNF-R have been demonstrated to have accurate diagnostic as well as prognostic significance in clinical sepsis and other critical illnesses (5-13). Therefore, accurate determination of sTNF-R in plasma or serum has become an important tool to gain information about a variety of pathological conditions that are characterized by TNF-mediated immune activation, both in the experimental and in the clinical setting.

The anti-inflammatory potential of adenosine in ischemia-reperfusion injury: established and putative beneficial actions of a retaliatory metabolite.

The endogenous metabolite adenosine has been recognized as a protective agent in the setting of ischemia-reperfusion. Because the formation of adenosine during ischemia is closely linked to ATP catabolism, and its actions antagonize the deleterious metabolic and cardiovascular consequences of ischemia, it has been named a "retaliatory" metabolite. During recent years, however, the insight into its diverse scope of anti-inflammatory actions has increased considerably. In this review, the beneficial metabolic and cardiovascular actions of adenosine in ischemia and reperfusion are briefly outlined, followed by an extensive discussion of the established and putative anti-inflammatory actions of adenosine in the inflammatory response to ischemia and reperfusion. It is demonstrated that adenosine interferes with activated neutrophil function, neutrophil-endothelial adhesive interactions, the production and release of various inflammatory mediators, the expression of adhesion molecules, and that it activates cellular antioxidant defense systems, thus providing protective effects at multiple levels in the pathogenesis of ischemia and reperfusion. Finally, several potential pharmacological strategies to enhance the "natural defense mechanism" provided by endogenous adenosine are presented.

Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors.

Adenosine, acting via A2 receptors, is a potent inhibitor of neutrophil oxidative burst, but its effects and mechanisms of action on neutrophil degranulation have been less well characterized. We, therefore, investigated the effects of adenosine and its receptor-specific analogues on neutrophil degranulation in stimulated human whole blood. Adenosine dose-dependently inhibited the LPS- and TNF-alpha-induced release of the azurophilic granule proteins bactericidal/permeability-increasing protein, elastase, and defensins to approximately the same extent, with a maximum inhibition of 70 to 80% and an IC50 ranging from 14 to 24 microM. The inhibitory effects of adenosine were partially blocked by the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, the A1/A2 antagonist 8(p-sulfophenyl)theophyline, and the A1/A3 antagonist xanthine amine congener, but not by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine. The highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and the nonselective agonist 2-chloroadenosine reduced degranulation more potently than the A1 agonist N6-cyclopentyladenosine. The inhibitory effects of N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-chloroadenosine were strongly reversed by xanthine amine congener, but were not affected by 8(p-sulfophenyl)theophyline. In addition, the adenosine kinase inhibitor GP515 attenuated degranulation via an adenosine-mediated mechanism. These data indicate that adenosine acts via A2 as well as A3 receptors to inhibit neutrophil degranulation and add to the anti-inflammatory potential of adenosine and adenosine-regulating agents in neutrophil-mediated tissue injury.

Adenosine kinase inhibitor GP515 attenuates hepatic leukocyte adhesion after hemorrhagic hypotension.

Adhesion of leukocytes to the vascular endothelium hallmarks a key event in neutrophil-mediated organ injury after ischemia-reperfusion. The autacoid adenosine has been shown to inhibit activated neutrophil function and to interfere with leukocyte-endothelial adherence. Its therapeutic use in ischemia-reperfusion, however, has been limited by severe cardiovascular side effects. We therefore investigated the effects of the adenosine kinase inhibitor GP515 in vivo on hepatic leukocyte-endothelial interactions in a rat model of hemorrhagic hypotension and resuscitation, using intravital microscopy. Rats were pretreated with either GP515 (0.25 mg/kg) or saline in a randomized and blinded manner and subjected to pressure-controlled hemorrhagic hypotension at a mean arterial pressure of 40 mmHg for 60 min followed by 5 h of resuscitation. Five hours after resuscitation in saline-treated animals, firm leukocyte-sinusoidal adhesion was strongly enhanced in the periportal and midzonal sublobular regions, and sinusoidal diameters were also markedly reduced. Compared with saline treatment, GP515 significantly attenuated shock and resuscitation-induced leukocyte adhesion in both sublobular regions. Moreover, although GP515 did not significantly affect macrohemodynamical and hematological parameters, it enlarged narrowed sinusoidal diameters and tended to improve sinusoidal blood flow. We propose that the adenosine-regulating agent GP515 has a therapeutic potential to attenuate ischemia-reperfusion-induced inflammation by capitalizing on the beneficial anti-inflammatory effects of endogenous adenosine.

Adenosine inhibits cytokine release and expression of adhesion molecules by activated human endothelial cells.

Ischemia induces excessive ATP catabolism with subsequent local release of its metabolite adenosine, an autacoid with anti-inflammatory properties. Because activation of the vascular endothelium is critical to the inflammatory host response during ischemia and reperfusion, the effects of adenosine on two major determinants of endothelial cell activation (i.e., the release of proinflammatory cytokines and the expression of adhesion molecules) were studied. Adenosine dose dependently inhibited the release of interleukin (IL)-6 and IL-8 by stimulated human umbilical vein endothelial cells (HUVEC). Expression of E-selectin and vascular cell adhesion molecule 1 (VCAM-1), but not intercellular adhesion molecule 1 (ICAM-1), by activated HUVEC was also reduced by adenosine. Inhibition of endogenous adenosine deaminase activity by erythro-9-(2-hydroxy-3-nonyl)adenine or 2'-deoxycoformycin strongly enhanced the inhibitory effects of exogenous adenosine on cytokine release and expression of E-selectin and VCAM-1. However, a clear role for specific adenosine receptors in the described inhibitory events could not be established. Together, these data imply that the vascular endothelium constitutes an important target for the anti-inflammatory actions of adenosine.

Low energy laser irradiation fails to modulate the inflammatory function of human monocytes and endothelial cells.

BACKGROUND AND OBJECTIVE: In view of the important regulatory role of cytokines in wound healing and inflammation, we investigated the effects of low energy laser irradiation on cytokine release by human peripheral blood monocytes (M phi) and human umbilical vein endothelial cells (HUVEC) in vitro. Also, the effects of laser light on the expression of endothelial adhesion molecules, another important feature of inflammatory and regenerative responses, were assessed. STUDY DESIGN/MATERIALS AND METHODS: Cells were irradiated with a pulsed GaAs-laser (904 nm) at energy densities 0 (= sham), 0.3, 3.0, or 9.0 J/cm2 and subsequently incubated in absence or presence of endotoxin (M phi) or the proinflammatory cytokines TNF alpha and IL-1 beta (HUVEC). RESULTS: Irradiation at any of the dosages used did not significantly affect spontaneous or endotoxin-induced release of TNF alpha, IL-6, and IL-8 by M phi. Similarly, secretion of IL-6 and IL-8 by resting or cytokine-activated HUVEC after either single or repeated laser treatment was unchanged as compared to sham-irradiated controls. Moreover, laser treatment did not induce de novo expression or upregulation of the endothelial adhesion molecules E-selectin, ICAM-1, and VCAM-1, and it failed to modify their expression in response to stimulation with TNF alpha or IL-1 beta. CONCLUSION: We conclude that with the specific laser parameters and dose-regimen used, low energy laserlight does not affect the inflammatory function of human monocytes and endothelial cells in vitro.

Differential regulatory effects of adenosine on cytokine release by activated human monocytes.

Adenosine is an endogenous nucleoside that can modulate the function of cells involved in the inflammatory response, such as polymorphonuclear leukocytes (PMN) and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia-reperfusion injury, a pathologic phenomenon that is associated with excessive ATP catabolism and subsequent local release of adenosine. The "retaliatory" metabolite adenosine has been shown to interfere with PMN function, thereby attenuating the deleterious consequences of ischemia and reperfusion. In this study, we demonstrate that adenosine inhibits the production of TNF-alpha, IL-6, and IL-8 by LPS-activated human monocytes with a differential potency. The A2 receptor-specific adenosine analogues 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) were most effective in attenuating LPS-induced cytokine production, whereas the A1-selective adenosine analogue N6-cyclopentyladenosine (CPA) was less effective, indicating that inhibition of cytokine production by adenosine is primarily an A2 receptor-mediated event. The observed inhibitory effects were not restricted to endotoxin-induced cytokine production, because adenosine also inhibited TNF-alpha production by monocytes stimulated with the proinflammatory cytokine IL-1 beta. Again, 2-chloroadenosine and NECA reduced IL-beta-induced TNF-alpha production more potently than CPA. In contrast, adenosine enhanced production of IL-6 and IL-8 by monocytes stimulated with IL-1 beta. Furthermore, only 2-chloroadenosine, but not NECA, strongly inhibited cytokine-induced IL-6 and IL-8 production. These results suggest an additional A2 receptor-mediated mechanism of retaliatory action of adenosine under pathologic conditions where cytokine production by activated mononuclear phagocytes is involved, such as ischemia-reperfusion injury and septic shock.

Anti-CD14 antibodies reduce responses of cultured human endothelial cells to endotoxin.

Lipopolysaccharide (LPS) activates both myeloid and endothelial cells. Whereas CD14 has been shown to be involved in LPS recognition by myeloid cells, the mechanism responsible for the strong response of endothelial cells to LPS remains to be elucidated. The role of CD14 in this process was studied using CD14-specific antibodies (Ab). Anti-CD14 Ab inhibited LPS-induced interleukin-6 (IL-6) release and E-selectin expression by cultured human umbilical vein endothelial cells (HUVEC). Messenger RNA encoding IL-6 and E-selectin was reduced in parallel. The inhibitory effect of anti-CD14 Ab was epitope dependent, maximal at low LPS concentrations and dropping with increasing LPS doses. Anti-CD14 Ab did not affect endothelial cell activation induced by IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA). IL-6 release and E-selectin expression of HUVEC were strongly reduced when LPS activation was performed in the absence of serum, indicating involvement of serum components in LPS activation of HUVEC. Nevertheless, anti-CD14 Ab also blocked LPS-induced HUVEC activation in the absence of serum. Although the role of serum components in LPS activation remains to be elucidated, CD14 seems to be a key mediator in LPS-induced activation of endothelial cells.