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.

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. Originally identified as a tumoricidal agent, TNF is now recognized as a major inflammatory cytokine with pleiotropic activities on many cell types and organs, which is involved in the local physiological host immune response to invading micro-organisms as well as in the pathophysiology of systemic inflammatory conditions, such as sepsis [1–4]. Highly elevated systemic levels of TNF, as observed during sepsis and septic shock, induce a wide range of immunological and metabolic sequelae, that result in tissue injury, eventually culminating in multiple organ dysfunction with high mortality. The accumulated evidence for the central involvement of TNF in the pathogenesis of septic shock has led many researchers, molecular biologists and clinicians alike, to investigate the complex mechanisms that regulate the production and release of TNF, and that determine its biological effects. The goals of such research efforts are to gain more insight into the pathophysiology of critical illnesses, to develop sensitive and rapid diagnostic tests, and ultimately to provide new therapeutic strategies from which the critically-ill may benefit.

This study was designed to investigate the effects of endogenous adenosine by the use of a novel adenosine kinase-inhibitor (GP-1-515) on pathological hepatic leukocyte adhesion following hemorrhagic shock.Anesthetized rats were prepared for invasive hemodynamical monitoring and subjected to hemorrhagic shock for 60 min. Five hours after adequate resuscitation the hepatic microcirculation was investigated in vivo using epifluorescence microscopy. The adenosine kinase inhibitor GP-1-515 (0.25 mg/kg i.v.) was given in a randomized and blinded manner before shock induction.GP-1-515 had no significant effect on central hemodynamics. Inhibition of adenosine kinase by GP-1-515 significantly reduced shock-induced permanent leukocyte adhesion to sinusoidal endothelium. Narrowing of sinusoids was partially prevented and sinusoidal blood flow was improved by GP-1-515The inflammatory process following hemorrhagic shock reflected by enhanced leukocyte adhesion is effectively attenuated by GP-1-515 due to increased endogenous adenosine levels.

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.

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.

Bouma, M. G.; Bauer, C.; Herrmann, I.; van den Wildenberg, F. A.J.M.; Firestein, G. S.; Marzi, I.; Buurman, W. A.

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.

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.

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 (MΦ) 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.

Adenosine is now used to treat cardiac arrhythmias and a variety of other potential therapeutic uses for adenosine and its receptor-specific analogues have been suggested. We will review here the evidence that adenosine may be useful in the treatment of inflammatory diseases although its potential for promotion of inflammation must be taken into account. The antiinflammatory effects of adenosine and its receptor analogues were first suggested in 1983. Originally shown to diminish neutrophil function via interaction with adenosine A(2) receptors, adenosine has a variety of effects on the cells involved in inflammation, which, in general, are antiinflammatory. In this series of reports we will discuss the effects of adenosine, acting at its receptors on macrophage/monocytes, and on the synthesis and secretion of the cytokines that orchestrate inflammation. In previous studies, the paradoxical capacity of adenosine to promote the inflammatory functions of neutrophils has been shown to result from A(1) receptor occupancy. We will discuss here the potential pro-inflammatory role of adenosine, acting at A(1) receptors, to enhance the adhesive capacity of vascular endothelium, a critical element in recruiting leukocytes to inflamed sites. Although prior studies have focussed on the potential for adenosine receptor-specific agonists to diminish inflammation it is also possible that endogenously released adenosine plays an antiinflammatory role. We will review the evidence that two commonly used and potent antiinflammatory agents, methotrexate and sulfasalazine, diminish inflammation via promotion of adenosine release. The expansion of the potential therapeutic uses of adenosine to include inflammatory diseases may permit the development of novel pharmacologic agents for the treatment of such diseases as rheumatoid arthritis. (C) 1997 Wiley-Liss, Inc.

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.

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-1beta, 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.