Dianne Cooper

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis

A growing body of evidence indicates that resolution of acute inflammation is an active process. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis, the phase in which disease declines. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we establish RvD2’s potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte–endothelial interactions in vivo by endothelial-dependent nitric oxide production, and by direct modulation of leukocyte adhesion receptor expression. In mice with microbial sepsis initiated by caecal ligation and puncture, RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from sepsis induced by caecal ligation and puncture and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Hypercholesterolemia promotes inflammation and microvascular dysfunction: role of nitric oxide and superoxide

Relatively brief periods (days) of hypercholesterolemia can exert profound effects on endothelium-dependent functions of the microcirculation, including dilation of arterioles, fluid filtration across capillaries, and regulation of leukocyte recruitment in postcapillary venules. Hypercholesterolemia appears to convert the normal anti-inflammatory phenotype of the microcirculation to a proinflammatory phenotype. This phenotypic change appears to result from a decline in nitric oxide (NO) bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide (O(2)(*)(-)), or both. A consequence of the hypercholesterolemia-induced microvascular responses is an enhanced vulnerability of the microcirculation to the deleterious effects of ischemia and other inflammatory conditions. Hence, therapeutic strategies that are directed towards preventing the early microcirculatory dysfunction and inflammation caused by hypercholesterolemia may prove effective in reducing the high mortality associated with ischemic tissue diseases. Agents that act to maintain the normal balance between NO and reactive oxygen species (ROS) in vascular endothelial cells may prove particularly useful in this regard.

15-epi-lipoxin A4–mediated Induction of Nitric Oxide Explains How Aspirin Inhibits Acute Inflammation

The established model for the mechanism of action of aspirin is the inhibition of prostaglandin synthesis. However, this has never fully explained aspirins repertoire of antiinflammatory properties. We found in acute pleuritis that aspirin, but not salicylate, indomethacin, or piroxicam, increased plasma nitric oxide (NO), which correlated with a reduction in inflammation. Inhibiting aspirin-elicited NO pharmacologically in this model nullified the antiinflammatory effects of aspirin. Moreover, aspirin was not antiinflammatory in either constitutive (eNOS) or inducible NO synthase (iNOS) knockout mice with IL-1β–induced peritonitis. It transpires that aspirin generates NO through its unique ability to trigger the synthesis of 15-epi-lipoxin A4. Aspirin and 15-epi-lipoxin A4 were shown to inhibit leukocyte trafficking in an NO-dependent manner using intravital microscopy on IL-1β–stimulated mouse mesentery. Not only did aspirin inhibit leukocyte–endothelial interaction in a manner similar to NO in wild-type mice but both aspirin and 15-epi-lipoxin A4 had markedly reduced effects on leukocyte–endothelial cell adherence in eNOS- and iNOS-deficient mice compared with wild type. Collectively, these data suggest that aspirin triggers the synthesis of 15-epi-lipoxin A4, which increases NO synthesis through eNOS and iNOS. This aspirin-elicited NO exerts antiinflammatory effects in the microcirculation by inhibiting leukocyte–endothelium interactions.

Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles

Polymorphonuclear leukocyte (PMN)-derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A(4) receptor or ALX. Intravenous delivery of human PMN-derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1beta. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1beta-induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.

Platelet-Leukocyte-Endothelial Cell Interactions After Middle Cerebral Artery Occlusion and Reperfusion

The adhesion of both leukocytes and platelets to microvascular endothelial cells has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds. The objectives of this study were to (1) assess the platelet–leukocyte–endothelial cell interactions induced in the cerebral microvasculature by middle cerebral artery occlusion (MCAO)/reperfusion, and (2) define the molecular determinants of the prothrombogenic and inflammatory responses in this model of focal I/R. MCAO was induced for 1 hour in wild-type (WT) mice, WT mice treated with a monoclonal antibody (mAb) to either P-selectin or GPIIb/IIIa, and in P-selectin−/−(P-sel−/−) chimeras. Isolated platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) were administered intravenously and observed with intravital fluorescence microscopy. Leukocytes were observed after intravenous injection of rhodamine 6G. One hour of MCAO followed by 1 hour of reperfusion resulted in the rolling and adhesion of leukocytes in venules, and after 4 hours of reperfusion, the adhesion of both leukocytes and platelets was detected. Although both the P-selectin and GPIIb/IIIa mAbs significantly reduced the adhesion of leukocytes and platelets at 4 hours of reperfusion, the antiadhesive effects of the P-selectin mAb were much greater. The leukocyte and platelet adhesion responses were significantly attenuated in both P-sel−/−→WT and WT→P-sel−/− bone marrow chimeras, compared with WT→WT chimeras. Neutropenia, induced by antineutrophil serum treatment, also reduced the recruitment of leukocytes and platelets after cerebral I/R. These findings implicate a major role for both platelet-associated and endothelial cell–associated P-selectin, as well as neutrophils in the inflammatory and prothrombogenic responses in the microcirculation after focal cerebral I/R.

Molecular Determinants of the Prothrombogenic and Inflammatory Phenotype Assumed by the Postischemic Cerebral Microcirculation

Background and Purpose— Circulating blood cells have been implicated in the pathogenesis of cerebral ischemia/reperfusion (I/R) injury and stroke. The objective of this study was to define the magnitude and molecular determinants of the platelet- and leukocyte–endothelial cell adhesive interactions induced by I/R in the mouse brain. Methods— Bilateral common carotid artery occlusion was induced for 1 hour in C57BL/6 mice, followed by either 40 minutes or 4 hours of reperfusion. Fluorescent platelets were administered intravenously, and the frontal brain surface was observed with intravital fluorescence microscopy. Leukocyte–endothelial cell adhesion was monitored with the use of rhodamine-6G. Results— Ischemia followed by 40 minutes of reperfusion resulted in the rolling (125.1±23.6/mm2) and firm adhesion (109.5±25.8/mm2) of leukocytes but not platelets in venules. However, with 4 hours of reperfusion, rolling (138.8±24.6/mm2) and firm adhesion (153.7±22.3/mm2) of platelets were detected, and this was accompanied by a more intense recruitment of rolling (374.5±54.6/mm2) and adherent (445.2±57.1/mm2) leukocytes. In mice deficient in either P-selectin (P-selectin−/−) or intercellular adhesion molecule-1 (ICAM-1) (ICAM-1−/−), the I/R-induced platelet–endothelial cell (by 80% and 60%, respectively) and leukocyte–endothelial cell (by 84% and 78%, respectively) interactions were significantly blunted compared with those of wild-type mice. Conclusions— These findings indicate that I/R promotes the adhesion of both platelets and leukocytes in cerebral venules, with the accumulation of adherent leukocytes preceding the recruitment of platelets. Both P-selectin and ICAM-1 contribute to the inflammatory and prothrombogenic state induced by cerebral I/R.

Oxidative stress promotes blood cell-endothelial cell interactions in the microcirculation

Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the capacity of the cell to detoxify these potentially injurious oxidants using endogenous antioxidant defense systems. Conditions associated with oxidative stress include ischemia/reperfusion, hypercholesterolemia, diabetes, and hypertension. The adhesion of circulating blood cells (leukocytes, platelets) to vascular endothelium is a key element of the pro-inflammatory and prothrombogenic phenotype assumed by the vasculature in these and other disease states that are associated with an oxidative stress. There is a growing body of evidence that links the blood cell-endothelial cell interactions in these conditions to the enhanced production of ROS. Potential enzymatic sources of ROS within the microcirculation include xanthine oxidase, NAD(P)H oxidase, and nitric oxide synthase. ROS can promote a pro-inflammatory/prothrombogenic phenotype within the microvasculature by a variety of mechanisms, including the inactivation of nitric oxide, the activation of redox-sensitive transcription factors (e.g., nuclear factor-ξB) that govern the expression of endothelial cell adhesion molecules (e.g., P-selectin), and the activation of enzymes (e.g., phospholipase A2) that produce leukocyte-stimulating inflammatory mediators (e.g., platelet-activating factor). The extensively documented ability of different oxidant-ablating interventions to attenuate blood cell-endothelial cell interactions underscores the importance of ROS in mediating the dysfunctional microvascular responses to oxidative stress.

Novel insights into the inhibitory effects of Galectin‐1 on neutrophil recruitment under flow

Galectin‐1 (Gal‐1) is a β‐galactoside‐binding protein endowed with anti‐inflammatory properties. The purpose of this study was to investigate the effects of endogenous and exogenous Gal‐1 on neutrophil recruitment onto TNF‐treated endothelium. The effect of human recombinant (hr)Gal‐1 on markers of neutrophil activation (CD11b expression, P‐selectin glycoprotein ligand 1, and L‐selectin shedding) was also assessed. Gal‐1 inhibited the platelet‐activating factor‐induced increase in CD11b expression in a concentration‐dependent manner, as assessed by flow cytometry. To determine the effects of Gal‐1 on neutrophil recruitment, an in vitro flow chamber was used: Preincubation of neutrophils with hrGal‐1 significantly decreased the extent of capture, rolling, and adhesion on activated endothelial monolayers. This inhibition was shared with the endogenous protein, as knockdown of endothelial Gal‐1 using small interfering RNA resulted in a significant increase in the number of cells captured and rolling. To verify the effects of Gal‐1 in an in vivo system, intravital microscopy of Gal‐1 null mice and their wild‐type counterparts was performed. Leukocyte adhesion and emigration were increased significantly in the cremasteric circulation of Gal‐1 null mice inflamed with IL‐1β. These findings indicate that Gal‐1 functions to limit neutrophil recruitment onto a TNF‐treated endothelium, a property that may underline its inhibitory effects in acute inflammation.

Carbon Monoxide-Releasing Molecules Modulate Leukocyte-Endothelial Interactions under Flow

Carbon monoxide (CO) generated by the enzyme heme oxygenase during the breakdown of heme is known to mediate a number of biological effects. Here, we investigated whether CO liberated from a water-soluble CO-releasing molecule (CO-RM) is capable of modulating leukocyte-endothelial interactions. Tricarbonylchoro(glycinato)ruthenium (II) (CORM-3), a fast CO releaser, proved to be anti-inflammatory in two distinct models of acute inflammation in vivo. In both cases, a significant reduction in neutrophil extravasation was observed. Subsequent in vitro static experiments showed that CORM-3 produced a direct effect on neutrophil (polymorphonuclear neutrophil; PMN) adhesion molecule expression; dose-dependently inhibiting platelet-activating factor stimulated CD11b up-regulation and L-selectin shedding, whereas no effect was observed on up-regulation of human umbilical vein endothelial cell (HUVEC) adhesion molecules intercellular adhesion molecule-1 or E-selectin nor on interleukin-8 chemokine production. In addition, when PMN interaction with HUVECs was studied, an inhibitory effect of CORM-3 on cell capture and rolling was observed. The effect of CORM-3 on PMN CD11b expression was mimicked by the incubation of PMN with the selective large potassium channel opener 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)-phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619), which suggests that CORM-3 actions in this instance are mediated, at least in part, via opening of this channel. In conclusion, we have reported that CORM-3 possesses acute anti-inflammatory effects in vivo and that these are probably the result of targeting PMN activation and rolling upon the endothelium.

Inhibitory control of endothelial galectin-1 on in vitro and in vivo lymphocyte trafficking

Galectin‐1 (Gal‐1) is a β‐galactoside‐binding protein, the expression of which is increased in endothelial cells on exposure to proinflammatory stimuli. Through binding of several receptors (CD7, CD45, and CD43) Gal‐1 is known to induce apoptosis of activated T lymphocytes, an effect thought to mediate the beneficial effects it exerts in various inflammatory models. The data presented here highlights another function for Gal‐1, that of a negative regulator of T‐cell recruitment to the endothelium under both physiological and pathophysiological conditions. We have shown, using siRNA to knockdown Gal‐1 in endothelial cells, that endogenous Gal‐1 limits T‐cell capture, rolling, and adhesion to activated endothelial cells under flow. Furthermore, the reverse effect is observed when exogenous human recombinant Gal‐1 is added to activated endothelial monolayers whereby a dramatic reduction in lymphocyte recruitment is seen. These findings are corroborated by studies in Gal‐1 null mice in which homing of wild‐type (WT) T lymphocytes is significantly increased to mesenteric lymph nodes and to the inflamed paw in a model of delayed‐type hyper‐sensitivity. In conclusion, mimicking endothelial Gal‐1 actions would be a novel strategy for controlling aberrant T‐cell trafficking, hence for the development of innovative anti‐inflammatory therapeutics.—Norling, L. V., Sampaio, A. L. F., Cooper, D., Perretti, M. Inhibitory control of endothelial galectin‐1 on in vitro and in vivo lymphocyte trafficking. FASEB J. 22, 682–690 (2008)