Hydrogen sulfide is an endogenous modulator of leukocyte-mediated inflammation.

Hydrogen sulfide (H2S) is increasingly recognized as an important signaling molecule in the cardiovascular and nervous systems. Recently, H2S donors were reported to induce neutrophil apoptosis and to suppress expression of some leukocyte and endothelial adhesion molecules. Using rats, we examined the possibility that H2S is an endogenous regulator of key inflammatory events at the leukocyte-endothelial interface. Via intravital microscopy, we observed that H2S donors (NaHS and Na2S) inhibited aspirin-induced leukocyte adherence in mesenteric venules (ED50 of 5.0 micromol/kg for Na2S), likely via activation of ATP-sensitive K+ (K(ATP)) channels. Inhibition of endogenous H2S synthesis elicited leukocyte adherence. Leukocyte infiltration in an air pouch model was also suppressed by H2S donors (NaHS, Lawesson's reagent, and N-acetylcysteine; ED50 of 42.7, 1.3, and 29.9 micromol/kg, respectively) and exacerbated by inhibition of endogenous H2S synthesis. Carrageenan-induced paw edema was suppressed by H2S donors (NaHS and Na2S; ED50s of 35 and 28 micromol/kg, respectively) to the same extent as by diclofenac and enhanced by an inhibitor of H2S synthesis. Suppression of edema formation by H2S donors was mimicked by a K(ATP) channel agonist and reversed by an antagonist of this channel. These results suggest that endogenous H2S is an important mediator of acute inflammation, acting at the leukocyte-endothelium interface. These findings have important implications for anti-inflammatory drug development.

Annexin-1 is an endogenous gastroprotective factor against indomethacin-induced damage.

Adherence of neutrophils to the vascular endothelium is an early and critical event in the pathogenesis of gastric injury induced by NSAIDs. Pretreatment with glucocorticoids has been shown to prevent NSAID-induced neutrophil adherence and, in turn, to protect the stomach from injury. Some of the anti-inflammatory effects of glucocorticoids, including inhibition of neutrophil adherence, are mediated via the release of annexin-1. In this study, we assessed the contribution of annexin-1 to the protective actions of a glucocorticoid (dexamethasone) against indomethacin-induced gastric damage. Dexamethasone pretreatment markedly reduced the extent of indomethacin-induced gastric damage in rats. Immunoneutralization of annexin-1 resulted in a reversal of the gastroprotective actions of dexamethasone. Similarly, pretreatment with either of two antagonists of the formyl peptide receptor family, to which annexin-1 binds, reversed the gastroprotective effects of dexamethasone. The inhibitory effects of dexamethasone on indomethacin-induced leukocyte adherence in the mesenteric microcirculation were abolished by pretreatment with an antibody directed against annexin-1 or with an antagonist of the formyl peptide receptors. These results demonstrate that annexin-1 mediates the gastroprotective effects of a glucocorticoid against NSAID-induced damage. We propose that in some circumstances, annexin-1 plays an important role as an endogenous mediator of mucosal defense.

TLR4 contributes to disease-inducing mechanisms resulting in central nervous system autoimmune disease.

Environmental factors strongly influence the development of autoimmune diseases, including multiple sclerosis. Despite this clear association, the mechanisms through which environment mediates its effects on disease are poorly understood. Pertussis toxin (PTX) functions as a surrogate for environmental factors to induce animal models of autoimmunity, such as experimental autoimmune encephalomyelitis. Although very little is known about the molecular mechanisms behind its function in disease development, PTX has been hypothesized to facilitate immune cell entry to the CNS by increasing permeability across the blood-brain barrier. Using intravital microscopy of the murine cerebromicrovasculature, we demonstrate that PTX alone induces the recruitment of leukocytes and of active T cells to the CNS. P-selectin expression was induced by PTX, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab. P-selectin blockade also prevented PTX-induced increase in permeability across the blood-brain barrier. Therefore, permeability is a secondary result of recruitment, rather than the primary mechanism by which PTX induces disease. Most importantly, we show that PTX induces intracellular signals through TLR4, a receptor intimately associated with innate immune mechanisms. We demonstrate that PTX-induced leukocyte recruitment is dependent on TLR4 and give evidence that the disease-inducing mechanisms initiated by PTX are also at least partly dependent on TLR4. We propose that this innate immune pathway is a novel mechanism through which environment can initiate autoimmune disease of the CNS.

A down-regulatable E-selectin ligand is functionally important for PSGL-1-independent leukocyte-endothelial cell interactions.

P-selectin glycoprotein-1 (PSGL-1) supports P-selectin-dependent rolling in vivo and in vitro. However, controversy exists regarding the importance of PSGL-1-dependent and -independent E-selectin rolling. Using antibodies against PSGL-1 and PSGL-1(-/-) mice, we demonstrated abolition of P-selectin-dependent rolling but only partial inhibition of E-selectin-mediated rolling in the cremaster microcirculation following local administration of tumor necrosis factor alpha (TNF-alpha). In vitro studies demonstrated that binding of recombinant mouse E-selectin chimera to PSGL-1(-/-) neutrophils was dramatically decreased in mice treated systemically but not locally with TNF-alpha. Further, PSGL-1 blockade abolished E-selectin-dependent rolling in wild-type mice following systemic TNF-alpha administration but not local TNF-alpha administration. Together, these data support an E-selectin ligand present on PSGL-1(-/-) neutrophils that is down-regulatable upon systemic but not local activation. To determine whether the PSGL-1-independent E-selectin ligand was physiologically important, we used a P- and E-selectin-dependent cutaneous contact hypersensitivity model. Binding studies showed no E-selectin ligand down-regulation in this model. The few cells that rolled on E-selectin ligand following PSGL-1 antibody administration or in PSGL-1 deficiency were sufficient to induce profound contact hypersensitivity. In conclusion, E-selectin mediates PSGL-1-dependent and independent rolling and the latter can be down-regulated by systemic activation and can replace PSGL-1 to support the development of inflammation.

Probucol restores the defective leukocyte-endothelial interaction in experimental diabetes.

Defective leukocyte-endothelial interactions have been observed in experimental type 1 diabetes. One of the mechanisms involved in the late complications of diabetes mellitus is the formation of free radicals species. Antioxidant treatment has been demonstrated to have beneficial effects on the complications observed in this pathology. Using intravital microscopy to visualize venules of the internal spermatic fascia, we demonstrated that the defective leukocyte-endothelial interactions in alloxan-induced diabetic rats could be corrected by probucol treatment. The defects were quantitated by the number of leukocytes rolling along the venular endothelium, sticking to the venular wall after topical application of zymosan-activated plasma (10%-0.1 ml) or leukotriene B4 (1 microM/0.1 ml) and migrated after the application of a local irritant stimulus (carrageenan, 100 microg/0.1 ml). Leukocyte counts, erythrocyte velocity and venular shear rate, unaltered in diabetic rats, were not modified by this treatment. Reactive oxygen species formation by endothelial cells increased in diabetic preparations, and the reduced expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and P-selectin in cross-sections of the whole testis of the animals, were both restored by the antioxidant agent. Therefore, antioxidant treatment improves leukocyte-endothelial interaction in diabetic rats at least in part by restoring the expression of adhesion molecules in venules of diabetic rats.