Inducible nitric oxide synthase‐deficient mice have enhanced leukocyte–endothelium interactions in endotoxemia

Abstract

Nitric oxide (NO) from constitutive NO synthase (NOS) has been postulated to be a homeostatic regulator of leukocyte-endothelial cell interactions. By contrast, the inducible NO synthase (iNOS) isoform has been invoked as a potential pathogenic enzyme in numerous inflammatory diseases. The objective of this study was to determine whether the iNOS isoform is also capable of functioning as a regulator of leukocyte recruitment. Mice received endotoxin (LPS, 30 microg/kg, i.v.); 2-4 h later, intravital microscopy was used to examine leukocyte rolling and adhesion in postcapillary venules of the cremaster muscle and the sinusoids and postsinusoidal venules of the hepatic microcirculation. Leukocyte recruitment into the lung was also examined. RT-PCR confirmed that this treatment induced iNOS mRNA expression in wild-type mice as early as 2 h after LPS treatment. Between 2 and 4 h after LPS administration, the number of rolling and adherent leukocytes in cremasteric postcapillary venules and of adherent cells in liver postsinusoidal venules of iNOS-deficient mice were significantly higher than in wild-type mice. Leukocyte accumulation in the lung (measured by myeloperoxidase assay) was also significantly elevated in iNOS-deficient animals. These effects could not be attributed to differences in systemic blood pressure, shear rates, circulating leukocyte numbers, or baseline levels of rolling and adhesion because these parameters were not different between the two groups. To establish whether the differences in leukocyte recruitment were related to the leukocytes per se, perfusion of iNOS+/+ or iNOS-/- septic blood over purified E-selectin (using parallel plate flow chambers) revealed much larger recruitment of iNOS-/- leukocytes. These results suggest that iNOS induced in response to LPS releases NO that is capable of reducing leukocyte accumulation by affecting leukocytes directly and raises the possibility that induction of iNOS is a homeostatic regulator for leukocyte recruitment.

In vivo regulation of PECAM-1 activity during acute endothelial dysfunction in the rat mesenteric microvasculature

“…In fact, among the three stimulating agents we investigated, L-NAME was found to be the most effective in terms of inducing transendothelial migration of leukocytes. This finding is of particular interest considering the complex role exerted by nitric oxide in the homeostasis of endothelial cells [18,19] and in the regulation of leukocyte-endothelium interaction [20][21][22]. In this regard, it is well known that an acute decrease in NO production by the vascular endothelium up-regulates endothelial cell adhesion molecule expression (i.e., P-selectin and ICAM-1), thus enhancing leukocyte-endothelial interaction [12,[23][24][25].…”

Airway epithelial cell signaling in response to bacterial pathogens

“…The lack of iNOS induced a significant increase in leukocyte rolling and neutrophil recruitment to the lungs. 87 Decreased iNOS expression also contributes to bacterial colonization and infection. Bacterial killing activity could be restored in airway epithelial cells derived from a patient with cystic fibrosis, which have reduced expression of iNOS, by transfection with human iNOS cDNA.…”

Asymmetric Dimethylarginine Produces Vascular Lesions in Endothelial Nitric Oxide Synthase–Deficient Mice

“…Reverse-transcriptase polymerase chain reaction (RT-PCR) for iNOS was performed with PC-701 thermocycler (Astec, Fukuoka, Japan) using a moloney murine leukemia virus (M-MLV) RT kit (Gibco) and a Takara Ex Tag kit (Takara, Tokyo, Japan). 32 PCR products were subjected to electrophoresis in 1% agarose gels stained with ethidium bromide and analyzed using an FLA-2000 fluoroimage analyzer (Fujifilm, Tokyo, Japan).…”