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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.03-0216fje. |
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Department of Anatomy, Histology and Forensic Medicine,
* Section of Histology and
Section of Anatomy, University of Florence, I-50139 Florence, Italy
2Correspondence: Dipartimento di Anatomia, Istologia e Medicina Legale. Sezione di Istologia, Università di Firenze, V.le G.Pieraccini, 6, I-50139 Florence, Italy. E-mail: daniele.bani{at}unifi.it
SPECIFIC AIMS
Neutrophil margination within blood vessels, an early finding during myocardial ischemia, depends on the endothelial expression of early- and late-phase adhesion molecules such as P-selectin, E-selectin, VCAM-1, and ICAM-1 that allows leukocyte extravasation, thereby initiating myocardial tissue injury. In previous studies, the peptide hormone relaxin (RLX) was found to protect against experimental myocardial injury due to ischemia and reperfusion and to reduce neutrophil extravasation into the inflamed tissues. This study addresses the role of RLX in down-regulating endothelial-neutrophil adhesion and the possible involvement of NO, a potent anti-adhesive molecule, in the mechanism of action of RLX, using an in vitro coculture model with rat coronary endothelial (RCE) cells and neutrophils activated by proinflammatory molecules (LPS).
PRINCIPAL FINDINGS
1. RLX reduces the adhesiveness of RCE cells to neutrophils upon activation with LPS
RCE cell cultures were pretreated for 24 h with purified porcine RLX (2500–3000 U/mg, a generous gift from Dr. O. D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign) at concentrations of 6, 60, and 600 ng/mL. During the last 4 h, the RCE cell cultures were activated with Escherichia coli lipopolysaccharide (LPS, 10 µg/mL). Meanwhile, neutrophils were isolated from whole blood of rats and stimulated with LPS for 30 min. Finally, neutrophils were added to RCE cell cultures and allowed to adhere for another 15 min at 37°C. By light microscopy, LPS-primed RCE cells not treated with RLX showed a marked increase in the amount of adherent neutrophils compared with unprimed control cells. RLX treatment caused a reduction of adherent neutrophils, which was fully appreciable at 60 and 600 ng/mL RLX, when statistical significance was reached (Fig. 1
). The inhibitory effect of RLX on endothelial adhesiveness to neutrophils appears to be a specific property of this hormone since it could not be reproduced by replacing authentic RLX with inactivated RLX (iRLX, 60 ng/mL) obtained by reaction with 1,2-cyclohexanedione, which is similar to RLX except for its inability to bind to RLX receptors.
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2. RLX reduces the expression of the surface adhesion molecules P-selectin and VCAM-1 by LPS-primed RCE cells
By immunocytochemistry, LPS-primed RCE cells showed an increase in staining intensity for P-selectin (1:100, raised in goat; Santa Cruz Biotechnology, Santa Cruz, CA, USA), VCAM-1 (1:100, raised in rabbit; Santa Cruz), and ICAM-1 (1:200, raised in mouse, Chemicon, Temecula, CA, USA) compared with the unprimed controls. RLX (60 ng/mL) appeared to decrease the staining intensity for P-selectin and VCAM-1, leaving ICAM-1 immunoreactivity unchanged. iRLX (60 ng/mL) had no effect on P-selectin and VCAM-1 immunostaining. Western blot (Fig. 2
) and RT-PCR analyses confirmed the immunocytochemical findings, for they showed that RLX causes a clear-cut, significant reduction of the expression of P-selectin and VCAM-1 protein and mRNA induced by LPS priming in RCE cells; ICAM-1 expression remained unaffected.
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In our experiments, E-selectin was never expressed by RCE cells, either in basal conditions or upon priming with LPS, regardless of the assay method used. Involvement of P-selectin, VCAM-1, and ICAM-1 in endothelial adhesiveness to neutrophils was confirmed by parallel adhesion experiments carried out in the presence of blocking antibodies against the noted adhesion molecules. In all cases, the tested antisera markedly reduced the number of neutrophils adherent to RCE cells, as judged by the neutrophil/RCE cell ratio (basal: 0.15±0.02; LPS: 0.43±0.06; LPS+anti-P-selectin, 60 µg/mL: 0.28±0.07; LPS+anti-VCAM-1, 60 µg/mL: 0.28±0.06; LPS+anti-ICAM-1, 40 µg/mL: 0.22±0.03).
3. Involvement of the NO biosynthetic pathway in the RLX-induced reduction of the adhesiveness of LPS-primed RCE cells to neutrophils
Administration of the highly specific NO-synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA, 10-4 M) to RCE cells treated with RLX (60 ng/mL) 5 h before addition of neutrophils significantly blunted the RLX-induced inhibition of RCE cell adhesiveness to neutrophils (Fig. 1)
. In control experiments, L-NMMA alone given before LPS priming had no effect on neutrophil adhesion. Similarly, L-NMMA blunted the RLX-induced decrease in the expression of P-selectin and VCAM-1 mRNA and protein by RCE cells (Fig. 2)
.
CONCLUSIONS AND SIGNIFICANCE
The present study provides the first experimental evidence that the peptide hormone RLX, acting through a NO-dependent mechanism, markedly reduces the adhesiveness of vascular endothelial cells to neutrophils upon activation by LPS as proinflammatory stimulus, an effect apparently exerted by a down-regulation of the expression of some early- and late-phase ECAMs, namely, P-selectin and VCAM-1. The number of neutrophils adhering to RCE cells upon LPS priming was significantly reduced by a 24 h incubation with RLX, with a maximal effect at the hormone concentration of 60 ng/mL. At these concentrations and time of exposure, RLX significantly reduced the expression of P-selectin and VCAM-1 mRNA and protein by RCE cells induced by a 4 h priming with LPS, as shown by RT-PCR, immunocytochemistry, and Western blot. Conversely, RLX did not substantially influence ICAM-1 expression, which could account for the residual amounts of neutrophils adherent to RCE cells observed in the RLX-treated cultures.
Activation of endothelial cells and leukocytes resulting in reciprocal adhesion and directed migration of the latter cells to the inflamed site is an early and crucial event in the inflammatory tissue injury. RLX appears to interfere with this phenomenon, thereby exerting anti-inflammatory effects. Parallel experiments carried out by us on isolated human neutrophils indicate that RLX, at the concentrations used in the present study, also reduces the activation and chemotaxis of these cells. This novel property of RLX may account for at least part of the previously reported beneficial effects of this hormone in animal models of inflammation, such as rats subjected to cardiac ischemia and reperfusion and sensitized guinea pigs with allergic lung disease due to antigen inhalation, in which a marked reduction of leukocytes infiltrating the inflamed tissue was observed upon systemic treatment with RLX.
In the present study, the inhibitory effect of RLX on endothelial adhesiveness to neutrophils clearly emerges as a specific property of this hormone since it could not be reproduced by replacing authentic RLX with iRLX, which is similar to RLX except for its inability to bind to RLX receptors. The moderate, not statistically significant reduction of neutrophils adherent to RCE cells observed upon iRLX treatment is likely to be attributed to a residual biological activity of the iRLX preparation, possibly due to incomplete inactivation by cyclohexanedione of some RLX molecules.
Conceivably, the mechanism by which RLX exerts its anti-adhesive effect relies on the stimulation of endogenous NO synthesis by RCE cells, which has been shown to depend chiefly on the up-regulation of NOS II expression. In fact, the present findings show that coincubation with the NOS inhibitor L-NMMA blunted the RLX-induced decrease in neutrophil adhesion and endothelial expression of P-selectin and VCAM-1. Indeed, NO is known as a physiological inhibitor of endothelial-leukocyte interaction and can suppress the expression of P-selectin and VCAM-1 by activated endothelial cells.
The present findings may have important functional implications, since they further support the hypothesis that RLX, secreted and bound specifically by the heart, could have a physiological role as an autocrine cardioprotective factor against the detrimental effects of ischemic disease. On these grounds, the presence of circulating RLX in women during fertile life may help explain the low incidence of coronary heart disease compared with age-matched men and postmenopausal women.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0216fje; doi: 10.1096/fj.03-0216fje 
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