Novel mode of action of iloprost: in vitro down-regulation of endothelial cell adhesion molecules

https://doi.org/10.1016/S0090-6980(01)00131-9Get rights and content

Abstract

Iloprost is a stable prostacyclin analog commonly employed in the treatment of peripheral vascular disease and also indicated in the treatment of patients affected by systemic sclerosis (SSc) in the presence of severe Raynaud’s phenomenon (RP). Several mechanisms of action of the drug other than vasodilation and antiplatelet effect have been demonstrated that may be involved in the exertion of its clinical efficacy. Aim of the present study was to investigate whether iloprost down-regulated lymphocyte adhesion to endothelium through a modulation of adhesion molecule expression on the surface of endothelial cells.

In the presence of iloprost, both lymphocyte adhesion and IL-1 stimulated expression of ICAM-1 and ELAM-1 exhibited a significant reduction, while unstimulated adhesion molecule expression was not significantly affected.

Our results confirm that iloprost is able to down-regulate lymphocyte adhesion to endothelial cells and indicate that endothelium itself could be target of iloprost administration. Attenuation of the inflammatory response through modulation of cellular interactions could be suggested as a potential mechanism of action of iloprost, when used in the treatment of pathological conditions characterized by endothelial activation.

Introduction

Ischemia and restoration of blood flow to an ischemic tissue initiate a series of events that culminate in inflammation. As demonstrated by both in vivo and in vitro studies, in response to ipoxia and reoxygenation endothelial cells produce chemotactic cytokines, such as interleukin (IL)-1, IL-8 and tumor necrosis factor (TNF)-α, and up-regulate their surface expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1, thus promoting the adhesion and the activation of leukocytes [1], [2], [3], [4].

Interaction of vascular endothelium with immune cells, through a well documented involvement of the adhesion molecule cascade, is one of the earliest changes in SSc, a connective tissue disorder characterized by microvascular alterations and fibrotic lesions in the skin and internal organs [5], [6]. Increased expression of ICAM-1, VCAM-1 and ELAM-1 is commonly found on endothelial cells of SSc skin biopsies, and it has been demonstrated that elevated levels of soluble adhesion molecules in the peripheral circulation correlate with clinical disease activity [7], [8], [9], [10]. In SSc, restoration of blood flow subsequent to intense vasospastic attacks characteristic of RP together with immune activation are thought to trigger the inflammatory response that culminates in tissue injury and fibrosis [11], [12].

Iloprost is a stable prostacyclin analog commonly employed in the treatment of peripheral vascular disease because of its vasodilating and platelet inhibitory effects [13]. Its use in the treatment of patients affected by SSc is indicated in the presence of severe RP, since it has been extensively demonstrated that the cyclic administration of iloprost induces a long-term reduction of both the frequency and the severity of the ischemic attacks with clinical improvement of cutaneous trophic lesions [14], [15], [16], [17]. Furthermore, one-year intermittent infusion of iloprost to SSc patients is also effective on microvascular and fibrotic manifestations of the disease, as assessed by capillaroscopic pattern and cutaneous fibrosis [18], [19].

Both the persistence of clinical improvement and the efficacy of the drug on clinical parameters other than RP cannot be explained only by the vasodilator and antiplatelet effects of the drug, which persist for no more than 2 h after the termination of the infusion [20], [21]. Several mechanisms of action of iloprost have been demonstrated to be possibly involved in the exertion of its clinical efficacy. They include enhancement of fibrinolysis [22], [23], increase in red cell deformability [24], inhibition of vascular smooth muscle cell proliferation [25], down-regulation of leukocyte adhesion molecules [26] and, as demonstrated in our previous in vitro and ex vivo studies, inhibition of lymphocyte production of proinflammatory cytokines, such as TNF-α, IL-1 and IL-6 [18], [27]. Furthermore, it has been demonstrated that prostacyclin or its stable analog partially inhibit the adhesion of polymorphonuclear cells and lymphocytes to endothelial cells [28], [29]. In the present study we explored whether iloprost exerts this inhibitory effect through a down-regulation of adhesion molecules on the surface of endothelial cells. Expression of ICAM-1, VCAM-1 and ELAM-1 on HUVECs under IL-1β stimulation was evaluated.

Section snippets

Cell isolation and culture

Single-donor HUVEC lines, obtained from four anonymous umbilical vein donors, were isolated by collagenase digestion of umbilical cord veins, grown in standard HUVEC medium consisting of M199 supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), 100 μg/ml crude extract of endothelial cell growth factor (ECGF), 50 μg/ml porcine intestinal heparin (Sigma Chemical, St. Louis, MO, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and passaged as previously described

Iloprost effects on lymphocyte adhesion

Alloactivated lymphocytes and confluent HUVECs were incubated for 24 h in the presence of 10−10 to 10−6 M iloprost before being processed for flow cytometric analysis. In the presence of iloprost lymphocyte adhesion exhibited a dose-dependent reduction (data not shown); as shown in Fig. 1 this effect was significant at 10−6 M (38.7 ± 2.1% CD45 positive cells vs basal 43.9 ± 2.2%; n = 5; P < 0.02).

Iloprost effects on cell adhesion molecules

Cultures of confluent HUVECs were treated with 10−10 to 10−6 M iloprost either in basal conditions

Discussion

Our results confirm that iloprost does inhibit lymphocyte adhesion to endothelial cells. This inhibitory effect is mild but dose-dependent and observed in all the performed experiments, at the same doses used in clinical treatments [36]. The ability of prostacyclin to reduce polymorphonuclear leukocyte adhesion to endothelial cells, as described by other authors [28], should be favorably considered when iloprost is used in the treatment of ischemic diseases whatever the etiology is, since it is

Acknowledgements

We wish to thank Santo Scrofani for expert technical assistance.

References (46)

  • J.R. Siebold

    Connective tissue diseases characterized by fibrosis

  • S. Sollberg et al.

    Elevated expression of β1 and β2 integrins, intercellular adhesion molecule 1, and endothelial leukocyte adhesion molecule 1 in the skin of patients with systemic sclerosis of recent onset

    Arthritis Rheum

    (1992)
  • H.N. Claman et al.

    Endothelial and fibroblastic activation in sclerodermathe myth of the “uninvolved skin”

    Arthritis Rheum

    (1991)
  • A.E. Koch et al.

    In situ expression of cytokines and cellular adhesion molecules in the skin of patients with systemic sclerosis

    Pathobiology

    (1993)
  • M.S. Gruschwitz et al.

    Correlation of soluble adhesion molecules in the peripheral blood of scleroderma patients with their in situ expression and with disease activity

    Arthritis Rheum

    (1995)
  • S.M. Grant et al.

    Iloprosta review of its pharmacodynamic, and pharmacokinetic properties, and therapeutic potential in peripheral vascular disease, myocardial ischemia, and extracorporeal circulation procedures

    Drugs

    (1992)
  • M. Rademaker et al.

    Comparison of intravenous infusions of iloprost and oral nifedipine in treatment of Raynaud’s phenomenon in patients with systemic sclerosisa double blind randomised study

    Br Med J

    (1989)
  • H.I. Torley et al.

    A double blind, randomised, multicentre comparison of two doses of intravenous iloprost in the treatment of Raynaud’s phenomenon secondary to connective tissue diseases

    Ann Rheum Dis

    (1991)
  • F.M. Wigley et al.

    Intravenous iloprost treatment of Raynaud’s phenomenon and ischemic ulcers seondary to systemic sclerosis

    J Rheumatol

    (1992)
  • J.J.F. Belch

    Prostaglandins in Raynaud’s phenomenon

  • S. Della Bella et al.

    Cytokine production in scleroderma patientseffects of therapy with either iloprost or nifedipine

    Clin Exp Rheumatol

    (1997)
  • D. Biasi et al.

    Iloprost as cyclic five-day infusions in the treatment of scleroderma. An open pilot study in 20 patients treated for one year

    Rev Rhum Engl Ed

    (1998)
  • D.A. Yardumian et al.

    Platelet function studies during and after infusions of ZK 36374, a stable prostacyclin analogue, to healthy volunteers

    Haemostasis

    (1986)

    • Cited by (66)

    • Wilderness Medical Society Clinical Practice Guidelines for the Prevention and Treatment of Frostbite: 2019 Update

      2019, Wilderness and Environmental Medicine
      Citation Excerpt :

      Although some of these techniques show potential, further studies are required to determine their exact role. Iloprost, a prostacyclin (PGI2) analogue, is a potent vasodilator that also inhibits platelet aggregation, down-regulates lymphocyte adhesion to endothelial cells,69 and may have fibrinolytic activity.70 Intravenous iloprost was first used for treatment of frostbite by Groechenig in 1994, in 5 patients with second- and third-degree frostbite.

    • Systemic sclerosis

      2019, Genomic and Precision Medicine: Infectious and Inflammatory Disease

    • Case Report: Severe Frostbite in Extreme Altitude Climbers—The Kathmandu Iloprost Experience

      2018, Wilderness and Environmental Medicine

    • Des-aspartate-angiotensin I, a novel angiotensin AT<inf>1</inf> receptor drug

      2015, European Journal of Pharmacology

    • The effects of iloprost on ischemia-reperfusion injury in skeletal muscles in a rodent model

      2014, Journal of Surgical Research

    • Systemic Sclerosis

      2013, Genomic and Personalized Medicine
    View all citing articles on Scopus
    1

    Present address: L.I.T.A., Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy.

    View full text
    Copyright © 2001 Elsevier Science Inc. All rights reserved.