Delivery of pharmacologically active dexamethasone into activated endothelial cells by dexamethasone–anti-E-selectin immunoconjugate
Introduction
During chronic inflammatory diseases such as asthma, rheumatoid arthritis, and inflammatory bowel disease, the endothelium plays an active role in leukocyte recruitment and infiltration [1]. These disorders are characterized by a continuously increased expression of inflammatory genes by a variety of cell types. Activated endothelial cells express adhesion molecules on their surface and secrete various cytokines leading to a multi-step cascade of leukocyte adhesion and subsequent transmigration of leukocytes into the inflamed tissue. Hence, manipulating the processes of endothelial cell activation may be advantageous for therapeutic outcome of these diseases.
Glucocorticoids are commonly used in anti-inflammatory therapy [2]. Once inside the cytoplasm of the cell, the glucocorticoid binds to a GR (Fig. 1). As a result, the receptor translocates into the nucleus where it can bind to glucocorticoid responsive elements found in promoters of various genes, thereby inducing their transcription [3]. The anti-inflammatory effects of glucocorticoids may partly be caused by enhanced expression of genes encoding anti-inflammatory proteins [4]. However, the most prominent pharmacological effect of glucocorticoids is inhibition of expression of inflammatory genes encoding cytokines, receptors, and cell adhesion molecules. This repression of gene activity is predominantly caused by an inhibitory interaction of GR with other transcription factors, such as nuclear factor kappa B (NFκB) and activator protein 1 (AP1) [2], [5]. Competition for common co-activators has also been proposed as a mechanism for this phenomenon [6], [7]. Similarly, induction of IκB expression by glucocorticoids, the natural inhibitor of NFκB, was reported for lymphocytes and monocytes [8] but this was not observed in endothelial cells [9], [10].
For cell-specific drug delivery, endothelial cells are attractive targets due to their direct contact with the blood. To deliver glucocorticoids into activated endothelial cells at the inflammatory site, Dexa was conjugated to a monoclonal antibody recognizing E-selectin, a cell adhesion molecule strongly upregulated by activated endothelium [11], [12]. Previously, it was demonstrated that this Dexa–AbhEsel conjugate was internalized by activated endothelium, not resting endothelium, via the lysosomal pathway [13]. According to the chemical conjugation strategy, the linkage between Dexa and the protein part of the construct will be degraded within the lysosomal compartment, resulting in a release of the free drug into the cytoplasm (Fig. 1). Here, the pharmacological effects of the intracellularly delivered conjugate in HUVEC are reported and compared to the effects mediated by free Dexa, which enters the cell via passive diffusion. Analyses of gene expression levels were performed by cDNA expression array studies and RT–PCR.
Section snippets
Preparation and characterization of Dexa–AbhEsel conjugate
Dexa was conjugated as described previously [13]via a succinate linker to AbhEsel (kindly provided by Dr. M. Gimbrone Jr., Harvard Medical School). Briefly, Dexa–hemisuccinate was synthesized and its identity was confirmed by mass spectrometry. Then Dexa–AbhEsel conjugate was prepared by coupling the carboxylic acid group of Dexa–hemisuccinate to primary amino-groups of the protein. The final product was purified by dialysis against PBS at 4°, filtered through a 0.2 μm filter and stored at −20°.
Uptake of Dexa–AbhEsel conjugate in HUVEC
The conjugate Dexa–AbhEsel used throughout this study contained an average of two Dexa molecules per molecule AbhEsel. Characterization of the conjugate and determination of the Dexa:antibody ratio was described previously [13]. The conjugate Dexa–AbhEsel showed specific binding to activated HUVEC, which was mediated by E-selectin, reaching a maximum after 4 hr and saturation at 10 μg/mL, a concentration corresponding to 100 nM Dexa [13], [19]. Since Dexa has to be released intracellularly before
Discussion
In the present study, the pharmacological activity of an immunoconjugate selectively delivering Dexa to activated endothelial cells was demonstrated. It is generally accepted that the anti-inflammatory effects of glucocorticosteroids are primarily based on repression of proinflammatory genes. However, different cell-types can respond differently to the inhibitory action of glucocorticoids, possibly caused by differences in relative abundance of transcription factors [4]. Conflicting results
Acknowledgements
We thank H.E. Moorlag of the Endothelial Cell Facility RuG/AZG, Groningen, The Netherlands for isolating and culturing the HUVEC; L. Meinkema, G. Harms, and A. van den Berg for helping setting up the cDNA array analysis; and L. Ashgarnegad, S. van Goor, and H. van der Schaar for technical assistance. S.A.Á., R.J.K., and M.E. are members of UNYPHAR, a network collaboration between the universities of Groningen, Leiden and Utrecht, and the pharmaceutical company Yamanouchi.
References (26)
- et al.
Nuclear integration of glucocorticoid receptor and nuclear factor-kB signaling by CREB-binding protein and steroid receptor coactivator-1
J. Biol. Chem.
(1998) - et al.
Glucocorticoid-mediated repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha synthesis
J. Biol. Chem.
(1996) - et al.
Protein measurement with the Folin Phenol reagent
J. Biol. Chem.
(1951) - et al.
Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase
Thromb. Res.
(1995) - et al.
Multifunctional cytokine expression by human coronary endothelium and regulation by monokines and glucocorticoids
Microvasc. Res.
(1998) - et al.
Signal transduction by tumor necrosis factor and its relatives
Trends Cell Biol.
(2001) - et al.
Production of monocyte chemoattractant protein-1 by bovine glomerular endothelial cells
Kidney Int.
(1995) - et al.
Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells
Cytokine
(2000) - et al.
Dexa inhibits tumor necrosis factor-alpha-induced expression of macrophage inflammatory protein-2 and adhesion of neutrophils to endothelial cells
Biochem. Biophys. Res. Commun.
(2000) - et al.
Adhesion molecules in inflammatory diseases
Drugs
(1998)
Therapeutic strategies for allergic diseases
Nature
Mechanism of gene expression by the glucocorticoid receptor: role of protein–protein interactions
Bioessays
Anti-inflammatory actions of glucocorticoids: molecular mechanisms
Clin. Sci. (Lond.)
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