Delivery of pharmacologically active dexamethasone into activated endothelial cells by dexamethasone–anti-E-selectin immunoconjugate

doi:10.1016/S0006-2952(03)00122-9

Biochemical Pharmacology

Volume 65, Issue 10, 15 May 2003, Pages 1729-1739

https://doi.org/10.1016/S0006-2952(03)00122-9 Get rights and content

Abstract

To deliver selectively anti-inflammatory agents into activated endothelial cells, drug-targeting conjugates were developed. Dexamethasone (Dexa) was covalently linked to a monoclonal antibody specifically recognizing E-selectin, which is strongly upregulated in endothelial cells at inflammatory sites. In the present study, the pharmacological effects of this Dexa–mouse antihuman E-selectin antibody (H18/7) (Ab_hEsel) conjugate were investigated and compared to the effects obtained by free Dexa in human umbilical vein endothelial cells. Flow cytometry and ELISA were performed to analyze the levels of cell adhesion molecules (ICAM-1 and VCAM-1) and secreted cytokines (IL-6 and IL-8). The studies were extended by analysis of a complex gene expression pattern, using a cDNA expression array containing 268 genes encoding human cytokines/cytokine-receptors. Fifty genes and 28 genes were upregulated (ratio≥2) upon incubation of human umbilical vein endothelial cells with TNFα for 6 and 24 hr, respectively. This gene expression profile was markedly altered when cells were activated with TNFα in the presence of Dexa (100 nM) or Dexa–Ab_hEsel conjugate (10 μg/mL conjugate corresponding to 100 nM Dexa). Relative and competitive RT–PCR analysis verified downregulation of TNFα-mediated expression of CD40L and IL-8 by Dexa and Dexa–Ab_hEsel, respectively. These results indicated a successful internalization and processing of Dexa–Ab_hEsel in activated endothelial cells, allowing the intracellularly delivered Dexa to exert its pleiotropic anti-inflammatory activity.

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–Ab_hEsel 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–Ab_hEsel conjugate

Dexa was conjugated as described previously [13]via a succinate linker to Ab_hEsel (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–Ab_hEsel 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–Ab_hEsel conjugate in HUVEC

The conjugate Dexa–Ab_hEsel used throughout this study contained an average of two Dexa molecules per molecule Ab_hEsel. Characterization of the conjugate and determination of the Dexa:antibody ratio was described previously [13]. The conjugate Dexa–Ab_hEsel 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.

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Delivery of pharmacologically active dexamethasone into activated endothelial cells by dexamethasone–anti-E-selectin immunoconjugate

Abstract

Introduction

Section snippets

Preparation and characterization of Dexa–AbhEsel conjugate

Uptake of Dexa–AbhEsel conjugate in HUVEC

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Thromb. Res.

Microvasc. Res.

Trends Cell Biol.

Kidney Int.

Cytokine

Biochem. Biophys. Res. Commun.

Adhesion molecules in inflammatory diseases

Drugs

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.)

Preparation and characterization of Dexa–Ab_hEsel conjugate

Uptake of Dexa–Ab_hEsel conjugate in HUVEC