Leukocyte adherence contributes to disruption of the blood–brain barrier during activation of mast cells

Abstract

The goal of this study was to examine the role of leukocytes in disruption of the blood–brain barrier during activation of mast cells using compound 48/80. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood–brain barrier (clearance of fluorescent-labeled dextran; molecular weight 10 000 daltons; FITC-dextran-10 K) was determined while suffusing with vehicle or compound 48/80 (10 or 25 μg/ml). During superfusion with vehicle (saline), clearance of FITC-dextran-10 K from pial vessels was modest and remained relatively constant during the experimental period (0.52±0.05 ml/s×10⁻⁶ at 80 min). In addition, diameter of pial arterioles remained constant (32±5 μm) while suffusing with vehicle. In contrast, topical application of compound 48/80 produced marked disruption of the blood–brain barrier to FITC-dextran-10 K. For example, suffusion with compound 48/80 (25 μg/ml) increased clearance of FITC-dextran-10 K about 4-fold to 2.26±0.25 ml/s×10⁻⁶ at 80 min. In addition, superfusion with compound 48/80 (25 μg/ml) constricted pial arterioles by 26±9% at 80 min. To determine a potential role for leukocyte adhesion to endothelium in disruption of the blood–brain barrier during suffusion with compound 48/80, we examined permeability during suffusion with compound 48/80 (25 μg/ml) in the presence of WT.3 (2 mg/kg i.v.), a monoclonal antibody directed against the functional epitope of the leukocyte adhesive glycoprotein (CD18; LFA-1β). We found that infusion of WT.3 markedly attenuated disruption of the blood–brain barrier to FITC-dextran-10 K in response to compound 48/80. The clearance of FITC-dextran-10 K during superfusion with compound 48/80 in the presence of WT.3 was 1.29±0.14 ml/s×10⁻⁶ at 80 min (P<0.05). Thus, the findings of the present study suggest that application of compound 48/80, to degranulate mast cells, activates the adhesion of leukocytes to cerebral venular endothelium which contributes to disruption of the blood–brain barrier.

Introduction

The adherence of leukocytes to the endothelium is a key event in the inflammatory process. Ischemia/reperfusion injury and stimulation with a variety of mediators have been shown to stimulate the interaction of circulating leukocytes with vascular endothelium in the peripheral circulation [16], [18], [20], [21], [22], [44]. Further, there is evidence to suggest that mast cells and products released by mast cells play a role in the recruitment of leukocytes to sites of vascular inflammation [7], [19]. The attraction of leukocytes to the endothelium requires the expression of adhesion molecules on leukocytes and/or endothelium. These adhesion molecules can act either by tethering leukocytes to endothelial cells or as signals to induce activation-dependent processes. Several types of proteins are important in the adhesion of leukocytes to endothelium [19], [24]. First, selectins (L-selectin, P-selectin and E-selectin), a family of glycoproteins, are important in the initial interaction of leukocytes to endothelium. Second, β₂ integrins (CD11/CD18 complexes) are important in the adhesion of leukocytes to endothelium. Third, a family of immunoglobulins (ICAM-1 and VCAM-1) appear to be the endothelial counterparts for the β₂ integrins and thus mediate the firm adhesion of leukocytes to the endothelium. Thus, the adherence of leukocytes to the endothelium plays an important role in the inflammatory process.

During cerebrovascular injury a number of vasoactive compounds, including inflammatory mediators, are released by brain tissue [3], [42]. Further, there appears to be an abundance of mast cells in brain tissue, which may degranulate and release vasoactive substances during cerebrovascular injury [5], [6], [14], [15], [43]. Thus, it is conceivable that the synthesis/release of inflammatory mediators and/or activation of mast cells, with the subsequent release of vasoactive products, may stimulate the activation of leukocytes to cerebrovascular endothelium and contribute to disruption of the blood–brain barrier during cerebrovascular trauma. However, few studies have examined the role of leukocyte–endothelium interactions on disruption of the blood–brain barrier. Thus, the goal of the present study was to examine whether activation of leukocytes contributes to disruption of the blood–brain barrier during degranulation of mast cells using compound 48/80. Compound 48/80 has been shown to degranulate mast cells as well as activate the leukocyte–endothelium complex [7], [18], [36]. In addition, to further examine the role of leukocyte adhesion in disruption of the blood–brain barrier during stimulation with compound 48/80 we examined the effects of a monoclonal antibody (WT.3) directed against the functional epitope of the leukocyte adhesive glycoprotein [13], [38], [39].