Abstract
Antileukoproteinase (ALP) is a physiological inhibitor of granulocytic serine proteases that has been shown to have anti-inflammatory properties in addition to its antiproteolytic activity. On the basis of its potential to block anti-collagen type II (CII) antibody-induced arthritis (CAIA) and to suppress the conformational activation of β2-integrins in leukocytes, the present study was undertaken to investigate its interference with leukocyte adherence to cytokine-activated endothelium. The potential of recombinant ALP to block the interactions of leukocytes with the endothelial lining was concomitantly investigated in vitro and in vivo. Thus, intravital fluorescence microscopic imaging of leukocyte rolling and firm adhesion to postcapillary venules were performed in the knee joints of DBA1/J mice after intravenous injection of anti-CII mAbs. An IL-1β-activated endothelial layer formed by a murine glomerular cell line (glEND.2) was used to assay the interaction with human leukocytes in vitro. Electromobility shift and luciferase reporter gene assays permitted the analysis of cytokine-induced activation of the NF-κB pathway. Fluorescence-activated cell sorting was applied to determine endothelial E-selectin expression. Leukocyte rolling and firm adhesion to the synovial endothelium in an early response to the anti-CII antibody transfer were significantly decreased in ALP-pretreated mice. Concomitantly, ALP suppressed the IL-1β-induced NF-κB activation and the upregulation of E-selectin expression in glEND.2 cells in vitro. These findings support the notion that the newly uncovered properties of ALP to interfere with cytokine signalling and upregulation of adhesion molecules in endothelial cells are likely to contribute to the therapeutic potential of ALP in immune-complex-induced tissue injury.
Highlights
ALP = antileukoproteinase; ANOVA = analysis of variance; CAIA = anti-collagen II antibody-induced arthritis; CFDA-SE = carboxyfluorescein diacetate succinimidyl diester; CII = collagen type II; DMEM = Dulbecco's modified Eagle's medium; EMSA = electrophoretic mobility-shift assay; FACS = fluorescence-activated cell sorting; hSA = human serum albumin; IL = interleukin; mAb = monoclonal antibody; NF = nuclear factor; PBS = phosphate-buffered saline
The results show the fraction of leukocytes rolling along the endothelium of postcapillary synovial venules in anticollagen II (CII) antibody-induced DBA1/J mice pretreated with either antileukoproteinase (ALP; 100 and 300 μg), human serum albumin or NaCl in comparison with healthy mice (HC) that did not receive the antibody challenge
The results show the number of leukocytes adherent to the endothelium of postcapillary synovial venules in antiCII antibody-induced DBA1/J mice pretreated with either ALP (100 and 300 μg), hSA (300 μg) or NaCl in comparison with healthy mice (HC) that did not receive the antibody challenge
Summary
The observed suppression of leukocyte rolling in vivo as a selectindependent interaction with the vessel wall [11] is reconcilable with the newly uncovered blocking effect of ALP on a crucial cytokine signalling pathway in endothelial cells [12] This regulatory potential and the inhibition of firm leukocyte adhesion in vivo by ALP, which probably reflects the already known interference with β2-integrin activation on leukocytes, complement each other in their anti-inflammatory effect. This synergism may contribute to the established capacity of ALP to block leukocyte infiltration and tissue injury in a variety of experimental models of inflammation, such as streptococcal cell wall arthritis [13], CAIA [5] or ischaemia/reperfusioninduced organ damage [14]
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