Neutrophil-Derived Heparin-Binding Protein (HBP/CAP37) Deposited on Endothelium Enhances Monocyte Arrest under Flow Conditions

Abstract

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca2+ that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.

Neutrophil-derived azurocidin alarms the immune system

“…In fact, it has been shown that azurocidin with preference binds to monocytes [25,26]. Once monocytes in flow recognize azurocidin presented on the endothelial surface, a mobilization of intracellular Ca 2ϩ is initiated, which is crucial for the azurocidin-mediated adhesion of monocytes [23]. Similar to PMN-derived elastase and proteinase-3 [27,28] monocyte adhesion stimulated by azurocidin was mediated via ␤ 2 -intergins (unpublished data).…”

“…Interestingly, azurocidin is only deposited by adherent but not rolling PMN, indicating that PMN activation via ␤ 2 -integrins is an important signal for discharge of secretory vesicles. The accumulation of azurocidin on the endothelium is reduced by treatment with heparinase and chondroitinase, suggesting that negatively charged proteoglycans in the endothelial glycocalyx act as primary binding sites [23,24]. A specific receptor for azurocidin on EC has not been identified.…”

“…Following treatment with azurocidin, a strong and transient enhancement of intracellular Ca 2ϩ was observed shortly, indicating a receptor-signaling mechanism to be involved in this event [23,26]. Early findings by Cai and Wright [28] pointed at a possible interaction between azurocidin and ␤ 2 -integrins (CD11/CD18).…”

“…Azurocidin, a major component of secretory vesicles, is strongly, positively charged [20] and may thus accumulate on the negatively charged EC surface. In this way, azurocidin becomes immobilized on the endothelium and thereby exposed to cells in the blood flow [23] (Fig. 1A).…”

Distinct Infiltration of Neutrophils in Lesion Shoulders in ApoE−/− Mice

“…Neutrophil granule proteins, which are released on neutrophil activation, may efficiently perpetuate lesion growth and contribute to destabilisation and rupture. 27,28 Granule contents activate endothelial cells leading to subsequent induction of adhesive 29 and chemotactic proteins 30 directed at recruiting other leukocytes. Neutrophil proteins may also influence antigen-presenting cells and T-lymphocytes to enhance antigen-presentation.…”