Neutrophil Adhesion to Fibrinogen and Fibrin Under Flow Conditions Is Diminished by Activation and L-Selectin Shedding

Abstract

The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by β2-integrins. However, the influence of physiologic flow conditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil activation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of ) neutrophil adhesion, real-time video-assisted microscopy and custom-made software were used. Under flow conditions, adherent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the adherence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10−7 mol/L) or 4β-phorbol, 12-myristate, 13-acetate (PMA; 100 ng/mL) resulted in approximately 50% inhibition of adhesion to fibrin and a more random distribution (cluster index <0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respectively. Under these conditions, anti–L-selectin antibodies or neuraminidase did not affect adhesion. These results indicate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin–mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interaction. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation.

P-selectin and MAC-1 mediate monocyte rolling and adhesion to ECM-bound platelets under flow conditions

“…Different types of adhesion were observed; primary tethering of monocytes to the platelet surface, rolling adhesion, and firm attachment. In recent publications secondary tethering of flowing leukocytes to already surface-adherent leukocytes was described as an important mechanism for enhanced leukocyte recruitment [27][28][29][30]. Although this type of monocyte interaction was also observed in our adhesion assay at higher shear stresses, most cells primarily adhered to the surface (results not shown).…”

“…Although this type of monocyte interaction was also observed in our adhesion assay at higher shear stresses, most cells primarily adhered to the surface (results not shown). Cluster or string formation, which is a result of secondary tethering, was less evident compared with neutrophil clusters observed on fibrinogen [30]. This is probably due to the high capacity of platelets to induce P-selectin-mediated primary tethering.…”

Dexamethasone enhances interaction of endogenous Annexin 1 with L‐selectin and triggers shedding of L‐selectin in the monocytic cell line U‐937

“…On the contrary, several studies investigating the role of L‐selectin shedding in leukocyte migration demonstrated an important role for L‐selectin in enabling leukocytes to respond and migrate effectively to chemotactic stimuli ( Jung et al , 1998 ; Jung & Ley, 1999; Hickey et al , 2000 ). Blockade of L‐selectin shedding decreased leukocyte rolling in vivo (reviewed in Ebnet & Vestweber, 1999) and diminished neutrophil adhesion ( Kuijper et al , 1997 ). While these latter studies appear paradoxical, it is possible that: first, L‐selectin is not critical for leukocyte migration while it may still have a functional role in capturing leukocytes; second, studies have shown that selectins have overlapping functions (Jung & Ley, 1999; Steeber et al , 1999 ).…”

Fibrin Matrices as (Injectable) Biomaterials: Formation, Clinical Use, and Molecular Engineering

“…These actions can be a result of cells directly interacting with fibrin; for example, platelets adhere through the high fibrin affinity of α II β 3 integrin and fibroblasts directly bind to fibrin fibers through α (v) β 3 binding to exposed RGD sites . Inflammatory cells such as neutrophils can adhere to fibrin through a mix of interactions involving P‐selectin and integrins, possibly more specifically through the binding of the αMβ2/Mac‐1 to sites in the γ chain, specifically to the 383–395 sequence that is cryptic in fibrinogen but becomes exposed upon immobilization or specific enzymatic cleavage. It is worth mentioning that adhesion to fibrin, however, appears to be strongly dependant on flow conditions; for example, when fibrin is forming in the flow of a fibrinogen‐containing medium, neutrophils can end up coating themselves in fibrin (but not in fibrinogen)…”