Scott I. Simon

Neutrophil Tethering on E-Selectin Activates β2 Integrin Binding to ICAM-1 Through a Mitogen-Activated Protein Kinase Signal Transduction Pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of β2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via β2 integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears ≥0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that ∼60% of the interacting cells remained firmly adherent, as compared with ∼10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through β2 integrins binding to ICAM-1.

L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase.

The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of β2-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules.

P-selectin mediates neutrophil adhesion to endothelial cell borders.

During an acute inflammatory response, endothelial P‐selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P‐selectin is stored in secretory granules (Weibel‐Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P‐selectin‐dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10‐4 M) or thrombin (0.2 U/mL) treatment induced preferential (≥ 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P‐selectin dependent. P‐selectin surface expression increased significantly after histamine treatment and P‐selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P‐selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders. J. Leukoc. Biol. 65: 299–306; 1999.

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organisms pathogenesis.

Quantitation and prevalence of tympanosclerosis in a pediatric otolaryngology clinic

OBJECTIVEnTo determine the incidence and prevalence of tympanosclerosis (TS) in patients seen in a pediatric otolaryngology clinic.nnnDESIGNnProspective observational study.nnnSETTINGnTertiary care, ambulatory care clinic.nnnPATIENTS AND OTHER PARTICIPANTSnWe conducted a prospective observational study to determine the incidence of TS in 218 patients seen consecutively in a pediatric otolaryngology clinic for otologic and nonotologic reasons. The patient age range was 3 weeks to 30 years; 63% were male and 37% female. Of the 218 patients, 37% (81) had undergone bilateral myringotomy and tube placement (BM&T), and 35% (21) of the 81 showed signs of TS; 63% (137) of the 218 patients had no history of otologic surgery, but 12% (15) of the 137 showed signs of TS.nnnMETHODnThe area of the tympanic membrane affected by TS was determined by otoscopy performed by one viewer, who drew the otoscopic findings on a standardized tympanic membrane template. The area of TS was quantified in terms of percentages by digital image analysis of the scaled drawings.nnnRESULTSnThere was a range of 0.5-59.9% involvement of the tympanic membrane with TS, with the median percentage of involvement being 4.95%. There was an increased percentage of TS with repeat BM&T.nnnCONCLUSIONnThis observational study shows that patients who have had BM&T have a higher incidence of TS than those who have not had the surgery. However our findings also show that 38% of the patients in this study who had TS had never undergone BM&T.

Induction of homotypic lymphocyte aggregation: evidence for a novel activation state of the beta1 integrin.

Intercellular adhesion of Jurkat lymphocytic cells was investigated by use of monoclonal antibodies 33B6 and 18D3, which bind to the β1 integrin receptor. 33B6 induced homotypic aggregation of Jurkat cells, whereas 18D3 inhibited this aggregation. Jurkat cells could be induced to aggregate at low 33B6 concentrations corresponding to 5% β1 integrin site occupancy, and the rate of aggregation was maximum at 30% occupancy. Simultaneous addition of mAb 18D3 and 33B6 demonstrated that the two antibodies mediate changes in the β1 integrin activation state that are competitive in nature. Aggregation through β1 integrin induced by 33B6 was reversed by subsequent addition of 18D3. To further examine the mechanism by which 33B6 and 18D3 affect cell adhesion function, we explored the binding of monoclonal antibody (mAb) 15/7. This mAb recognizes an activation epitope of the β1 integrin and has been shown to sustain cell adhesion to vascular cell adhesion molecule 1 (VCAM‐1) and fibronectin. Activation of Jurkat cells with Mn2+ caused a 2.5‐fold increase in 15/7 binding but did not increase binding of 33B6. 33B6 partially blocked 15/7 binding to β1 integrin on unstimulated and Mn2+‐activated Jurkat cells. 18D3 did not affect mAb 15/7 binding. These results indicate that 33B6 and 18D3 modulated homotypic aggregation by inducing a novel activation state of the very late activation integrin distinct from the state recognized by 15/7, which supports cell binding to VCAM‐1 and fibronectin.

Co-operative signaling between leukocytes and endothelium mediating firm attachment

This chapter focuses on the interactions of leukocytes and endothelial cells that are necessary for adhesion sufficiently stable to withstand the shear forces of flowing blood. Some aspects of the interplay between shear rates and the functions of specific adhesion molecules will be discussed, as will possible signaling steps in the cascade of events leading to stable adhesion (though signaling pathways will not be discussed). In addition, simple observations of cell interactions will be presented to emphasize the potential influence of surface topography on the sequence of events that control firm adhesion and its localization.

Molecular Mechanisms of Neutrophil Adhesion Studied by Inducing a High Avidity State at β2-Integrin

The immune host response of neutrophils is dependent upon adhesion to endothelium, chemotaxis into the periferral tissue, and phagocytosis of microorganisms. Essential to these adhesion dependent functions are the Leu-CAM family of β2-integrin adhesive molecules which consist of 3 distinct alpha subunits (LFA-1 CD11a, Mac-1 CD11b, and P150.95 CD11c), noncovalently linked to a common beta subunit (CD18) (for review see ref. 1). The physiologic role of these adhesion molecules is elucidated in patients presenting with a deficiency in leukocyte integrins. These patients suffer from opportunistic infections due to an inability of their neutrophils to firmly adhere and migrate across the endothelium and fight tissue borne infections (2). Normal neutrophils isolated from blood respond to the binding of chemotactic formyl peptide to its surface receptor by an increase in the adhesive avidity of CD18 to bind a multitude of ligands including ICAM-1, fibrinogen, and an as yet uncharacterized ligand on the neutrophil plasma membrane (3). At saturating doses of chemotactic peptide (>500 nM) Mac-1 is rapidly mobilized to the plasma membrane from intracellular granular stores, increasing the surface expression ≈10 fold over constitutive levels (3). However, recent studies from our laboratory and others indicate that only a portion of these sites participate in adhesion. Stimulation of neutrophils in a step-wise fashion by binding nanomolar concentrations of formyl peptide results in newly mobilized Mac-1 which participate in adherence-dependent locomotion (4). It has been recently reported that as little as 10% of the surface Mac-1 on activated cells participate in binding to fibrinogen and ICAM-1 (5). The high avidity state of Mac-1 appears to be mediated by a change in the structure or conformation of the epitope.

Mechanico-transduction through selectins signals integrin mediated neutrophil arrest under shear flow

Evidence is provided for activation of neutrophils rolling on E-selectin, independent of signaling through G-proteins. A cooperative mechanism in which L-selectin and PSGL-1 were required for optimal activation was found. Neutrophil emigration appears to be regulated by shear and signals transduced through selectins during tethering and rolling.

Multistep processes in neutrophil homotypic aggregation and tissue injury

This review will deal with two broad aspects of neutrophil function — the mechanisms of neutrophil aggregation, and the mechanisms of neutrophil mediated tissue injury using the liver as a specific example. Much of the work on neutrophil aggregation has been performed in vitro, which allows dissection of the molecular mechanisms involved, and much of the work regarding tissue injury reveals tissue specific features that could not have been predicted by studies of isolated neutrophils in vitro.