Effects of endothelial basement membrane on neutrophil adhesion and migration

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Abstract

The influence of the sub-endothelial basement membrane (BM) on the adhesion and migration of leukocytes is not well-defined. We therefore investigated the behaviour of human neutrophils on purified BM proteins and on BM deposited by short- or long-term cultures of endothelial cells (EC). The adhesion, but not migration velocities, of neutrophils activated with interleukin-8 was dependent on the coating concentrations of purified collagen, laminin or fibronectin. In contrast, adhesion was similar on matrices deposited by 3-day or 20-day cultures of EC, but neutrophils migrated more slowly on the distinct BM that formed over 20 days. In addition, while adhesion on all surfaces was greatly reduced when neutrophils were treated with antibody against β2-integrins, antibody against β1-integrins only inhibited adhesion to the 20-day BM. Thus, the native BM has distinct effects on integrin usage and migration by neutrophils, which are not reproduced by purified proteins or matrix deposited early during endothelial culture.

Introduction

Recruitment of neutrophils into inflamed tissue requires them to adhere to the luminal surface of the vascular endothelium, and to migrate through the endothelial cell monolayer and the subendothelial basement membrane. The adhesion molecules and chemotactic agents that regulate the initial stages of the adhesion and migration of neutrophils have been well described [1], [2]. However, the mechanisms by which neutrophils interact with the BM are not well-defined. Recent studies indicate that, in the mouse at least, crossing the BM is a separately regulated stage during extravasation that requires upregulation of β1-integrins [3]. Comparable information in humans is lacking, largely because of lack of experimental models. The BM is a complex structure containing mainly collagen type IV, fibronectin, laminin, entactin and heparan sulphate proteoglycans (HSPG) [4]. Huber and Weiss [5] showed that after 21 days of culture, human umbilical vein endothelial cells (HUVEC) deposited a BM which resembled that found in vivo. We previously reported that transmigrated neutrophils moved at a slower velocity underneath 20-day cultures of HUVEC, compared to shorter term cultures [6], suggesting that behaviour of neutrophils was influenced by the fully-formed BM. To study this possibility directly, here we compared the adhesion and migration of neutrophils on matrix deposited by EC monolayers after 3 or 20 days, and on purified matrix proteins.

Previous studies of neutrophil behaviour on purified proteins or substrate deposited by short-term endothelial cultures, have often given contradictory results. For instance, resting neutrophils adhered more efficiently to laminin than type IV collagen [7], but the opposite was found for neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) [8]. Neutrophils activated with PMA adhered to collagen type IV and fibronectin through β2-integrins [8], [9], but migration of neutrophils stimulated with formyl peptide (fMLP) through collagen gels was mainly dependent on β1-integrins in one study [10], and the importance of β2-integrins was dependent on gel concentration in another [11]. Blockade of β1- and β2-integrins was required to inhibit adhesion to subendothelial matrix deposited by HUVEC over 24 h or to laminin for neutrophils stimulated with PMA or tumour necrosis factor-α[9]. However, blockade of β2-integrins alone was sufficient to inhibit adhesion of neutrophils activated with formyl peptide, platelet-activating factor or activated complement fragment C5a [9]. More recent studies found that binding of PMA-treated neutrophils to laminin was blocked by antibodies against αmβ2-integrin but not affected by antibodies against β1-integrins [12]. Thus, adhesion, migration and integrin-usage by neutrophils may depend on the substrate and activating agent used, and purified proteins may not accurately mimic the BM. Here, we describe behaviour of neutrophils (either unstimulated or activated with interleukin-8, IL-8) on fully-formed BM for the first time, and show that BM influences integrin usage and migration in a manner not predicted by studies on purified proteins or on less well-developed endothelial matrix.

Section snippets

Preparation of surfaces coated with endothelial cell BM or purified proteins

HUVEC were isolated and maintained as previously described [6]. Confluent primary cultures were passaged into six-well tissue culture plates (Falcon; Becton Dickinson Labware, NJ, USA), and cultured for 3 or 20 days. EC monolayers were then detached from their substrate using phosphate buffered saline (PBS) containing 0.5% Triton X-100 and 20 mM NH4OH (all Sigma). The method preserved the basement membrane intact and completely removed the endothelial monolayer [6]. In some experiments, the

Neutrophil adhesion and migration on purified proteins

Adhesion and migration were studied for resting or IL-8-activated neutrophils settled on surfaces coated with albumin or with different concentrations (0.02–20 μg/ml) of purified LN-1, Coll-IV or FN before blocking with albumin. Adhesion of unstimulated neutrophils to any of the proteins was negligible (<1% in all assays), but adhesion was increased by treatment of neutrophils with IL-8. Throughout the study, in the presence of IL-8, neutrophil adhesion on albumin was 7.6% ± 0.8 (mean ± SEM 27

Conclusions

Our results show that the adhesion, integrin usage and migration of neutrophils are directly modified by contact with basement membrane structures resembling those found in microvessels. The comparisons of adhesion and migration on endothelial matrix deposited over different times are the first to our knowledge. Levels of adhesion did not vary greatly between these surfaces, but integrin usage by neutrophils and migration velocity did change as a complete basement membrane evolved. Behaviour of

Acknowledgment

This work was supported by a Grant from the British Heart Foundation. Umbilical cords were collected with the assistance of the Birmingham Women’s Health Care NHS Trust.

References (27)

Cited by (11)

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    We previously found that pre-treatment of neutrophils with function-blocking antibodies against β1- or β3-integrins had no effect on the ability to cross endothelial monolayers or motility underneath them in short-term cultures [23]. However, neutrophil β1-integrins did influence adhesion to BM from 20-day cultures [17], and leukocyte integrins of each of these families are capable of binding matrix proteins [24]. Thus, evaluation of the roles of different integrins in migration through the subendothelial space requires models where realistic BM is present.

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