β1- and β2-integrins: central players in regulating vascular permeability and leukocyte recruitment during acute inflammation

Abstract

The integrin family, an indispensable part of cell-cell and cell-matrix interactions, consists of a group of heterodimeric adhesion receptors formed by α- and β-integrin subunits. Their wide expression and unique bidirectional signaling pathways allow them to play roles in a variety of biological activities including blood clot formation, cell attachment, and migration. Evidence suggests that integrins are essential regulators of the initiation of acute inflammation, especially two key aspects of this process i.e., vascular permeability and leukocyte recruitment. This mini-review discusses the importance of integrins at the onset of the acute inflammatory response and outlines research advances regarding the function of integrins and their modulators at different stages of this process. Insights into the fine-tuning of integrin signaling during acute inflammation may inspire the design of new drugs for inflammatory diseases.

INTRODUCTION

Acute inflammation is an immediate and innate cascade of cellular defenses in response to infection or injury, mainly characterized by edema and leukocyte infiltration (mostly neutrophils). Although inflammation is initiated to limit tissue damage, excessive activation of the inflammatory response can have devastating consequences and lead to acute tissue injury, toxic shock, and multiorgan dysfunction as recently observed in patients with COVID-19.

The onset of acute inflammation can be temporally divided into two phases, namely, a vascular phase and a cellular phase, which are responsible for vascular permeability and leukocyte extravasation, respectively. In the present mini-review, we consider the family of integrins as mediators of both phases of acute inflammation and discuss how they, together with their regulators, influence the inflammatory response.

INTEGRIN FAMILY

Integrins are a family of transmembrane cell surface adhesion receptors consisting of at least 24 transmembrane heterodimeric pairs that are generated from 18 α-subunits and 8 β-subunits by noncovalent association. According to ligand specificity, they can be broadly separated into four groups, namely, RGD-binding receptors, leukocyte-specific receptors, laminin-binding receptors, and collagen-binding receptors (Fig. 1A). As well-known bidirectional signaling receptors, integrins possess the ability to integrate intracellular and extracellular information by inside-out and outside-in signaling pathways (1). Owing to their important roles in mediating both cell-cell and cell-extracellular matrix interactions, much effort has been devoted to explore their involvement in a variety of diseases.

Figure 1.

Integrins classification and bidirectional activation during leukocyte-endothelial cell interaction. A: integrin classification: integrins are broadly classified into RGD-binding receptors, laminin-binding receptors, leukocyte-specific receptors, and collagen-binding receptors. B: integrin bidirectional activation during leukocyte-endothelial cell interaction (inside-out signaling): selectins and chemokines presented on the endothelial surface trigger talin and kindlin recruitment to the integrin β-cytoplasmic tail inside the leukocyte, leading to an allosteric change toward a high-affinity conformation with the head-piece extended and ligand-binding site open. Outside-in signaling: subsequent ligation of integrin induces salt-bridge dissociation and integrin clustering, connecting the cytoplasmic tail to the cytoskeleton. RGD, arginine-glycine-aspartate. Created with BioRender; published with permission.

ROLE OF β1-INTEGRINS IN VASCULAR PERMEABILITY

Endothelium integrity is the structural foundation of normal endothelial barrier permeability, which is controlled by endothelial cell-cell adhesions including tight junctions and adherens junctions (AJs). Exposure to proinflammatory stimulation disrupts AJs and tight junctions, resulting in vascular leakage (2). Although endothelial adhesion to the matrix is also critical for barrier integrity, to date, only few recent studies provide insights into the role of endothelial β1-integrins in mediation of endothelial barrier function during inflammation. β1-integrins are localized in focal adhesions (FAs), where they are complexed with vinculin and talin, both of which have also been linked to AJ stability, suggesting cross talk between FAs and AJs during inflammation (3). In an LPS-induced murine endotoxemia model, both heterozygous deletion of endothelial cell (EC) β1-integrin and pharmacological inhibition with a β1-integrin blocking antibody significantly decreased vascular leakage after the onset of acute inflammation (4). In the same study, stimulation of ECs with inflammatory agents such as IL-1β, thrombin, or LPS led to the disruption of vascular endothelial (VE)-cadherin junctions, and β1-integrin-mediated induction of actin stress fibers which could be inhibited by β1-integrin antibodies. The authors proposed that inflammation-induced centripetal translocation of β1-integrins to tensin-1-rich fibrillar adhesions, which differ from peripheral talin-1-positive focal adhesions in stable monolayers, destabilizes EC junctions by remodeling the cytoskeleton and generating more centripetal pulling forces.

Another proposed pathway connecting β1-integrin to adherens junctions is through talin-1. The binding of the talin-1 N-terminal domain to the cytoplasmic tail of β-integrin has been demonstrated as a key step in integrin activation (5). Although the significance of talin-dependent activation of integrins has been demonstrated in hematopoietic cells (6, 7), its role in ECs of established blood vessels cannot be verified by gene ablation, given the embryonic lethality of Talin1 EC-specific deletion in mice (8). However, by conditionally deleting Talin1 in the endothelium of adult mice, researchers detected disrupted AJs, pronounced intestinal vascular leakage, and death within 3 wk, which were proposed to be the result of impaired β1-integrin activation (9). The interconnection between talin1 and β1-integrin in ECs was more directly demonstrated by showing that β1-integrin blockade phenocopied talin1 depletion in wild-type ECs, whereas antibody-mediated activation of β1-integrin rescued the EC barrier function in talin-deficient ECs (9). Moreover, transfection of talin-deficient HUVECs with the talin1 integrin-activating domain, rather than the integrin-binding domain, restored junctional VE-cadherin organization. The morphological and functional changes displayed by talin-deficient ECs are associated with increased cell contractility (10), which is consistent with increased actin stress fiber formation and myosin light chain phosphorylation in talin-deficient ECs (9).

At first glance, the results of studies by Pulous et al. (9) and Hakanpaa and Kiss (4) appear conflicting, even though these studies were performed in distinct organs (intestine vs. lung). However, taken together, they raise the interesting possibility that the localization (peripheral vs. central) and state of activation of β1-integrin, along with its intracellular binding partners (tensin vs. talin) and the signaling pathways which they activate, determine the role of integrin signaling in EC monolayer integrity and vascular permeability during inflammation (Fig. 2).

Figure 2.

β1-integrin and its cytoplasmic binding partners play primary roles in vascular permeability. In quiescent endothelium, monolayer integrity is maintained by VE-cadherin in adherens junctions and talin-1 in focal adhesion complexes. Upon inflammation, β1 integrins translocate to α5β1-integrin and tensin-1-positive fibrillar adhesions, leading to increased tension and VE-cadherin junction disassembly. Talin-1 deficiency causes VE-cadherin internalization and inactivation of β1-integrin contributing to high contractility and vascular leakage. VE-cadherin, vascular endothelial cadherin. Created with BioRender; published with permission.

ROLE OF β1- AND β2-INTEGRINS IN LEUKOCYTE RECRUITMENT

The cellular phase of inflammation consists of leukocyte recruitment, adhesion, and transmigration through the endothelium to the site of injury. Leukocyte recruitment is orchestrated by adhesive molecules, chemokines, cytokines, and other regulators and usually occurs in postcapillary venules. This cascade of events consists of sequential adhesive interaction between leukocytes and ECs, including selectin-dependent tethering, chemokine-induced leukocyte activation and integrin-mediated adhesion, crawling and transmigration (Fig. 3) (11). Although the overall importance of integrins at each step is undeniable, many of the signaling pathways involved are still poorly understood. Given that leukocyte recruitment during acute inflammation is mainly characterized by an overwhelming influx of neutrophils and monocytes/macrophages, we therefore focus on integrins expressed in these two cell types.

Figure 3.

Leukocyte adhesion cascade and integrin regulation. The leukocyte adhesion cascade is a multistep process consisting of slow rolling/tethering, arrest, crawling, and transmigration with multiple soluble and intracellular regulators that are described in detail in the text. Created with BioRender; published with permission.

Rolling and Tethering

Inflammatory cytokines stimulate surface expression of E-selectin and P-selectin on ECs, allowing for interaction with their cognate counter-ligands on leukocytes, slowing their rolling velocity. Selectin-driven rolling allows endothelial-bound chemokines such as the chemokine (C-X-C motif) ligand 1 (CXCL1) to activate leukocytes. This in turn stimulates inside-out signaling of integrins and causes a conformational change of leukocyte integrins from an inactive “bent and closed” state to an active “extended and open” state, leading to the next phase of leukocyte recruitment—adhesion (12, 13).

Adhesion

Activated integrins exhibit high binding affinity to ligands on the EC surface, which then triggers outside-in signaling, enhancing the avidity of integrins by inducing clustering (Fig. 1B). β2-Integrins on neutrophils, αLβ2-integrin (a.k.a lymphocyte function associated antigen-1, LFA-1) and αMβ2 (a.k.a. macrophage-1 antigen, Mac-1), are vital for their neutrophil influx into injured tissue, whereas α4β1-integrin (a.k.a. very late antigen-4, VLA-4) is essential for monocyte adhesion (11). In addition, deficiency in αXβ2-integrin attenuates the ability of monocytes/macrophages to adhere and to kill pathogens, indicating its critical role in their function (14).

Crawling

After adhering to ECs, leukocytes actively move to cellular junctions and start transmigration within seconds. The pattern of monocyte crawling in vivo varies under different situations: on unstimulated venules, monocytes crawl in an LFA-1-dependent manner, but after TNF-α-induced upregulation of adhesion molecules on endothelial cells, crawling becomes Mac-1-dominated. In contrast, neutrophil crawling is not dependent on LFA-1 in inflamed venules, but similar to monocytes, knockout of Mac-1 significantly decreases the percentage of crawling neutrophils along with decreased distance and velocity (15). Once at the junction, leukocytes can start transmigration from the vascular lumen into the inflamed tissue.

Transmigration and Migration

The mechanisms of integrin-mediated leukocyte transmigration have been mostly studied in neutrophils but are applicable to monocytes. At the preferred site of transmigration, the endothelial cell-cell junctions are temporally disrupted owing to leukocyte integrin engagement with intercellular adhesion molecule 1 (ICAM-1) (16), creating a space through which leukocytes can pass. Neutrophils move through this space in a Mac-1 and LFA-1-dependent manner (17). Hyper-responsive neutrophils, defined by their expression of α3β1-integrin (a.k.a. very late antigen-3, VLA-3), tend to migrate beyond the EC layer during sepsis and are associated with Toll-like receptor-induced inflammatory responses and cytokine production (18). Infiltrated neutrophils can release myeloperoxidase (MPO) during migration, leading to polyunsaturated fatty acid oxidation and formation of 2-(ω-carboxyethyl) pyrrole (CEP) adducts of extracellular matrix proteins. These CEP adducts can be recognized by αMβ2- and αDβ2-integrins on macrophages and in turn augment their migration during inflammation (19).

Regulators of Integrins during Leukocyte Recruitment

Abundant evidence shows that either genetic deletion or pharmacological inhibition of integrins can reduce leukocyte extravasation and protect from pathological inflammation (12). Moreover, many extracellular and cellular proteins have been demonstrated to influence integrin-mediated leukocyte recruitment.

Extracellular modulators.

Growth differentiation factor 15 (GDF-15), a member of the transforming growth factor-β (TGF-β) family, is the first cytokine known to counteract the extravasation of myeloid cells by rapidly interfering with chemokine-induced integrin activation (20). On myeloid cells, it binds to activin receptor-like kinase 5 (ALK-5) and TGF-β receptor II (TGF-βRII) heterodimer. After binding to its receptors, GDF-15 inhibits activation of Rap-1 (21), a GTPase required for the transition of the integrin complex to its active conformation. In addition, the EC secreted glycoprotein, developmental endothelial locus 1 (DEL-1), inhibits leukocyte adhesion during inflammation by competing with ICAM-1 on endothelial cells for binding to the LFA-1 integrin on neutrophils (22). In mice, genetic deletion of DEL-1 gives rise to elevated neutrophil infiltration in LPS-induced lung inflammation and experimental autoimmune encephalomyelitis (23). Recently, the matricellular protein vitronectin was shown to complex with plasminogen activator inhibitor-1 on the endothelial surface and then to interact with low-density lipoprotein receptor-related protein-1 (LRP-1) on neutrophils, leading to clustering of β2-integrins on the neutrophil surface and stabilization of adhesion (24).

Cellular modulators.

In addition to the secreted modulators discussed above, some recently described intracellular proteins add to the complexity of integrin regulation. These generally fall into the category of G-protein-related proteins and kinases or cytoskeletal elements, and act to enhance the formation of integrin complexes or increase the activation state of integrins. GIV, a guanine nucleotide-exchange modulator of the trimeric GTPase Gαi, has been shown to maximize β1-integrin activation and clustering in a cancer metastasis model by binding to the integrin adaptor kindlin-2, resulting in augmented cell adhesion, spreading, and invasion (25). Integrin-linked kinase (ILK) also binds kindlin-2 in the β2-integrin complex, thus inhibiting the conformational change of β2-integrin to the high-affinity state, ultimately impairing neutrophil adhesion and extravasation (26). Deficiency of the Rho GTPase-activating protein 15 (ARHGAP15) leads to the prolongation of chemokine-dependent leukocyte adhesion, which enhances Mac-1 affinity, resulting in defective crawling capacity and infiltration (27).

Cytoskeletal-associated proteins, such as nonmuscle myosin light chain kinase (MYLK), also take part in neutrophil transmigration. In an LPS-induced lung injury model, MYLK was shown to be essential for the activation of protein tyrosine kinase 2 (Pyk2), which mediates full activation of β2-integrins and is thus required for neutrophil recruitment (28). Moreover, coronin 1A, an actin-binding protein, can regulate the aggregation of high-affinity LFA-1 in focal zones of adherent cells by interacting with the cytoplasmic tail of β2-integrins, thus promoting the transition of neutrophil rolling to firm adhesion (29).

Finally, endomucin, a glycoprotein expressed on the luminal surface of venous and capillary ECs, is a novel player in integrin regulation. Although glycoproteins in general and heparan sulfate proteoglycans in particular block leukocyte adhesion, new evidence shows that deficiency of endomucin facilitates firm neutrophil adherence to quiescent endothelium via LFA-1-ICAM-1 binding and that endomucin is downregulated concurrently with ICAM-1 upregulation in inflammatory conditions (30). Conversely, adenovirus-induced overexpression of endomucin prevents neutrophil adhesion in vitro.

CONCLUDING REMARKS

As discussed in this review, integrins and their interacting partners play central roles in both the vascular and cellular phases of acute inflammation. Both temporal and spatial factors influence integrin participation in various subpopulations of cells and at different stages of the leukocyte adhesion cascade. The balance of activation and inhibition of integrins and how their downstream pathways interact with each other is an important area for further investigation. The recent focus on pathways and molecules that regulate integrin activation is an important area of research and is likely to identify new therapeutic targets to mitigate the inflammatory response. Thus, even though integrin blockade may significantly modulate the initiation and/or progression of inflammatory disease and some antibodies have been adopted clinically, better understanding of the mechanisms governing this process and identification of their modulators is essential for the development of therapeutic agents with fewer side effects.

GRANTS

This work is supported by National Heart, Lung, and Blood Institute Grants HL95070, HL151133, and HL152167.

DISCLOSURE

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Z.O. prepared figures; Z.O. drafted manuscript; Z.O., E.V.D., B.L., and K.K.G. edited and revised manuscript; Z.O., E.V.D., B.L., and K.K.G. approved final version of manuscript.