LEUKOCYTE TRANSMIGRATION ACROSS ENDOTHELIAL AND EXTRACELLULAR MATRIX PROTEIN BARRIERS IN LIVER ISCHEMIA/REPERFUSION INJURY

Abstract

Purpose of review

Hepatic ischemia reperfusion injury (IRI) linked to leukocyte recruitment and subsequent release of cytokines and free radicals remains a significant complication in organ transplantation. The aim of this review is to bring attention to advances made in our understanding of the mechanisms of leukocyte recruitment to sites of inflammatory stimulation in liver IRI.

Recent findings

Leukocyte transmigration across endothelial and extracellular matrix (ECM) barriers is dependent on adhesive events, as well as on focal matrix degradation mechanisms. While adhesion molecules are critical for the successful promotion of leukocyte transmigration by providing leukocyte attachment to the vascular endothelium, matrix metalloproteinases (MMPs) are important for facilitating leukocyte movement across vascular barriers. Among different MMPs, MMP-9, an inducible gelatinase expressed by leukocytes during hepatic IRI, is emerging as an important mediator of leukocyte traffic to inflamed liver.

Summary

It is generally accepted that the understanding of the molecular mechanisms involved in leukocyte recruitment will lead to the development of novel targeted therapeutic approaches for hepatic IRI and liver transplantation. Here, we review mechanisms of leukocyte traffic in liver IRI and the role of some of the proteins that are thought to be important for this process.

INTRODUCTION

Hepatic ischemia reperfusion injury (IRI) is a pathophysiological process in which the hypoxic insult is further accentuated by restoration of blood flow to the compromised organ. Hepatic IRI occurs in all transplanted livers, in trauma, shock, and in elective surgery where blood supply to liver is temporary interrupted. In human orthotopic liver transplantation (OLT), IRI is a major determinant of postoperative allograft dysfunction and morbidity as it causes up to 10% of early transplant failures, and increases the risk of acute and chronic rejections [1–3]. Furthermore, liver IRI limits the supply of organs available for transplantation [4;5*].

The mechanisms of ischemic damage in the liver associated with leukocyte adhesion/migration and with release of cytokines/free radicals play a central role in post-IR organ injury. They lead to a decline in liver function and, potentially, to an increase in organ immunogenicity, which may result in graft loss [2;6]. While the deleterious effects of cytokines and reactive oxygen species (ROS) released by leukocytes have been fairly documented, the mechanisms of leukocyte recruitment to sites of inflammation in the liver are far from being understood.

Overall, the process of leukocyte recruitment across endothelial and extracellular matrix (ECM) barriers involves complex cascades of adhesive and focal matrix degradation events leading to leukocyte tethering and rolling, firm adhesion and, finally, transmigration from the vasculature [7]. Liver is a venous driven vascular bed with slow flow rates and the recruitment of leukocytes to inflamed liver may require distinct adhesive and deadhesive mechanisms as compared with other organs with higher flow rates.

SELECTINS

The selectins (E-, L-, and P- selectin) are a family of glycoproteins that mediate low-affinity endothelial-leukocyte interactions, thus promoting the tethering and rolling of leukocytes through interactions with specific carbohydrate residues [8;9]. The three members of the selectin family share a similar structure containing an N-terminal lectin-like domain, an EGF-like domain, a variable number of consensus repeats, a single transmembrane domain, and a short cytoplasmic tail [10].

L-selectin (CD62L) is constitutively expressed on many leukocytes [11]. It regulates lymphocyte homing into the peripheral lymph nodes through interactions with peripheral lymph node addressins (PNAds), which are constitutively expressed on high endothelial venules (HEV) and induced in different chronically inflamed venules [12*]. P-and E- selectins, on the other hand, are mostly inducibly expressed in both acutely and chronically stimulated endothelium; P-selectin is also present on platelets [13;14]. The expression of these molecules on the vascular endothelium is induced upon exposure to a variety of proinflammatory stimuli; among the factors that induce selectin expression by endothelial cells are shear stress, several cytokines, and complement activation products [15;16]. Leukocytes are considered to first tether to and roll on P- and E-selectins expressed on activated endothelial cells [14]. Their roll on P- and E-selectins is mediated through interactions with P-selectin glycoprotein ligand-1 (PSGL-1), and other carbohydrate ligands, expressed on various leukocyte subsets [14;17–19*]. P- and E-selectins have been shown to play important roles in leukocyte recruitment in a variety of pathological conditions [20–22]; however, their role on leukocyte recruitment in liver IRI remains not fully understood. While a number of reports show that P-selectin blockade is beneficial in liver IRI [23–25], others minimize its role in the recruitment of leukocytes to sites of inflammation in liver [26–28]. In this regard, studies performed in mice deficient in P-selectin, or in both P- and E-selectins, demonstrated a minimal role for selectins in leukocyte recruitment into the inflamed liver microvasculature [27]. A selectin/rolling dependent leukocyte recruitment may not be necessary in the low shear conditions that prevail in the hepatic microvascular bed [29;30*]. Thus, leukocytes moving slowly through the narrow sinusoids may be able to interact directly with other adhesion molecules early expressed on the endothelium without the need of an initial selectin-mediated arrest. In view of the observations that steatosis further decreases sinusoidal blood flow by approximately 50% [31;32], this concept is perhaps even more relevant for marginal fatty livers, which are highly susceptible to hepatic IRI [33]. While there is an indication that selectin blockade have a beneficial role in liver IRI, future studies are still needed to further explain the apparent minimal contribution of selectins to leukocyte recruitment after inflammatory stimulation in liver.

CHEMOKINES

Chemokines are a large family of mostly 8- to 12-kDa proteins, which are essential in regulating directional leukocyte traffic [34]. They can be subdivided in four families according to the position of their cysteine residues (reviewed in Charo et al. [35]. The majority of chemokines belong to the CC (CCL1–28) and CXC (CXCL1-16) subfamilies. The C and CX₃C subfamilies have only 2 members (XCL1 and XCL2) and 1 member (CX₃CL1), respectively [36]. Exposure of leukocytes to chemokines released by inflamed tissues is particularly important for the activation of leukocyte integrins [37], which are key mediators of leukocyte firm adhesion to the vascular endothelium. There are a growing number of reports supporting a role for CC and CXC chemokines in the liver pathophysiology [38*]. Several CC chemokines have been shown to be major attractants for T cells, B cells and monocytes [35]. CCL2 [also known as monocyte chemotactic protein-1 (MCP-1)] is one of the best characterized chemokines in liver; CCL2 is a ligand for the receptor CCR2 and is secreted by several cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs) [39]. CCL2 is highly expressed in livers after toxic or biliary injury and it has been shown that mice with a targeted deletion of the CCR2 gene develop reduced levels of hepatic fibrosis [40]. CCL2 expression has been detected in relatively high levels in hepatic IRI [41]. CXCL-1 [also known as keratinocyte-derived chemokine (KC)] and CXCL-2 [also known as macrophage inflammatory protein-2 (MIP-2)] are considered to be potent neutrophil chemoattractants in liver IRI [42;43]. The murine chemokines CXCL-1 and CXCL-2 bind to the chemokine receptor CXCR2 and are the functional murine homologues of the human IL-8 [44;45]. In our own studies in liver IRI, we found a modest correlation between neutrophil infiltration and the expression of the CXCL-2 chemokine [46], which is primarily induced by TNF-α [47]. The CXCL9 and CXCL10 chemokines are believed to mediate the infiltration of virus-specific T cells in liver [48] and their receptor CXCR3 has been detected on liver infiltrating leukocytes [49]. The CXCL16 chemokine is able to support lymphocyte adhesion by inducing conformational activation of β1 integrins, and its receptor CXCR6 is expressed on liver infiltrating lymphocytes [50]. Moreover, a recent study has provided evidence that CXCL16 may be involved in the recruitment of inflammatory cells in cholestatic liver disease [51*]. During the last several years, chemokines have emerged as important mediators in liver diseases; however, there is still much to be learned about the complexity of chemokine networks involved in leukocyte traffic in liver IRI.

INTEGRINS AND THEIR LIGANDS

It is well accepted that leukocytes have to acquire strong adhesion interactions to the vessel wall to migrate across the vascular endothelium [52]. The firm adhesion of leukocytes to the endothelium is mediated primarily by integrins. Integrins are αβ transmembrane adhesion receptors that mediate cell-cell and cell-ECM adhesion [53]. Each integrin contains one α and one β–subunit, and the 24 integrins identified in mammals are formed from combinations of 18 α-subunits and 8 β-subunits [54*]. The most characterized integrins expressed on leukocytes belong to the β1 and β2 integrin families. Of the β1 integrins, α4 (CD49d) has a central role; it interacts with the connecting segment-1 (CS-1), which is located within the V region of fibronectin (FN) [55], and with a recently described segment, PEDGIHELFP, located in the EIIIA fibronectin splicing domain [56]. In addition, it also binds to the endothelial Vascular Cell Adhesion Molecule-1 (VCAM-1: QIDSPL), recognizing yet a different sequence [57]. The α4β1 integrin, in the absence of the α5β1 integrin, is also able to interact with the RGD sequence, which is present in the cell adhesion domain of FN [55]. Cellular FN is a key ECM protein expressed by sinusoidal endothelial cells very early after liver injury [58], and its vascular expression precedes leukocyte recruitment in hepatic IRI [59]. In addition to its widely reported role on leukocyte adhesion and migration [60], FN is capable of mediating platelet adhesion [61], and may contribute to complement activation [62;63]. VCAM-1, the other ligand for the α4β1 integrin, is mostly detected on large-vessel endothelial cells after liver IRI. α4 integrin is particularly interesting because of its ability to support both leukocyte rolling and adhesion [64]. Clinical trials documented that a humanized α4-integrin antibody have been effective in inflammatory conditions such as multiple sclerosis (MS) [65], and inflammatory bowel disease [66]. Interestingly, it has been recently shown that the anti-α4 integrin antibody infused into MS patients reduced the ex-vivo adhesion of their leukocytes to activated human brain endothelial cells under flow conditions [67*]. Moreover, additional experiments blocking CS1 FN and VCAM-1 interactions, showed that the ligand of α4 integrin on the activated endothelial cells was FN and not VCAM-1 [67]. Our studies in rats also support an important role for the α4β1-CS1 FN interactions in leukocyte recruitment after hepatic IRI [59;68]. Others, using human HSEC and flow-based adhesion in vitro assays, have shown that lymphocyte adhesion to hepatic sinusoids can also be inhibited by blocking VCAM-1 [69]. The β2 integrin family is considered to play a role in neutrophil extravasation from the hepatic microcirculation into the parenchyma [70]. Intercellular adhesion molecule-1 (ICAM-1), a major endothelial-cell ligand for β2 integrins, is constitutively expressed on the liver vascular endothelium. However, hepatic IRI has been shown to be only moderately or not at all improved by anti-ICAM therapies [71]. Observations that leukocyte recruitment within the hepatic sinusoids is likely selectin-independent [30] have further attracted attention to integrins. However, there is still much to be unveiled about the role of integrins and their ligands in hepatic IRI.

MATRIX METALLOPROTEINASES

Leukocyte transmigration across endothelial and ECM barriers is dependent on the expression cell-activating chemokines, adhesive events, as well as on focal matrix degradation mechanisms. Interactions between ECM components and cell adhesion receptors regulate leukocyte functions; therefore, enzymatic degradation of ECM can alter leukocyte behaviors [72]. Leukocyte migration across ECM proteins is dependent on matrix degradation not only for facilitating “matrix permeability”, but also for generating ECM-derived fragments, which are biologically active and can be highly chemotactic for leukocytes [73]. The matrix metalloproteinases (MMPs) are a family of >24 specialized zinc-dependent proteases that play key roles in the responses of cells to their microenvironment [74–76*]. It is generally accepted that while MMP-facilitated degradation of ECM proteins is essential in physiological processes, such as remodeling and tissue repair, MMP inappropriate, prolonged, or overexpression has harmful consequences.

Among the different MMPs, a specific subset, the gelatinases, MMP-2 and MMP-9 (also known as gelatinase A and B or 72-kDa and 92-kDa type IV collagenases, respectively) are of particular interest. MMP-2 and MMP-9 are activated in damaged livers, and are thought to play a key role in liver injury [77]. MMP-9 is an inducible gelatinase expressed mostly by leukocytes, whereas MMP-2 is generally expressed constitutively. MMP-2 is thought to be derived largely from stromal cells, and not usually expressed by leukocytes [78]. These MMPs are characterized by the presence of fibronectin-like domain of three type II repeats, which facilitate enzyme binding to ECM substrates [79]. Gelatinases are responsible for the turnover and degradation of several ECM proteins, including FN and type IV collagen, the major component of basement membranes [79;80]. Indeed, MMP-9 expression has been linked to several pathological conditions that require disruption of the basement membrane, such as tumor invasion [81], inflammation [82], arthritis [83], multiple sclerosis [84], systemic lupus erythematosus [85], cerebral IRI [86], and traumatic brain injury [87].

In human orthotopic liver transplantation, MMP-9 has been detected in the serum of patients after surgery [88–90]; MMP-9 serum levels were found to be significantly increased in a few minutes after reperfusion [89] and remained elevated for several days after transplantation [88]. In rat livers, MMP-9 has been shown by our group to be upregulated after 6h following OLT [68], and by others after 3h of IRI [91]. Further studies from our laboratory have shown that MMP-9 is a critical mediator of leukocyte migration in liver IRI [92*]. MMP-9 deficiency and specific anti-MMP-9 antibody therapy clearly depressed the infiltration of CD4, Ly-6G, and Mac-1 leukocytes in periportal areas after IRI, significantly ameliorating hepatic IRI. A beneficial effect for MMP-9 inhibition has also been recently shown in a distinct model of small-for-size liver graft IRI [93*].

There is a growing body of evidence that, despite overlapping activities, MMP-2 and MMP-9 may have distinct biological functions [94]. For example, it has been demonstrated that MMP-2 and MMP-9 regulate platelet aggregation in opposite ways [95]. Moreover, while MMP-2−/− mice develop exacerbated experimentally-induced arthritis and acute colitis, MMP-9−/− mice show significantly reduced signs of these diseases [96;97]. The substrate specificities of MMP-2 and MMP-9 are similar but not identical [98], and this may account, in part, for the different roles that these gelatinases may have. In this regard, it has been shown that whereas MMP-9 is not able of cleaving monocyte chemoattractant protein-3 (MCP-3), MMP-2 is, and that the cleaved molecule acts as a general chemokine antagonist depressing inflammation [99]. Moreover, the same MMP can have opposing effects based upon the cell type in which is expressed [100].

The regulation of MMP activity is complex and it takes place at transcriptional, post-transcriptional, and protein levels [72]. Tissue inhibitors of metalloproteinases (TIMPs) regulate the proteolytic activity of MMPs. There are at least four identified members (TIMP 1–4) in the TIMP family, which vary in tissue specific expression and in their ability to inhibit various MMPs [81]. TIMP-1, which inhibits MMP-9 with high affinity, has been detected in the serum of OLT patients [89]. Our unpublished studies (Duarte & Coito) suggest that TIMP-1 inhibition leads to increased levels of MMP-9 activity and leukocyte recruitment after hepatic IRI. There is a growing body of evidence supporting the view that cell attachment to ECM proteins and subsequent degradation are related events. Indeed, studies from our laboratory have shown that fibronectin interactions with its two α4β1 and α5β1 integrin receptors, expressed on leukocytes, are capable of regulating MMP-9 expression by leukocytes in hepatic IRI [68;101*]. Others have demonstrated that MMP-9 activation involves nitric oxide (NO)-mediated metalloproteinase S-nitrosylation S [86]. In liver IRI, the inability of nitric oxide synthase (iNOS) deficient mice to generate iNOS-derived NO profoundly inhibited MMP-9 activity as well as the recruitment of leukocytes [102*]. In addition, we also found that pro-inflammatory cytokines such as IFN-γ and IL-6 are capable of regulating MMP-9 activity by cultured neutrophils [102]. It is important to note that the ECM proteolysis mediated by metalloproteinases may not only facilitate leukocyte migration, but may also lead to detachment of liver cells resulting in apoptosis, a phenomenon called “anoikis” [103]. Indeed, MMP-9−/− deficient livers demonstrated significantly decreased numbers of hepatocytes undergoing apoptosis as compared with respective MMP-9 +/+ controls after IRI [102]. Thus, it is reasonable to postulate that MMP-9+ leukocytes infiltrating livers after IRI can cause parenchyma cell detachment from ECM and, consequently promote apoptosis/anoikis of these cells. Figure 1 illustrates the concept of a central role for MMP-9 in hepatic IRI [68;92;101;102].

Figure 1. Schematic representation of the role of MMP-9 in hepatic IRI.

Interactions between activated α4β1 and α5β1 integrins expressed on leukocytes and cellular fibronectin, newly synthesized by endothelial cells after injury, favor the induction of MMP-9 expression by leukocytes. Cytokines and other pro-inflammatory factors, such as iNOS-derived NO produced during the acute phase of IRI, can also mediate MMP-9 activation. MMP-9 assisted focal matrix degradation facilitates leukocyte transmigration into the liver. In addition, MMP-9+ leukocytes infiltrating livers after IRI can cause parenchyma cell detachment from ECM and, consequently promote apoptosis/anoikis of these cells leading to tissue injury.

Taken together, these studies emphasize the need for further exploring the individual functions of MMPs to support the development of potential therapeutic approaches to treat successfully liver IRI and other inflammatory diseases.

Conclusions

This review focuses primarily on the transmigration of leukocytes across the vascular endothelium and ECM barriers during liver IRI, and on the proteins that are thought to be important for this process. The understanding of leukocyte migration mechanisms remains a major challenge for the development of targeted therapies to treat pathological conditions that require modulation of immune responses [104–106]. While studies continue unraveling the complexity of mechanisms potentially involved in leukocyte traffic in hepatic IRI, recent developments support an important role for leukocyte-expressed MMP-9 as a key mediator of leukocyte transmigration and activation leading to liver injury.

Abbreviations

CS-1

connecting segment-1

ECM

extracellular matrix

FN

fibronectin

IRI

ischemia/reperfusion injury

KO

knockout

MMP

matrix metalloproteinase

OLT

orthotopic liver transplantation

ROS

reactive oxygen species

TIMP

tissue inhibitor of metalloproteinases