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REVIEW |
CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada
* corresponding author, ron.zohar{at}utoronto.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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KEY WORDS: osteopontin • mucosa • immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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). The increased expression of OPN is associated with increased cell mobilization, survival, and activity and is reflected in elevated concentrations of OPN in tissue fluids and plasma, which has potential diagnostic and prognostic value for cancer progression and tumor burden (Tuck et al., 2003), as well as for inflammatory disease progression in the joints, cardiac and nervous systems, and in the intestines (Reinholt et al., 1990; Gassler et al., 2002; Ohshima et al., 2002a.b; Koguchi et al., 2003; Tamura et al., 2003; Vogt et al., 2003).
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). However, there have been few studies of OPN in the normal or diseased mucosal tissues. In a recent report, an exacerbation of intestinal tissue destruction has been observed in a model of acute colitis induced by dextran sodium sulphate (DSS) in OPN-null mice (Batista-da-Silva et al., under review). These findings are in contrast to the attenuation of inflammatory disease models observed in closed, non-luminal tissues in OPN-null mice, emphasizing the importance of protective functions of OPN in mucosal disease. This review attempts to provide insights into the various known and putative roles of OPN in the complex interplay between the local and immune cell systems involved in mucosal barrier defense, resistance to infection, and tissue repair that are relevant to periodontal, intestinal, respiratory, and urogenital diseases. |
OPN STRUCTURE AND RELATED FUNCTIONS |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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). Since several detailed reviews on OPN have been published recently (Giachelli and Steitz, 2000; Sodek et al., 2000; Denhardt et al., 2001a,b; O’Regan, 2003), we include only a brief description of the salient features of OPN and focus on those properties of OPN that are particularly pertinent to mucosal defense. OPN is expressed by a single gene in a cluster of SIBLING family proteins that share structural and functional properties (Fisher and Fedarko, 2003) on the long arm of chromosome 4 in humans and chromosome 5 in mice. OPN is synthesized as a ~ 34-kDa nascent protein that is extensively modified by phosphorylation and glyco-sylation and sulphation prior to its secretion as a largely unstructured 44- to 75-kDa protein (Sodek et al., 2000). The heterogeneity of the secreted protein reflects differences in these post-translational modifications, which have been related to different functional activities associated with mineralization (Nagata et al., 1989; Sodek et al., 2000) and cell attachment and signaling (Ashkar et al., 2000; Zhu et al., 2001; Suzuki et al., 2002; Weber et al., 2002; Chellaiah et al., 2003). However, most of the known functional activities of OPN can be attributed primarily to highly conserved structural motifs involved in binding mineral and cell-surface CD44 and integrin receptors (Fig. 2).
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vß3 integrin, which is highly expressed in macrophages and osteoclasts, have been well-documented. Up-regulation of IL-12 in macrophages is mediated by RGD signaling through the vß3 and requires the OPN to be phosphorylated (Ashkar et al., 2000; Weber et al., 2002), as shown also for attachment and signaling in osteoclasts (Chellaiah et al., 2003), and for the migration of osteoclasts (Suzuki et al., 2002) and macrophages (Zhu et al., 2004). In this regard, it is interesting to note that phosphorylation of human OPN occurs in clusters of 3–5 sites, and that these phosphorylation sites are absent from the RGD region, as well as from regions of glycosylation (Christensen et al., 2005). In addition, a second cryptic integrin-binding site is recognized by the 4ß1 and 9ß1 integrins, which are preferentially expressed by leukocytes and lymphocytes (Bayless et al., 1998; Smith and Giachelli, 1998). This motif is contiguous with the RGD sequence, and, in human OPN, has the sequence "SLVVGLR" ("SLAYGLR" in murine OPN; Fig. 2), which is exposed by thrombin digestion of OPN at the conserved arginine, and is considered essential for the development of arthritis in a mouse model (Yamamoto et al., 2003).
CD44 has been identified as a receptor for OPN through which chemotactic (Weber et al., 1996) and cytokine responses of macrophages (Ashkar et al., 2000) have been demonstrated. Activities mediated by the CD44 receptor have implicated the amino-terminal peptide sequence (Fisher and Fedarko, 2003), a cryptic site near a central thrombin cleavage site (Lin and Yang-Yen, 2001), and an unidentified region in the C-terminal half of the molecule (Weber et al., 2002). The amino-terminal peptide has strong chemotactic activity for macrophages that can be blocked by CD44 antibodies and antibodies to the amino-terminus of OPN (Batista-da-Silva et al., unpublished observations), while Thr 147 in the murine OPN cryptic motif may be important for OPN signaling through the CD44 receptor in B-cells (Lin and Yang-Yen, 2001) (Fig. 2). Notably, chemotaxis of macrophages toward formyl-met-leu-phe (fMLP) in vivo is inhibited by antibodies to OPN (Giachelli et al., 1998). However, the interaction between OPN and CD44 may not be direct (Smith and Giachelli, 1998), and, for CD44, variant forms may require non-RGD-dependent mediation by the ß1 integrin (Katagiri et al., 1999).
The pleiotropic effects of OPN mediated by the different receptors provide the basis for its emergence as a cytokine that is highly expressed in various pathologies involving inflammation and cancer progression and metastasis, as well as in the pathophysiological processes of wound healing (Giachelli et al., 1998; Liaw et al., 1998; Giachelli and Steitz, 2000; O’Regan and Berman, 2000; Sodek et al., 2000; Lekic et al., 2001; Leali et al., 2003). Moreover, the frequent up-regulation of OPN in a variety of cells in response to a broad range of adverse conditions suggests that it functions as a stress-related glycoprotein. In the immune system, an increased transcription of OPN (identified as early T-lymphocyte activation 1; eta-1) in T-cells (Patarca et al., 1989) was subsequently linked to its suppression of T-lymphocyte function and enhancement of B-lymphocyte proliferation and antibody production (Lampe et al., 1991; Weber et al., 1996). OPN is now recognized as a key cytokine involved in immune cell recruitment and type-1 (Th1) cytokine expression at sites of inflammation (Ashkar et al., 2000; Chabas et al., 2001; Jansson et al., 2002), and it has been proposed that it be considered a new interleukin family member (IL-28; G Weber, International Conference on Osteopontin and Related Proteins, 3rd ICORP Meeting, San Antonio, May, 2002; Weber, 2002). Macrophages are central to the innate immune response (Fig. 3) and are a primary target of OPN. Ligation of CD44 receptors and the ß3-integrin by separate domains of OPN attract macrophages and stimulate cytokine and metalloproteinase production, respectively (Weber, 2002). OPN has also been reported to bind and activate pro-stromelysin (pro-MMP3) (Fedarko et al., 2000). Since the expression of this metalloproteinase is increased in wound healing, this surprising activity of OPN may be significant for the resolution of inflammatory diseases.
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vß3 and CD44 receptors (Fedarko et al., 2000). Localization of Factor H to the cell surface is believed to afford protection against immune surveillance and complement-mediated cell lysis, which could have particular significance for the survival of cells at sites of inflammation. The association of OPN with Factor H may also be a way to control OPN pro-inflammatory effects in the various body fluids.
In addition to the various secreted forms of OPN, the existence of an intracellular form of OPN (iOPN) that co-localizes with CD44 in migrating fibroblasts, macrophages, and osteoclasts has been reported (Zohar et al., 2000; Suzuki et al., 2002; Zhu et al., 2004). This novel form of OPN—which appears to modulate cytoskeleton-related functions, including cell motility and survival (Zohar et al., 2004), and mediates IFN expression in plasmacytoid dendritic cells (Shinohara et al., 2006)— introduces a further layer of complexity in deciphering the roles of different forms of OPN.
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OPN AND INFLAMMATORY DISEASES |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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vß3 activation and signaling through Ras and src tyrosine kinase, followed by activation of NF-
B (Scatena et al., 1998). In fibroblastic cells, the lack of OPN expression leads to caspase-independent necrosis, promoted by oxidants (Zohar et al., 2004). In contrast to programmed cell death, where dying cells undergo phagocytosis and clear the way for new tissues and cells, necrotic cell death is strongly associated with an exacerbation of inflammation and an increase of tissue destruction. Cell necrosis is a rapid form of cell death, in which damage and rapid permeabilization of cell membranes lead to the release of intracellular contents. The released cellular components act as irritants, recruiting more phagocytes (PMN, macrophages) and aggravating inflammation, and resulting in tissue destruction (Ina et al., 1999; Paleolog, 2003; Kurtovic and Segal, 2004).
In many inflammatory models, the formation of granulation tissue and the intensity of inflammatory reactions are dramatically reduced in the absence of OPN expression (O’Regan et al., 2001). Thus, the development of inflammatory diseases—such as lung (Miyazaki et al., 1995; O’Regan et al., 2001) and kidney (Noiri et al., 1999) fibrosis, arthritis (Yumoto et al., 2002) and multiple sclerosis (Chabas et al., 2001; Jansson et al., 2002)—is markedly suppressed in OPN-null mice. However, the critical role of OPN has been questioned in arthritis and experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, because the development of inflammation and its attenuation in the absence of OPN expression appear to be mouse-strain-specific, and, in some studies, concerns over the removal of flanking genes in back-crossed OPN-null mice have been raised (Blom et al., 2003; Jacobs et al., 2004). Consequently, the demonstration that arthritis-associated inflammation (Yamamoto et al., 2003) and concanavalin A-induced hepatitis (Diao et al., 2004) can be suppressed by antibodies directed at the cryptic "SLAYGLR" sequence in mice provides important alternative evidence of the pivotal role of OPN in the inflammatory response, by an approach that is not subject to compensatory mechanisms that can arise in knock-out mice. Importantly, in all of these pathologies, OPN expression by activated macrophages, T-cells, and fibroblastic cells is increased as part of the inflammatory response and in the subsequent repair process (Fig. 1), which also supports the significant contribution OPN makes in the progression and sequelae of inflammatory diseases. High levels of OPN expression are also a hallmark of monocytic granulomatous reactions in the context of tuberculosis and silicosis (Nau et al., 1999; O’Regan et al., 1999).
While most studies have generally concluded that OPN is a key regulator of cell-mediated inflammation, the potential role of OPN in protecting tissues from disease and/or excessive inflammatory destruction has largely been ignored. The apparent detrimental effects of OPN relate to its pro-inflammatory effects on macrophages, which respond by increased production of the Th1 cytokines IL-12 and IFN-
and decreased production of the Th2 cytokine IL-10, thereby polarizing the immune response to the Th1 pathway (Ashkar et al., 2000). These combined effects promote a protracted inflammation that is characteristic of autoimmune diseases, in which OPN may also contribute prominently (Lampe et al., 1991; Ashkar et al., 2000; Chabas et al., 2001). Notably, in OPN-null mice, the Th1 cytokines are reduced, while the Th2 cytokines are elevated. The protective effects of OPN may be more evident in mucosal inflammation, in which the patency of an epithelial lining is important for mediating injury and antigen presentation. However, while skin wound repair has been analyzed in OPN-null mice (Liaw et al., 1998), few studies have examined the role of OPN in mucosal disease. Following the creation of incision wounds in skin, the expression of OPN is increased within 6 hrs. However, in the absence of OPN expression, matrix production is impaired, and cell debris accumulates at the wound site (Liaw et al., 1998). Since neutrophil infiltration is an immediate response to mucosal injury that leads to the development of an acute inflammation, these observations suggest that, in OPN-null mice, there is an increased destructive activity of neutrophils due to slow clearance by macrophages, which may also have reduced phagocytic activity. In addition to the delayed resolution of the innate immune response, fibroblast function also appears to be compromised. This interpretation is consistent with the increased tissue destruction and markedly elevated (Fig. 1) PMN activity that we have observed in an acute colitis model in OPN-null mice, in which little epithelial regeneration is observed during remission in the absence of OPN expression (Batista-da-Silva et al., submitted).
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DETERMINANTS OF MUCOSAL IMMUNITY |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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Noxious irritation/damage of the epithelial barrier can be mediated by epithelial cells through special recognition receptors such as death Toll-like receptors, by specialized epithelial cells (microfold; M cells), or, more directly, by the penetration of bacteria or their products (Otte et al., 2003; Shi and Walker, 2004). Penetration of the epithelial barriers results in activation of resident neutrophils (polymorphonuclear leukocytes; PMNs) and macrophages; these are professional phagocytes, which provide the "immediate innate immune response" and non-specifically engulf foreign material and bacteria (Fig. 3). Cytokines and chemokines released from the injured epithelium as well as the leukocytes increase neutrophil and macrophage infiltration into the tissue, and thereby initiate the inflammatory response. Long-term residents of the subepithelial tissue, the antigen-presenting cells (APC)/dendritic cells, which are specialized macrophages, initiate the more specific "adaptive immune responses". The adaptive system is based on a specific recognition between APC and naïve T-cells, resulting in the differentiation of Th1, T-helper, and cytotoxic (NKT) T-cells, which control the immune reaction. Notably, many studies have correlated OPN expression with epithelial barrier changes (Gassler et al., 2002), with macrophage (Giachelli and Steitz, 2000; O’Regan et al., 2001; Sodek et al., 2002; Zhu et al., 2004), neutrophil (Alstergren et al., 2004), and lymphocyte activities (Ashkar et al., 2000), and with the function of reparative fibroblasts (Sodek et al., 2002).
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OPN AND THE EPITHELIAL BARRIER |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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In the gastrointestinal tract, a layer of columnar epithelial cells separates the underlying mucosa from the lumen and provides a reservoir for macrophages and T- and B-lymphocytes, which are concentrated in an organized subepithelial network along the gut and can be identified in focal areas, such as the Peyer’s patches in the distal small intestine. Moreover, the epithelial layer also contains specialized cells, such as the microfold (M) cells, capable of recognizing specific bacteria and antigens and transferring them to specialized APCs that reside in the vicinity of the epithelial layer (Neurath et al., 2002; Mowat, 2003; Acheson and Luccioli, 2004). The MHC class-II and Toll-like receptors on specialized epithelial cells may be involved directly with antigen presentation to underlying CD4+ T-cells (Acheson and Luccioli, 2004) (Figs. 3, 4).
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) is required for maintaining the epithelial barrier in the intestines (Gassler et al., 2002), and as a defense against tubulo-interstitial destruction in kidney diseases (Sibalic et al., 1997; Rangan et al., 2001). Moreover, increased OPN expression by tubular epithelium has been shown to have a critical role in mediating monocyte infiltration into the interstitium, leading to tubulo-interstitial alterations secondary to primary glomerulonephritis (Okada et al., 2000). OPN also has the potential to regulate specific functions of epithelial cells involved in the barrier defense process (Fig. 3), such as the expression of MHC-II and Toll receptors, which aid in antigen presentation and inflammatory signaling (Mowat, 2003; Basu and Fenton, 2004; Iwasaki and Medzhitov, 2004). For example, while the physical epithelial barrier can arrest microbial infection of mucosal surfaces by Candida (Steele et al., 2000), the Toll-like receptor 2 is required for the innate host defense against the spirochete Borrelia burgdorferi (Wooten et al., 2002). In airway systems, defensins secreted by neutrophils, originally thought to be important only for killing bacteria, can induce the differentiation of epithelial cells into mucosal-lining cells, which are characterized by their mucin secretion (Aarbiou et al., 2004) and enhancement of wound repair. Epithelial enterocytes in the gastrointestinal tract lack the typical microvilli associated with mucous-secreting cells, associate specifically with invading bacteria for transfer to adjacent dendritic cells, and take part in the initiation of the adaptive defense process (Debard et al., 2001; Mowat, 2003) (Fig. 4). The ability of the epithelial barrier to resist stress and trauma, and to regenerate, is important for the subsequent repair of the diseased tissues. Death of epithelial cells, therefore, is an important event associated with mucosal damage and occurs through up-regulation of the Fas ligand, and in response to TGF-ß1 expression and an increase in oxidative burst (Barkla and Gibson, 1999; Ophascharoensuk et al., 1999; Hagimoto et al., 2002; Kruidenier et al., 2003). In view of the role of OPN in cell survival, its presence may be important for supporting programmed cell death and preventing rapid necrotic death, which can result in intense inflammation and loss of epithelial barrier and protection.
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OPN IN MUCOSAL INFLAMMATION |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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, 4).
Innate immunity (Fig. 3) is the first line of defense for the broken epithelial barrier. IL-8 released from the injured epithelial cell recruits neutrophils and macrophages, which are capable of non-specific and specific phagocytosis—the macrophages utilizing mannose receptor or the CD14 (Toll 4) receptor for lipopolysaccharide. Upon phagocytosis, neutrophils and macrophages will continuously produce oxidative products such as hydrogen peroxide and nitric oxides. These cells, especially the macrophages, release mediators, which amplify the inflammatory reaction. The mediators include TNF-, IL-1, MCP-1, Rantes, and TGF-ß1, and degradative hydrolytic enzymes responsible for connective tissue degradation, such as the matrix metalloproteinases (MMPs). Other immediate changes associated with the innate immune response occur in response to increased blood flow to the area, with a local decrease in blood velocity. This is accompanied by the expression of adhesion molecules (e.g., VCAM-1) on the endothelial cells, which increases adhesion of leukocytes and vessel permeability, thereby facilitating extravasation of leukocytes to the inflamed tissues mediated by their integrin receptors, LFA-1 and Mac-1. The effect of innate immunity on the inflammatory progression can be demonstrated in glomerulonephritis models, in which OPN expression is increased (Eddy and Giachelli, 1995; Heinzelmann et al., 1999; Kitching et al., 2002), together with the up-regulation of ICAM-1s IL-1, TGF-ß1 MCP-1, and IL-8, and a decrease in IL-10 expression.
The short-lived immune response protects the injured organ prior to the development of the adaptive response (Fig. 4). The presentation of antigen by differentiated macrophages to naïve T-cells stimulates the differentiation of lymphocytes, including memory, helper, regulatory, and cytotoxic (NKT) T-cells, which augments further macrophage activity, including the clearance (phagocytosis) of apoptotic PMNs (Haslett et al., 1994). In response to IL-4, Th2 cells, in co-operation with Th1 cells, stimulate proliferation and differentiation of antibody-producing B-cells, while IL-10 secreted by Th2 cells regulates Th1 activity and thereby indirectly controls macrophage activity. During controlled infection, regulatory T-helper cells secrete IL-4 and TGF-ß1, which regulate the activity of the effector lymphocytes and stimulate fibroblast proliferation required for tissue remodeling (Fig. 5). Notably, increased secretion of TGF-ß1 by fibroblasts stimulates the proliferation of effector T-cells as well as antibody-secreting B-cells. In this respect, the activation of the adaptive response is more significant in mucosal defense, since mucosal tissues are challenged continuously by pathogens and noxious agents. The adaptive immune system is important in mucosal immunity, as well as in maintaining tolerance toward resident flora and nutrition products. Stimulation of lymphocyte growth and differentiation is achieved through the stimulation of the dendritic cells, which migrate to lymph-associated areas, such as the mucosal-associated lymphoid tissues (MALT; Fig. 4), and not in the infected inflamed area. Differentiated effector T-cells express selectins, which help them target the inflamed area and extravasate in a similar fashion to the PMNs. Migrating T-cells will be predominantly Th1 cells, which will stimulate further macrophage and T-cytotoxic cell activities.
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vß3 receptor, while the secretion of the Th2 cytokine IL-10 is suppressed by OPN signaling through the CD44 receptor. In the absence of OPN expression, the Th2 cytokine response is predominant, and OPN-null mice display an increased susceptibility to intracellular pathogens, including Rickettsia tsutsugamushi, Mycobacterium tuberculosis, Listeria monocytogenes, and herpes simplex virus-type 1 (Patarca et al., 1993; Nau et al., 1999; Ashkar et al., 2000). The impact of bacterial infection is particularly relevant in mucosal immunity. For intracellular pathogens, the generation of a cytotoxic T-cell response is critical, since the immune system faces a challenge in that the pathogens are hidden within host cells. Resistance to Rickettsia infection is mediated by a marked induction of OPN, leading to an early monocyte influx into infected sites and rapid acquisition of macrophage bactericidal activity. Susceptibility to Rickettsial infection, in contrast, reflects delayed and weak OPN responses and is characterized by an early accumulation of neutrophils at sites of infection. However, because extracellular pathogens, such as the spirochete B. burgdorferi, express lipoproteins that are highly immuno-stimulatory and pro-inflammatory, the requirement of an OPN-mediated stimulation of the Th1 response is circumvented (Craig-Mylius et al., 2005). Despite the direct effects of OPN on T effector cells, its importance, especially in mucosal immunity, may be mediated through the control of differentiated macrophage (i.e. dendritic cells) activity (Figs. 3, 4). OPN has been shown to affect macrophage activities such as chemotaxis, phagocytosis, bone resorption, cytotoxic activity, granuloma formation, and fibrotic repair (Denhardt et al., 1995; Miyazaki et al., 1995; McKee and Nanci, 1996; Rollo and Denhardt, 1996; Rollo et al., 1996; Giachelli et al., 1998; Zohar et al., 2000; O’Regan et al., 2001; Ide et al., 2003; Zhu et al., 2004; Ogawa et al., 2005). Different macrophage progeny are needed for the innate immunity response, functioning as unspecific phagocytic cells, but perhaps more importantly as dendritic (APC) cells (Figs. 3, 4). The dendritic cells signal the activation of lymphocyte proliferation and their differentiation into specific immune-effector cells (i.e., T-cell subclasses and B-cells), pro-inflammatory soluble mediators, and, eventually, myofibroblasts, matrix, and repair. Thus, a lack of macrophages or a malfunction in monocyte differentiation will result in an impaired specific immune response and/or repair.
Notably, OPN may not be needed for basic activity of isolated lymphocytes or macrophages in culture, but may be required for their interaction and co-operative effects in promoting the transition from innate to adaptive responses and the initiation of repair (Fig. 5).
Neutrophils (PMNs)
Also termed granulocytes or PMNs, the neutrophils provide the immediate and non-specific line of defense for broken mucosal surfaces. The release of cytokines such as IL-8 by damaged epithelial cells recruits PMNs to sites of injury to initiate the innate defense. In some situations, mucosal immunity may involve defense through the epithelial and PMN cells only, such as in the arrest of superficial Candida infections (Steele et al., 2000; Schaller et al., 2004). PMNs which attack invading bacteria or foreign material release cytotoxic products that, unless controlled, may result in destruction of the host tissue (Jaeschke and Smith, 1997; Heinzelmann et al., 1999). The infiltration of the subepithelial tissues by PMNs is an important stage in the inflammatory process of mucosal damage in inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis (Nikolaus et al., 1998; Ina et al., 2002; Kruidenier et al., 2003; Le’Negrate et al., 2003; Kurtovic and Segal, 2004). In normal self-restricting mucosal defense processes, PMNs are usually cleared within 24 hours of injury, due to the invading macrophages, which engulf apoptotic neutrophils and arrest further neutrophil mobilization and tissue destruction (Haslett et al., 1994; Hart et al., 2000). The lack of neutrophil clearance by macrophages may result in the persistence of PMNs in inflamed tissues, which can result in increased destruction (Batista-da-Silva et al., submitted). That a similar situation is seen in TNF-null mice, in which experimental colitis is aggravated due to hyperactivation of neutrophils (Naito et al., 2003), is of particular interest, in that TNF expression is suppressed in response to DSS-induced colitis in OPN-null mice (Batista-da-Silva et al., under review).
While the release of OPN by neutrophils can contribute to the recruitment of macrophages, few studies have reported on OPN expression by neutrophils. Chitosan and G-CSF have been shown to increase OPN mRNA expression in PMNs, although the increase in the release of OPN into the medium was modest (Ueno et al., 2001). Immunocytochemical staining of OPN in PMNs (Fig. 3) confirms the presence of OPN throughout the cytoplasm and, in contrast to macrophages, shows no particular association with CD44, which is characteristically concentrated in the uropods of polarized PMNs. Also, in contrast to macrophages (Zhu et al., 2004), the cell-surface expression of CD44 is not influenced by OPN, which may reflect differences in the migratory characteristics of these cells (Alstergren et al., 2004).
Macrophages
Macrophages participate in both immediate innate and adaptive immune responses (Figs. 3, 4). Differential expression of OPN by macrophages and T-cells determines the relative levels of the immediate and delayed-type hypersensitivity responses, which are neutrophil-dependent and neutrophil-independent, respectively. In immediate responses, the macrophages are attracted and activated by cytokines secreted by neutrophils, thereby promoting a protracted inflammatory reaction that frequently results in excessive fibrosis and scar formation (Fig. 5). Macrophages, therefore, may be the most versatile cells in mucosal protection. Macrophages infiltrating the injured mucosal tissues in early stages help the PMNs phagocytose invading pathogens, while controlling the amount of damage by PMNs and attracting the more specific adaptive pathways of immune cell-mediated protection of lymphocytes. Notably, in healthy subepithelial tissues of the gut, respiratory tract, and the urogenital systems, differentiated macrophages reside for long periods as dendritic cells, which respond to noxious insults by initiating the adaptive immune response. As noted previously, not only do the macrophages/APCs in mucosal surfaces activate the adaptive immune response, but they also maintain immune tolerance toward non-pathogenic normal flora (Mowat, 2003). The cell-mediated immune response is characterized by the formation of granuloma tissue, which occurs during wound healing, and, consistent with the effects of OPN on macrophage function, granuloma formation is impaired in OPN-null mice (O’Regan et al., 1999, 2001; Ashkar et al., 2000; Morimoto et al., 2004).
Macrophages produce large amounts of OPN, which is further up-regulated by LPS stimulation (Gao et al., 2004). The OPN produced by macrophages may act as an opsonin, facilitating phagocytosis (McKee and Nanci, 1996), as well as cell adhesion, through RGD binding. While immunostaining of OPN displays a perinuclear distribution typical of secreted proteins, in migrating macrophages, OPN is also seen co-localizing with CD44 at the cell membrane of cell processes (Fig. 5) as an intracellular form of OPN (iOPN). The iOPN and its association with CD44 were originally identified in migrating fibroblasts (Zohar et al., 2000) and subsequently characterized by pulse-chase labeling studies in macrophages (Zhu et al., 2004). In the absence of OPN expression, the formation of cell processes associated with the migration of macrophages and their chemotaxis to fMLP and MCP-1, which act through G-protein-coupled receptors, are impaired. The impairment appears to be related to decreased expression of CD44 (Zhu et al., 2004), which is regulated by OPN functioning through the vß3 integrin in macrophages (Marroquin et al., 2004) and osteoclasts (Chellaiah et al., 2003) and has been shown to be crucial for the polarization and migration of neutrophils (Alstergren et al., 2004).
In addition to its effects on macrophage recruitment and activation, MCP-1 also has important effects on OPN expression and cytokine synthesis by macrophages that affect myofibroblast accumulation in healing infarcts (Dewald et al., 2005). In macrophages stimulated with LPS and IFN-, nitric oxide (NO) directly up-regulates endogenous OPN, which acts as a negative feedback regulator of iNOS to reduce nitric oxide synthesis (Takahashi et al., 2000; Guo et al., 2001; Speyer et al., 2003). Recent studies have shown that the development of atherosclerosis is prevented by Liver X Receptor (LXR) ligands, which inhibit cytokine- and LPS-induced OPN expression in macrophages by targeting an AP1-element in the OPN promoter (Ogawa et al., 2005). Thus, expression of OPN in macrophages is required not only for recruitment, but also to orchestrate the specific adaptive and non-specific innate defense pathways.
Lymphocytes
OPN is considered to be an important lymphocyte mediator secreted by activated T-lymphocytes that induces macrophage migration and suppresses the production of reactive oxygen species, while enhancing immunoglobulin production and proliferation of B-lymphocytes (Weber et al., 1996). In the original studies of lymphocytes, OPN was identified as a T-lymphocyte activation-1 (Eta1) cytokine (Patarca et al., 1993) that was subsequently shown to be required for cell-mediated immunity and the development of the Th1 pathway and macrophage activity (Ashkar et al., 2000; Chabas et al., 2001; Jansson et al., 2002; O’Regan, 2003). The function of tissue resident macrophages/dendritic cells is not only to destroy pathogens but also to mediate the adaptive immune response by carrying pathogen antigens through the lymph circulation to peripheral lymphoid organs, where they present them to näive lymphocyte precursors through specialized MHC receptors (Fig. 4). Failure of dendritic cell activation may result in improper adaptive immune response or immune-tolerance to antigens.
Lymphocyte function in mucosal protection (Figs. 3, 4) is part of the more specific adaptive immunity. Lymphocyte activation and differentiation will usually occur via two routes:
to encourage killing by macrophages (Mowat et al., 2003; Watford et al., 2003; Dakic et al., 2004; Nagler-Anderson et al., 2004).
In CD4+ T-cells, OPN mRNA is expressed in Th1, but not in Th2, polarized cells (Nagai et al., 2001). OPN promotes adhesion of activated T-cells, and this activity is enhanced following proteolytic cleavage of OPN by thrombin (O’Regan et al., 1999). At low concentrations, OPN promotes chemotaxis but not chemokinesis of T-cells, while activated T-cell adhesion is enhanced at higher concentrations, especially following thrombin cleavage of OPN (O’Regan et al., 1999). OPN also co-stimulates T-cell proliferation and increases CD3-mediated T-cell production of IFN- and CD40 ligand (O’Regan and Berman, 2000). At low concentrations, OPN promotes chemotaxis, but not chemokinesis, of T-cells. However, this response is inhibited at higher OPN concentrations.
A variety of inflammatory and autoimmune diseases—including multiple sclerosis, rheumatoid arthritis, and atherosclerosis—is critically regulated by NKT cells, which also secrete OPN. The OPN augments NKT cell activation and triggers neutrophil infiltration and activation. Consistent with this role of OPN in NKT cell function, OPN-null mice, similar to NKT cell-deficient mice, are refractory to Con A-induced hepatitis. However, a neutralizing antibody, specific for a cryptic integrin-binding epitope of OPN that is exposed by thrombin cleavage, ameliorates the development of hepatitis in normal mice (Diao et al., 2004). In contrast, over-expression of OPN in the liver of transgenic mice resulted in massive liver necrosis and monocyte infiltration, due to an imbalance of the Th1 and Th2 immune responses (Mimura et al., 2004), presumably caused by the attraction of macrophage precursors by OPN and its promotion of the Th1 response.
Impaired OPN–/– macrophage differentiation and functions (Weber et al., 2002; Zhu et al., 2004) may be responsible for deficient healing in OPN-null mice (Liaw et al., 1998; Rittling et al., 1998). OPN induces T-cell proliferation, adhesion, and chemotaxis, especially in relation to granulomatous lesions (O’Regan et al., 1999). Moreover, soluble OPN may modulate the differentiation and proliferation of CD4+ and CD8+ lymphocytes (Higuchi et al., 2004). Over-expression of OPN in transgenic mice increased the numbers of CD4+ T-cells, but not CD8+ T-cells, in the spleen, particularly in the lymph nodes. Skin sensitization with 2,4-dinitrofluorobenzene (DNFB) in these mice increased the number of CD4+ cells and recruitment of CD8+ cells. However, peritoneal sensitization with DNFB (Higuchi et al., 2004) resulted in increases in the numbers of CD8+ T-cells in the peritoneal exudate, with no difference in the numbers of CD4+ T-cells. These results suggest that different responses might be anticipated in mucosal immunity, where there is an epithelial barrier involvement in the transfer of the inflammatory signals. In this regard, studies of colitis in OPN-null mice support the suppression of CD4+ and CD8+ cell numbers of the inflamed OPN-null spleens (Batista-da-Silva et al., submitted). These observations also suggest a direct effect of OPN on T-cells, or, alternatively, indirect effects due to lack of functional macrophages or differentiated dendritic cell stimulation. This is accompanied by a decrease of IL-12, IFN-, and the oxidative burst necessary for the development of proper protective inflammatory reaction.
IFN- treatment of Th-1 cells has been shown to induce OPN mRNA and protein expression in a time-dependent and dose-dependent manner (Li et al., 2003), consistent with the contribution of OPN to Th1 activation. These effects may also be dependent on the ability of macrophages to differentiate into dendritic cells necessary for the T-cellular response (Weiss et al., 2001) and their mobilization from the regional lymph nodes. Up-regulation of CD40L by OPN in T-lymphocytes (O’Regan and Berman, 2000) provides mechanistic support for the association of OPN with polyclonal B-cell proliferation and humoral autoimmune disease (Weber et al., 1996). During stimulation of the lymphoid centers, specific dendritic cells, through interaction with CD4 (naïve) lymphocytes, present antigen to B-cells, which have antigen-specific receptors that enable them to internalize large amounts of specific antigens, proliferate, and produce specific antibodies. Interestingly, an important mechanism for IgA production is that promoted by Th3 secreted TGF-ß1, which controls IgA switching and secretion in vivo (Borsutzky et al., 2004). B-cells then migrate into the surrounding mucosa and release secretory IgA through the epithelial lining of the mucosa. The secretory IgA binds directly to pathogens and prevents their attachment to epithelial cells; it also coats antigens, promoting their engulfment by macrophages. Although it is unclear how TGF-ß1 and its associated receptors control B-cell activity, the Toll death receptors and G-protein-coupled receptors appear to be involved (Cazac and Roes, 2000; Roes et al., 2003).
TGF-ß1 and OPN are important mediators of extracellular matrix formation and wound healing (Bendeck et al., 2000; Sodek et al., 2000; Denhardt et al., 2001a,b), and the matrix-promoting and immunoregulatory activities of TGF-ß1 have been studied quite extensively (Kitani et al., 2003; Wahl and Chen, 2003). However, the role of OPN and its relation to TGF-ß1-mediated activities are largely unknown. TGF-ß1 modulates differentiation of naïve CD4 lymphocytes (Wang and Mosmann, 2001), and over-expression of TGF-ß1 reduces the susceptibility of mice to colitis by blunting Th3 immune and humoral responses (Egger et al., 1998). TGF-ß1 expression during inflammation and immune reactions also stimulates IL-10 secretion in Th1 cells, regulates T-lymphocyte activity, and induces OPN expression in immune as well as reparative fibroblasts (Overall et al., 1991) (Fig. 5).
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OPN AND THE INITIATION OF HEALING |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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). OPN has also been reported to activate MMP-3 (Fedarko et al., 2000), the expression of which is increased markedly in inflamed colons of patients with ulcerative colitis and Crohn’s disease, and correlates with the loss of mucosal integrity, suggesting an important role for stromelysin and its possible activation by OPN in the process of destruction and tissue remodeling in inflammatory bowel diseases (Heuschkel et al., 2000; von Lampe et al., 2000). Myofibroblasts, which are typically associated with reparative granulation tissue formation (Desmoulière, 1995; Neubauer et al., 2001), are characterized by an enhanced expression of -smooth muscle actin (-SMA) in stress fibers (Jelaska and Korn, 2000; Thibault et al., 2001; Tomasek et al., 2002; McKaig et al., 2003). During matrix formation, myofibroblasts respond to TGF-ß1 by increased expression of extracellular matrix (ECM), including collagen, and selective suppression of MMPs (Overall et al., 1991). Since TGF-ß1 also increases -SMA, it is conceivable that the myofibroblast phenotype induced in fibroblasts is mediated through the up-regulation of TGF-ß1, which also increases the expression of OPN, integrins, and -actinin (Kawano et al., 2000; Mazzali et al., 2002) and promotes integrin-mediated collagen gel contraction (McKaig et al., 2003). TGF-ß1 and OPN also down-regulate apoptosis of fibroblasts and thus increase matrix deposition (Desmoulière, 1995; Jelaska and Korn, 2000; Zohar et al., 2004). Indeed, connective tissue wound healing and fibrosis, subsequent to inflammatory disease, are impaired in OPN-null mice (Liaw et al., 1998; Trueblood et al., 2001).
That OPN is expressed by epithelial and mucosal cells, as well as by immune cells in the intestines, has been shown (Qu and Dvorak, 1997; Gassler et al., 2002). However, despite the perceived importance of OPN in cell-mediated immune responses and wound healing, there have been few studies of OPN in the normal or diseased intestine. In support of the proposed functions of OPN in protecting the intestine from pathogens and in the development of inflammatory bowel diseases (Masuda et al., 2003), our preliminary studies indicate that destruction of the intestinal tissues is exacerbated in OPN-null mice subjected to experimental colitis (Batista-da-Silva et al., submitted). The loss of mucosal integrity and inflammatory destruction as seen in patients with ulcerative colitis and Crohn’s disease correlates with the production of IL-1ß, IL-6, TNF-, IL-10, TGF-ß1, and activated MMPs (Fedarko et al., 2000; Heuschkel et al., 2000; von Lampe et al., 2000; McKaig et al., 2003). Impairment of these mediators’ activity and overall granulation tissue formation will alter the process of tissue remodeling and repair in inflammatory bowel diseases.
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SUMMARY |
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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ACKNOWLEDGMENTS |
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Received March 31, 2005; Accepted November 8, 2005
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TOPABSTRACTINTRODUCTIONOPN STRUCTURE AND RELATED...OPN AND INFLAMMATORY DISEASESDETERMINANTS OF MUCOSAL IMMUNITYOPN AND THE EPITHELIAL...OPN IN MUCOSAL INFLAMMATIONOPN AND THE INITIATION...SUMMARYREFERENCES |
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