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Immunomodulatory effects of lactoferrin on antigen presenting cells
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Biochimie 91 (2009) 11e18 www.elsevier.com/locate/biochi
Review
Immunomodulatory effects of lactoferrin on antigen presenting cells Patrizia Puddu a, Piera Valenti b, Sandra Gessani a,* a
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy b Department of Public Health Sciences, Sapienza, University of Rome, Rome, Italy Received 18 March 2008; accepted 9 May 2008 Available online 21 May 2008
Abstract Lactoferrin (Lf) is an 80 kDa iron-binding protein of the transferrin family that is abundantly expressed in most biological fluids. It is now recognized that this glycoprotein is a key element in the mammalian immune system, playing an important role in host defence against infection and excessive inflammation. Although the mechanisms underlying Lf immunomodulatory properties have not been fully elucidated yet, evidence indicates that the capacity of this molecule to directly interact with antigen presenting cells (APCs), i.e. monocytes/macrophages and dendritic cells (DCs), may play a critical role. At the cellular level, Lf modulates important aspects of APC biology, including migration and cell activation, whereas at the molecular level it affects expression of soluble immune mediators, such as cytokines, chemokines and other effector molecules, thus contributing to the regulation of inflammation and immunity. While the iron-binding property was originally believed to be solely responsible for the plethora of host defence activities ascribed to Lf, it is now known that other mechanisms contribute to the broad spectrum of anti-infective and anti-inflammatory properties of this protein. Recent results suggest that at least some of the immunomodulatory effects of Lf rely on its capacity to form complexes with lipopolysaccharide (LPS). This review focuses on the effects of Lf on APC biology and function, highlighting known and putative mechanisms that underlie Lf immunomodulatory effects. The importance of LPS-binding capacity of Lf and LPS receptors, as well as of Lf-induced type 1 interferon (IFN) expression in some of these effects is also discussed. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Lactoferrin; Macrophage; Dendritic cell; Immunomodulation; Lipopolysaccharide
1. Introduction Antigen presenting cell (APC) function is central to the development of effective immune responses against pathogens.
Abbreviations: Lf, lactoferrin; LfR, lactoferrin receptor; APC, antigen presenting cell; DC, dendritic cell; MD-DC, monocyte-derived dendritic cell; LC, Langerhans cell; Mf, macrophage; PMf, peritoneal macrophage; BMMf, bone marrow macrophage; PBM, peripheral blood monocyte; NK, natural killer cell; Th, T helper cell; LPS, lipopolysaccharide; sCD14, soluble CD14; TLR4, toll-like receptor 4; LRP, low-density lipoprotein receptor-related protein; LAL, Limulus assay; NO, nitric oxide; MHC, major histocompatibility complex; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; MR, mannose receptor; HIV-1, human immunodeficiency virus 1; BCG, bacillus Calmette Guerin; ADCC, antibody-dependent cellular cytotoxicity; IL, interleukin; TNF, tumour necrosis factor; IFN, interferon. * Corresponding author. Tel.: þ39 06 49903169; fax: þ39 06 49903641. E-mail address: [email protected] (S. Gessani). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.05.005
Among APCs, macrophages (Mf) and dendritic cells (DCs) are of critical importance for the maintenance of tissue homeostasis and innate response to pathogens, as well as in linking innate to adaptive immune response. Mf are highly phagocytic cells which play a central role in the control of infections, either by direct intracellular killing of microorganisms or by secreting cytokines inhibiting their replication, as well as in type II inflammation and tissue repair processes [1,2]. DCs are a heterogeneous population of cells highly specialized for antigen recognition that play a key role in the immune system by virtue of their capacity to control both immunity and tolerance induction [3,4]. Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein abundantly found in exocrine secretions of mammals, in particular in milk and fluids of the digestive tract, and released by mucosal epithelia and neutrophils during inflammation [5]. Lf is a key element in the host defence system [6e8]. It
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is now well recognized that this molecule plays a direct antimicrobial role in secretions and at the surface of epithelia, by limiting the proliferation and adhesion of microbes and/or by killing them. These properties are mainly related to its ability to sequester iron in biological fluids or to destabilize the membranes of microorganisms. However, iron-independent microbicidal activities, requiring direct interaction between Lf and microbial surface components, have been subsequently demonstrated [5]. In addition to the antimicrobial properties of Lf, its ability to modulate the overall immune response and to protect against viral infections and septic shock has been largely described [6e8]. In this respect, it is noteworthy that Lf concentrations are elevated locally in inflammatory disorders including neurodegenerative diseases [9], inflammatory bowel disease [10], arthritis [11], and allergic inflammation [12]. Although the cellular and molecular mechanisms accounting for the immunomodulatory effects of Lf are far from being fully elucidated, both in vitro and in vivo studies suggest the existence of multiple mechanisms that include modulation of cytokine/chemokine production, regulation of reactive oxygen species production, and of immune cell recruitment. It is now clear that at least some of the Lf biological activities do not merely depend on its iron-binding capacity, but may arise from its interaction with a variety of molecules. In this respect, the capacity of Lf to influence either negatively or positively cytokine production relies, at least in part, on its ability to bind and sequester both lipopolysaccharide (LPS) and its receptor CD14, as well as CpG bacterial DNA, thus preventing the downstream activation of proinflammatory pathways, septic shock and tissue damage [13e16]. However, Lf can also favour the activation, differentiation, and proliferation of immune cells, and this promoting activity has been related to a direct effect of Lf on immune cells through the recognition of specific Lf binding sites [17,18]. 2. Cell determinants involved in Lf interaction with antigen presenting cells Cells of the immune system, like virtually any other cells in the organism, can bind Lf. The cationic nature of this molecule accounts for its propensity to bind to anionic molecules, resulting in a massive binding to mammalian cells [18]. Several Lf binding sites have been found on target cells, revealing considerable variations among species, tissues and cell types [19]. A number of studies reported the direct binding of Lf to monocytes/Mf and DCs. Indeed, mouse peritoneal Mf (PMf) was the first cell type reported to express Lf receptors (LfRs) in mammals [20], and human monocytes were also shown to bind human Lf in a reversible, saturable and specific manner [21]. At the surface of cells, the sulphated chains of proteoglycans are responsible for the low-affinity, high density binding of Lf [5]. Other important receptors are represented by low-density lipoprotein receptor-related proteins (LRPs), frequently referred to as scavenger receptors, widely expressed on several cell types, including Mf [22]. Lf has also been shown to bind to surface nucleolin [23], a multifunctional shuttling protein present in nucleus, cytoplasm, and on the
surface of some cell types, including macrophages [24]. Finally, at least four species of human Lf binding proteins have been found on the plasma membrane of THP-1, a human monocytic leukaemia cell line [25]. The LfR expressed on monocytes/Mf is most likely involved in the effects of human Lf on the inflammatory and immune responses of animals [26]. In this respect, it has been shown that human Lf interacts directly with CD14, an LPS receptor involved in the activation of the immune system that exists both as a soluble protein (sCD14) found in serum [27], and membrane protein highly expressed on the surface of monocytes/Mf [28]. Human Lf binds to sCD14 complexed to LPS or lipid A and protects animals from septic shock induced by LPS [29e32], thus indicating that CD14 is one of the LfR expressed on monocytes/Mf. In contrast to monocytes/Mf, no specific studies have been carried out yet to define the nature of LfRs expressed in DCs. However, the observation that the adjuvant effect of Lf in skin Langerhans cells (LCs) is inhibited by blocking the mannose receptor (MR), suggests that this receptor may represent a putative site for Lf interaction with this subset of DCs [33]. In addition, it has been recently shown that Lf binds to the DC-specific ICAM3-grabbing non-integrin (DC-SIGN), a C-type lectin receptor expressed by DCs [34], thus suggesting that receptors belonging to the C-type lectin family may play an important role in Lf interaction with DCs. The capacity of Lf to interact with C-type lectin receptors apparently depends on its sugar composition. In this respect, it has been demonstrated that although bovine and human Lf both interact with the MR, this interaction occurs to a different extent and strongly relates to the different composition in relevant sugars [34]. Likewise, it has been reported that human Lf, in contrast to bovine Lf, does not bind DC-SIGN, as assessed by the lack of capacity of this protein to prevent the DC-SIGN-mediated human immunodeficiency virus-1 (HIV-1) transfer to CD4þ T lymphocytes [34,35]. The presence of specific Lf receptors on immune cells suggests that the modulation of host responses by Lf may be related to a direct effect via receptor-mediated signalling pathways. In this respect, it has been reported that Lf downregulation of LPS-induced interleukin (IL)-6 secretion in monocytes involves Lf translocation to the nucleus and inhibition of NF-kB activation [36]. 3. Effects of Lf on cells of the monocyte lineage Tissue Mf play a master role in homeostasis and immune defence against infections, providing a rapid response against pathogen threats. In particular, Mf clear exogenous antigens and apoptotic cells through phagocytosis and intracellular killing, trigger tissue leukocyte infiltration, and collaborate with T and B cells by locally releasing cytokines and other effector molecules, including nitric oxide (NO), as well as by taking part to tissue repair processes. In this respect, Mf display a peculiar plasticity as they can be committed to undergo a different differentiation program depending on the cytokines released in the microenvironment [2]. In this view, Mf can be classified in two main groups named M1 (classically
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activated Mf) and M2 (alternatively activated Mf). M1 Mf exert strong endocytic and antimicrobic activities and elicit a potent T helper (Th)1 cellular response mediated by IL-12 production, whereas M2 Mf support Th2-associated effector functions such as high endocytic clearance capacity, trophic factor synthesis and reduced proinflammatory cytokine secretion, playing a role in resolution of inflammation. In this context, Lf could represent a crucial factor in directing Mf activation and, consequently, in polarizing the immune response. A variety of studies have indicated that Lf can selectively interact with monocyte and Mf, and modulate their functions during inflammatory and infectious processes. The results of these studies are summarized in Table 1. In particular, it has been reported that human Lf treatment positively affects human adherent monocyte cytotoxic activity, whereas it has no effect on nonadherent lymphocytes [37]. In contrast to the enhancement of cytotoxicity, Lf exhibited concentration-dependent inhibitory effect on antibody-dependent cellular cytotoxicity (ADCC). Again, this appeared to be an effect on monocytes because Lf did not alter nonadherent cell ADCC activity [37]. Some studies investigated the effect of Lf on the capacity of mouse PMf and human peripheral blood monocytes (PBM) to up-take and kill intracellular pathogens. These reports showed that human Lf-treated cells exhibit an increased phagocytosis and intracellular killing of Trypanosoma cruzi [38]. The presence of iron was found to be required for human Lf to stimulate intracellular killing by PMf but not to enhance the up-take of particles and microorganisms [39]. Likewise, oral administration of bovine Lf stimulated PMf activities in mice injected with inactivated Candida albicans [40]. In particular, Lf administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of NO by PMf. In keeping with these results, induction of NO release by bovine Lf was also observed in rat BMMf [41]. Furthermore, bovine Lf administration enhanced the ability of PMf to inhibit hyphal growth in C. albicans injected mice [40]. In keeping with these results, Tanida and colleagues reported that administration of Lf peptides prolonged the survival periods of C. albicans
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inoculated mice by up-regulating neutrophil and macrophage functions [42]. Lastly, bovine Lf was also shown to modulate APC response to bacillus Calmette Guerin (BCG) by significantly increasing the up-take of microorganisms during infection of murine BMMf and human monocytic cell lines [43]. Furthermore, it was also reported that BCG infected BMMf exposed to bovine Lf significantly up-modulated the expression of major histocompatibility complex (MHC) class II (I-A), but not class I (H-2k), surface molecules [43]. Likewise, bovine Lf stimulated CD40 expression in mouse RAW 264.7 cell line and PMf [44]. A variety of studies have indicated that Lf can modulate basal or LPS-induced cytokine production in different models of primary monocyte/Mf and monocytic cell lines. In this respect, we as well as other groups demonstrated that exposure of Mf to bovine Lf results in the production of several cytokines/ chemokines, including IL-6, IL-8, IL-12, tumour necrosis factor (TNF)-a and interferon (IFN)-b [41,44e46]. Interestingly, the bovine Lf stimulated IFN-b expression was responsible for the antiviral effect observed in murine resting PMf [45]. Although exposure of na€ıve macrophages to Lf results in the induction of some cytokines/chemokines, the effects of this molecule on activated Mf (i.e. LPS-treated or pathogen infected) are more complex. In particular, it has been demonstrated that LPS-induced production of IL-6, TNF-a, IL-1-b and IL-8 is inhibited by Lf, both bovine and human or its fragment lactoferricin B, in various human monocytic cell lines [36,47,48]. Furthermore, it has been observed that addition of bovine Lf to spleen Mf and J774A.1 murine cell line stimulated with suboptimal LPS concentrations increased production of IL-12 whereas the secretion of IL-10 was decreased [49]. These observations support the hypothesis that Lf could sustain Mf polarization versus a Th1 immune response. In keeping with this hypothesis, Guillen and colleagues reported an enhanced Th1 response to Staphylococcus aureus infection in huLf-transgenic mice, characterized by an enhanced in vivo production of IFN-g and TNF-a and reduced IL-5 and IL-10 secretion. Similarly, huLf-transgenic mice exhibited enhanced severity of Th1-driven collagen-induced arthritis [50].
Table 1 Immunomodulatory effects of lactoferrin on APCs Function
Effect
Lf origin
Model species
Cell types
Reference
Cytotoxic activity Antibody-dependent cytotoxic activity Pathogen up-take and intracellular killing Expression of surface molecules Cytokine production LPS-induced cytokines/chemokines IFN mediated antiviral state Production of Th1 cytokines Cell migration Viral transmission DC activation
j k j j j k j j k k j
Hu Hu Hu/Bo/P Bo Bo Hu/Bo/F Bo Bo Rec Bo Rec
Hu Hu Hu/Mu/Ra Mu Mu/Ra Hu/mu Mu Mu Mu/Hu Hu Hu
PBM PBM PBM, PMf, BMMf, cell lines PMf, BMMf, cell lines PMf, BMMf, cell lines cell lines PMf spleen Mf, BMMf, cell lines LCs MD-DCs MD-DCs
[37] [37] [38e43] [43,44] [41,44e46] [36,47,48] [45] [49] [78,79] [34] [83]
Summary of the principal experimental observations showing the immunomodulatory effects of Lf of different origins (murine, human, bovine or recombinant) on both primary cells and cell lines of the monocyte/DC lineage. Hu ¼ human; Mu ¼ murine; Ra ¼ rat; Bo ¼ bovine; P ¼ Lf synthetic peptide; F ¼ Lf fragment; Rec ¼ recombinant Lf.
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Overall, these results indicate that Lf can differently affect several Mf functions, ultimately leading to attenuation of excessive inflammation and stimulation of host response against pathogen threats. However, further studies are strongly needed to define molecular and cellular determinants involved in the immunomodulatory activity of this molecule. Structurally, Lf contains a highly basic region close to the N-terminus which binds to a variety of anionic biological molecules. Human Lf binds specifically and with a high affinity to the lipid A region of bacterial LPS [13,51], sCD14 and the sCD14/LPS complex [14]. Two N terminal basic clusters of human Lf, residues 1e5 and 28e34, are responsible for the binding to anionic molecules, such as LPS [51,52], heparin, or cell-surface heparan sulphates [53e55]. The LPS-binding property of Lf play an important role in the immunomodulatory activity of this molecule, as suggested by numerous in vitro and in vivo studies. In particular, the protective effect of exogenous Lf against endotoxin shock in various animal models was extensively reported [29e32] as well as its in vitro capacity to inhibit the LPS-induced IL-8, E-selectin and ICAM-1 expression in human endothelial cells [14,56], and reactive oxygen species production in neutrophils [15,57]. However, Na and co-workers recently reported that when LPS and purified Lf were mixed, and formed a complex, induction of proinflammatory mediators rather than inhibition of LPS challenge was observed in RAW 264.7 cells and PMf harvested from C3H/HeN mice [58]. Comparative studies carried out with LPS responsive and LPS hypo-responsive mice demonstrated a strong dependency of the LfeLPS complex triggered signals on toll-like receptor 4 (TLR4), leading to the conclusion that the immunomodulatory properties of Lf could be due, at least in part, to LPS binding [58]. Lf binds to the lipid A portion of LPS via chargeecharge interaction. The portion of Lf that binds to anionic molecules, including lipid A, is limited to its N-terminus arginine rich domain [52]. Thus, it is likely that bound LPS can still expose the unbound part of lipid A that is recognized by LPS receptors such as TLR4. Such a LfeLPS complex recognition would result in Mf activation [58]. Of note, the lipid A backbone is also the epitope being recognized in the Limulus assay (LAL), the standard method for detection of endotoxin contamination, thus explaining why the LfeLPS complex is found to be LAL positive [58,59]. Collectively, these results suggest that lipid A can be recognized even after LfeLPS complex has been formed, and that this complex retains the capacity to activate Mf through TLR4. However, the intimate relationship between Lf and LPS does not completely account for the different biological activities ascribed to this molecule. In keeping with these results, we have recently reported that the capacity of Lf to induce a type I IFN mediated antiviral state, but not TNF-a production, relies on the function of TLR4 in responding cells [45]. Our results showing that TLR4 is not essential for Lf-induced production of TNF-a by murine PMf, strongly suggest that this molecule induces Mf activation via TLR4dependent and -independent mechanisms. Accordingly, it has been recently reported that Lf-induced IL-6 secretion and CD40 expression in murine PMf was achieved via TLR4-
independent and TLR4-dependent mechanisms, respectively, thus indicating potentially separate pathways for Lf-mediated Mf events in innate immunity [44]. Thus, Lf binding to LPS may represent an important aspect, but does not entirely account for all immunomodulatory effects of this molecule. This aspect could be mostly relevant in those cell types, such as the Mf, in which TLR4 function is of critical importance in the regulation of their activity. Originally described for its antiviral activity, type I IFN has recently been shown to exert important effects on the immune system, including promotion of cellular and humoral responses, by virtue of its adjuvant effects on APCs [60e62]. Early in viral infection, IFNs interfere with viral replication and activate natural killer (NK) cells. Later in the adaptive immune response, these cytokines up-regulate MHC class I antigen expression and promote cross-priming, thus favouring the killing of virally infected cells by cytotoxic lymphocytes [63,64]. The induction of type I IFN by virus-infected cells occurs very rapidly, within a few hours post-infection, and represents the earliest antiviral response in the host [65]. Although every cell or tissue of the body can produce and secrete IFN upon stimulation, a specialized cell type, the blood plasmacytoid DC, has been shown to be the greatest IFN producer on viral challenge [66,67]. IFN production is transcriptionally activated upon recognition of a virus, or its molecular patterns, by TLRs of DCs [68]. In turn, the IFN secreted stimulates DC maturation and subsequent expression of proinflammatory cytokines and co-stimulatory molecules, leading to the transition to an adaptive antiviral immunity [67]. In addition to DCs, Mf are considered important elements in natural resistance against infection, also involved in early IFN production [69]. These cells are intimately related to the IFN system, and the existence of a so-called ‘‘IFN-macrophage alliance’’ has been postulated [69]. Several studies have revealed that low levels of type I IFN, mainly IFN-b, are spontaneously expressed in resting mouse PMf and are responsible for the natural antiviral state of these cells [70,71]. The antiviral activity of Lf has been largely demonstrated for a number of viruses [6]. Several studies indicated that Lf may exert its antiviral effects through inhibition of viral entry rather than stimulation of the immune cells [6]. In this regard, we have recently demonstrated that bovine Lf also exerts indirect effects on viral replication by stimulating the production of antiviral factors, such as type I IFN, which in turn allows to restrict viral replication [45]. Interestingly, a number of studies have previously suggested that Lf could somehow act on the IFN system by promoting its expression. In this regard, it has been demonstrated that oral administration of bovine Lf modulates the expression of immunity related genes in small intestines of mice. Interestingly, a significant up-modulation of transcripts coding for IFN-b was observed in animals that received Lf with respect to control mice [72]. Likewise, oral administration of bovine Lf was reported to increase the expression of IFN-a and IFN-b mRNA in Peyer’s patches and mesenteric lymph nodes, as well as the levels of IL-18 protein in the portal circulation [73]. Of note, administration of liposomal Lf also induced a significant up-modulation of IFN-a levels in healthy human
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Fig. 1. Immunomodulatory activity of Lf on APCs. The plethora of Lf activities relies on its capacity to bind iron but also to interact with molecular and cellular components of both host and pathogens. A schematic representation of the effects of Lf interaction with APC receptors leading to the control of excessive inflammation and enhancement of immune response is shown.
volunteers [74]. It is well known that type I IFN induces NK cell activation by enhancing their cytotoxic activity and IFNg production [75]. Interestingly, in studies describing that oral Lf administration stimulates type I IFN production, an increased NK cell activity was also found [73]. Overall, these studies suggest the importance of Lf-mediated type I IFN induction in at least some of the immunomodulatory effects ascribed to this molecule. Although it remains to be proved that APCs are in vivo targets of Lf activity, their marked capacity to produce type I IFN in response to different stimuli and their intimate relationship with these cytokines, argue in favour of a role of these cells in the Lf-mediated effects on the IFN system. Lastly, these results provide further evidence that some of the immunomodulatory effects ascribed to Lf may be related to its capacity to stimulate IFN production. 4. Effects of Lf on dendritic cell subsets Although the immunomodulatory effects of Lf on monocytes/Mf have been largely investigated, limited information is currently available on the activity of this compound on DCs (Table 1). Some studies have been carried out to evaluate the role of Lf in the regulation of epidermal LCs migration. LCs, a subset of DCs, are considered to play pivotal roles in the induction and regulation of cutaneous immune responses [76]. Consistent with the anti-inflammatory properties of Lf, in a mouse model of cutaneous inflammation, it was found that the intradermal injection of recombinant
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murine Lf significantly inhibits allergen-induced LC migration and DC accumulation [77]. This inhibitory effect was secondary to the inhibition of TNF-a local production. Finally, immunohistochemical analysis demonstrated that recombinant human Lf was indeed present into the skin, associated primarily with keratinocytes [78]. Additional evidence on the capacity of Lf to influence cutaneous immune and inflammatory processes was provided by subsequent studies assessing the effect of Lf on chemical allergen- and cytokine-induced LC migration in healthy human volunteers [79]. This study demonstrated that Lf, when administered topically, is able to inhibit the subsequent migration of LCs. In keeping with the results obtained in the mouse model, inhibition of LC migration was dependent on the de novo synthesis of TNF-a. Since Lf is expressed constitutively in both mouse and human skin [78,80], these results suggest that moderating inflammatory reactions secondary to regulation of local cytokine production might represent a physiological function of Lf. Subsequent studies examining the contribution of IL-1b to human LC migration provided further evidence on the role of Lf in the regulation of LC migration and inflammation. In particular, it was reported that recombinant IL-1b administered intradermally to healthy human volunteers provides a stimulus for LC migration and stimulates TNF-a production at the site of inoculum [81]. Prior topical application of human recombinant Lf inhibited the IL1b-mediated LC migration and also impaired TNF-a production [81]. Overall, these studies indicate that topical Lf may potentially represent a novel therapeutic in the treatment of skin inflammation where TNF-a is an important mediator. However, a recent study argues against Lf as an anti-inflammatory drug, because of its lack of inhibitory effects on monocytederived DC (MD-DC) maturation [82]. In this report, a panel of anti-inflammatory drugs were assessed for their capacity to revert nickel-induced maturation and cytokine secretion in MD-DCs. The results of these experiments indicated that all tested anti-inflammatory drugs, except talactoferrin, a recombinant human Lf, inhibit DC maturation, as assessed by impaired CD86 up-modulation and CXCL8 production [82]. In addition, Spadaro and colleagues very recently reported that the same preparation of Lf-induced MD-DC maturation and promoted Th1 type immune responses [83]. Another interesting aspect of Lf interaction with DCs is represented by the DC-SIGN, a C-type lectin receptor expressed by DCs that interacts with a variety of pathogens, including HIV-1, hepatitis C virus, Ebola virus, cytomegalovirus, Dengue virus, mycobacterium, Leishmania, and C. albicans [84]. In this regard, it has been reported that bovine Lf prevents MD-DC-mediated HIV-1 transmission by blocking DC-SIGN interaction with the viral surface glycoprotein gp120 [34]. Likewise, the efficacy of Lf to prevent HIV-1 capture by DCs was reported for both R5 and X4 HIV strains [85]. Conversely, Lf-reacting natural antibodies enhanced HIV attachment to DCs by forming complexes with the virus. HIV particles were found to directly interact with Lf following its interaction with Lf-reacting natural antibodies. Thus, these results are consistent with a model in which Lf would appear as
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a double-edge sword that could have beneficial or detrimental effects depending on both cellular and molecular microenvironments [85]. 5. Concluding remarks Lf, a natural compound of mammalian secretions, exerts a plethora of biological activities. Its protective effects range from direct antimicrobial activities against a variety of pathogens, including bacteria, viruses, fungi and parasites, to anti-inflammatory and anti-tumour activities. These multiple functions rely not only on the capacity of Lf to bind iron but also on its marked capacity to interact with molecular and cellular components of both host and pathogens. In this context, Lf interaction with APCs, cells of critical importance in the maintenance of immune system homeostasis, may play a pivotal role (Fig. 1). Understanding the molecular mechanisms underlying the capacity of Lf to reduce excessive inflammation and stimulate host immune responses, as well as identifying cell targets and receptors involved in Lf immunomodulatory activities hold the promise of a better exploitation of Lf therapeutic potential. Acknowledgements This work was supported by a research grant from the Italian Ministry of Health (COSM2) to S.G, PRIN 2004 and 2001 to P.V. References [1] S. Gordon, The macrophage: past, present and future, Eur. J. Immunol. 37 (Suppl. 1) (2007) S9eS17. [2] F.O. Martinez, A. Sica, A. Mantovani, M. Locati, Macrophage activation and polarization, Front. Biosci. 13 (2008) 453e461. [3] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Annu. Rev. Immunol. 18 (2000) 767e811. [4] R.M. Steinman, Some interfaces of dendritic cell biology, Apmis 111 (2003) 675e697. [5] D. Legrand, A. Pierce, E. Elass, M. Carpentier, C. Mariller, J. Mazurier, Lactoferrin structure and functions, Adv. Exp. Med. Biol. 606 (2008) 163e194. [6] P. Valenti, G. Antonini, Lactoferrin: an important host defence against microbial and viral attack, Cell Mol. Life Sci. 62 (2005) 2576e2587. [7] D. Legrand, E. Elass, M. Carpentier, J. Mazurier, Lactoferrin: a modulator of immune and inflammatory responses, Cell Mol. Life Sci. 62 (2005) 2549e2559. [8] P.P. Ward, S. Uribe-Luna, O.M. Conneely, Lactoferrin and host defense, Biochem. Cell Biol. 80 (2002) 95e102. [9] T. Kawamata, I. Tooyama, T. Yamada, D.G. Walker, P.L. McGeer, Lactotransferrin immunocytochemistry in Alzheimer and normal human brain, Am. J. Pathol. 142 (1993) 1574e1585. [10] K. Uchida, R. Matsuse, S. Tomita, K. Sugi, O. Saitoh, S. Ohshiba, Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer, Clin. Biochem. 27 (1994) 259e264. [11] E. Decoteau, A.M. Yurchak, R.E. Partridge, T.B. Tomasi Jr., Lactoferrin in synovial fluid of patients with inflammatory arthritis, Arthritis Rheum. 15 (1972) 324e325.
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Biochimie 91 (2009) 11e18 www.elsevier.com/locate/biochi
Review
Immunomodulatory effects of lactoferrin on antigen presenting cells Patrizia Puddu a, Piera Valenti b, Sandra Gessani a,* a
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy b Department of Public Health Sciences, Sapienza, University of Rome, Rome, Italy Received 18 March 2008; accepted 9 May 2008 Available online 21 May 2008
Abstract Lactoferrin (Lf) is an 80 kDa iron-binding protein of the transferrin family that is abundantly expressed in most biological fluids. It is now recognized that this glycoprotein is a key element in the mammalian immune system, playing an important role in host defence against infection and excessive inflammation. Although the mechanisms underlying Lf immunomodulatory properties have not been fully elucidated yet, evidence indicates that the capacity of this molecule to directly interact with antigen presenting cells (APCs), i.e. monocytes/macrophages and dendritic cells (DCs), may play a critical role. At the cellular level, Lf modulates important aspects of APC biology, including migration and cell activation, whereas at the molecular level it affects expression of soluble immune mediators, such as cytokines, chemokines and other effector molecules, thus contributing to the regulation of inflammation and immunity. While the iron-binding property was originally believed to be solely responsible for the plethora of host defence activities ascribed to Lf, it is now known that other mechanisms contribute to the broad spectrum of anti-infective and anti-inflammatory properties of this protein. Recent results suggest that at least some of the immunomodulatory effects of Lf rely on its capacity to form complexes with lipopolysaccharide (LPS). This review focuses on the effects of Lf on APC biology and function, highlighting known and putative mechanisms that underlie Lf immunomodulatory effects. The importance of LPS-binding capacity of Lf and LPS receptors, as well as of Lf-induced type 1 interferon (IFN) expression in some of these effects is also discussed. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Lactoferrin; Macrophage; Dendritic cell; Immunomodulation; Lipopolysaccharide
1. Introduction Antigen presenting cell (APC) function is central to the development of effective immune responses against pathogens.
Abbreviations: Lf, lactoferrin; LfR, lactoferrin receptor; APC, antigen presenting cell; DC, dendritic cell; MD-DC, monocyte-derived dendritic cell; LC, Langerhans cell; Mf, macrophage; PMf, peritoneal macrophage; BMMf, bone marrow macrophage; PBM, peripheral blood monocyte; NK, natural killer cell; Th, T helper cell; LPS, lipopolysaccharide; sCD14, soluble CD14; TLR4, toll-like receptor 4; LRP, low-density lipoprotein receptor-related protein; LAL, Limulus assay; NO, nitric oxide; MHC, major histocompatibility complex; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; MR, mannose receptor; HIV-1, human immunodeficiency virus 1; BCG, bacillus Calmette Guerin; ADCC, antibody-dependent cellular cytotoxicity; IL, interleukin; TNF, tumour necrosis factor; IFN, interferon. * Corresponding author. Tel.: þ39 06 49903169; fax: þ39 06 49903641. E-mail address: [email protected] (S. Gessani). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.05.005
Among APCs, macrophages (Mf) and dendritic cells (DCs) are of critical importance for the maintenance of tissue homeostasis and innate response to pathogens, as well as in linking innate to adaptive immune response. Mf are highly phagocytic cells which play a central role in the control of infections, either by direct intracellular killing of microorganisms or by secreting cytokines inhibiting their replication, as well as in type II inflammation and tissue repair processes [1,2]. DCs are a heterogeneous population of cells highly specialized for antigen recognition that play a key role in the immune system by virtue of their capacity to control both immunity and tolerance induction [3,4]. Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein abundantly found in exocrine secretions of mammals, in particular in milk and fluids of the digestive tract, and released by mucosal epithelia and neutrophils during inflammation [5]. Lf is a key element in the host defence system [6e8]. It
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is now well recognized that this molecule plays a direct antimicrobial role in secretions and at the surface of epithelia, by limiting the proliferation and adhesion of microbes and/or by killing them. These properties are mainly related to its ability to sequester iron in biological fluids or to destabilize the membranes of microorganisms. However, iron-independent microbicidal activities, requiring direct interaction between Lf and microbial surface components, have been subsequently demonstrated [5]. In addition to the antimicrobial properties of Lf, its ability to modulate the overall immune response and to protect against viral infections and septic shock has been largely described [6e8]. In this respect, it is noteworthy that Lf concentrations are elevated locally in inflammatory disorders including neurodegenerative diseases [9], inflammatory bowel disease [10], arthritis [11], and allergic inflammation [12]. Although the cellular and molecular mechanisms accounting for the immunomodulatory effects of Lf are far from being fully elucidated, both in vitro and in vivo studies suggest the existence of multiple mechanisms that include modulation of cytokine/chemokine production, regulation of reactive oxygen species production, and of immune cell recruitment. It is now clear that at least some of the Lf biological activities do not merely depend on its iron-binding capacity, but may arise from its interaction with a variety of molecules. In this respect, the capacity of Lf to influence either negatively or positively cytokine production relies, at least in part, on its ability to bind and sequester both lipopolysaccharide (LPS) and its receptor CD14, as well as CpG bacterial DNA, thus preventing the downstream activation of proinflammatory pathways, septic shock and tissue damage [13e16]. However, Lf can also favour the activation, differentiation, and proliferation of immune cells, and this promoting activity has been related to a direct effect of Lf on immune cells through the recognition of specific Lf binding sites [17,18]. 2. Cell determinants involved in Lf interaction with antigen presenting cells Cells of the immune system, like virtually any other cells in the organism, can bind Lf. The cationic nature of this molecule accounts for its propensity to bind to anionic molecules, resulting in a massive binding to mammalian cells [18]. Several Lf binding sites have been found on target cells, revealing considerable variations among species, tissues and cell types [19]. A number of studies reported the direct binding of Lf to monocytes/Mf and DCs. Indeed, mouse peritoneal Mf (PMf) was the first cell type reported to express Lf receptors (LfRs) in mammals [20], and human monocytes were also shown to bind human Lf in a reversible, saturable and specific manner [21]. At the surface of cells, the sulphated chains of proteoglycans are responsible for the low-affinity, high density binding of Lf [5]. Other important receptors are represented by low-density lipoprotein receptor-related proteins (LRPs), frequently referred to as scavenger receptors, widely expressed on several cell types, including Mf [22]. Lf has also been shown to bind to surface nucleolin [23], a multifunctional shuttling protein present in nucleus, cytoplasm, and on the
surface of some cell types, including macrophages [24]. Finally, at least four species of human Lf binding proteins have been found on the plasma membrane of THP-1, a human monocytic leukaemia cell line [25]. The LfR expressed on monocytes/Mf is most likely involved in the effects of human Lf on the inflammatory and immune responses of animals [26]. In this respect, it has been shown that human Lf interacts directly with CD14, an LPS receptor involved in the activation of the immune system that exists both as a soluble protein (sCD14) found in serum [27], and membrane protein highly expressed on the surface of monocytes/Mf [28]. Human Lf binds to sCD14 complexed to LPS or lipid A and protects animals from septic shock induced by LPS [29e32], thus indicating that CD14 is one of the LfR expressed on monocytes/Mf. In contrast to monocytes/Mf, no specific studies have been carried out yet to define the nature of LfRs expressed in DCs. However, the observation that the adjuvant effect of Lf in skin Langerhans cells (LCs) is inhibited by blocking the mannose receptor (MR), suggests that this receptor may represent a putative site for Lf interaction with this subset of DCs [33]. In addition, it has been recently shown that Lf binds to the DC-specific ICAM3-grabbing non-integrin (DC-SIGN), a C-type lectin receptor expressed by DCs [34], thus suggesting that receptors belonging to the C-type lectin family may play an important role in Lf interaction with DCs. The capacity of Lf to interact with C-type lectin receptors apparently depends on its sugar composition. In this respect, it has been demonstrated that although bovine and human Lf both interact with the MR, this interaction occurs to a different extent and strongly relates to the different composition in relevant sugars [34]. Likewise, it has been reported that human Lf, in contrast to bovine Lf, does not bind DC-SIGN, as assessed by the lack of capacity of this protein to prevent the DC-SIGN-mediated human immunodeficiency virus-1 (HIV-1) transfer to CD4þ T lymphocytes [34,35]. The presence of specific Lf receptors on immune cells suggests that the modulation of host responses by Lf may be related to a direct effect via receptor-mediated signalling pathways. In this respect, it has been reported that Lf downregulation of LPS-induced interleukin (IL)-6 secretion in monocytes involves Lf translocation to the nucleus and inhibition of NF-kB activation [36]. 3. Effects of Lf on cells of the monocyte lineage Tissue Mf play a master role in homeostasis and immune defence against infections, providing a rapid response against pathogen threats. In particular, Mf clear exogenous antigens and apoptotic cells through phagocytosis and intracellular killing, trigger tissue leukocyte infiltration, and collaborate with T and B cells by locally releasing cytokines and other effector molecules, including nitric oxide (NO), as well as by taking part to tissue repair processes. In this respect, Mf display a peculiar plasticity as they can be committed to undergo a different differentiation program depending on the cytokines released in the microenvironment [2]. In this view, Mf can be classified in two main groups named M1 (classically
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activated Mf) and M2 (alternatively activated Mf). M1 Mf exert strong endocytic and antimicrobic activities and elicit a potent T helper (Th)1 cellular response mediated by IL-12 production, whereas M2 Mf support Th2-associated effector functions such as high endocytic clearance capacity, trophic factor synthesis and reduced proinflammatory cytokine secretion, playing a role in resolution of inflammation. In this context, Lf could represent a crucial factor in directing Mf activation and, consequently, in polarizing the immune response. A variety of studies have indicated that Lf can selectively interact with monocyte and Mf, and modulate their functions during inflammatory and infectious processes. The results of these studies are summarized in Table 1. In particular, it has been reported that human Lf treatment positively affects human adherent monocyte cytotoxic activity, whereas it has no effect on nonadherent lymphocytes [37]. In contrast to the enhancement of cytotoxicity, Lf exhibited concentration-dependent inhibitory effect on antibody-dependent cellular cytotoxicity (ADCC). Again, this appeared to be an effect on monocytes because Lf did not alter nonadherent cell ADCC activity [37]. Some studies investigated the effect of Lf on the capacity of mouse PMf and human peripheral blood monocytes (PBM) to up-take and kill intracellular pathogens. These reports showed that human Lf-treated cells exhibit an increased phagocytosis and intracellular killing of Trypanosoma cruzi [38]. The presence of iron was found to be required for human Lf to stimulate intracellular killing by PMf but not to enhance the up-take of particles and microorganisms [39]. Likewise, oral administration of bovine Lf stimulated PMf activities in mice injected with inactivated Candida albicans [40]. In particular, Lf administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of NO by PMf. In keeping with these results, induction of NO release by bovine Lf was also observed in rat BMMf [41]. Furthermore, bovine Lf administration enhanced the ability of PMf to inhibit hyphal growth in C. albicans injected mice [40]. In keeping with these results, Tanida and colleagues reported that administration of Lf peptides prolonged the survival periods of C. albicans
13
inoculated mice by up-regulating neutrophil and macrophage functions [42]. Lastly, bovine Lf was also shown to modulate APC response to bacillus Calmette Guerin (BCG) by significantly increasing the up-take of microorganisms during infection of murine BMMf and human monocytic cell lines [43]. Furthermore, it was also reported that BCG infected BMMf exposed to bovine Lf significantly up-modulated the expression of major histocompatibility complex (MHC) class II (I-A), but not class I (H-2k), surface molecules [43]. Likewise, bovine Lf stimulated CD40 expression in mouse RAW 264.7 cell line and PMf [44]. A variety of studies have indicated that Lf can modulate basal or LPS-induced cytokine production in different models of primary monocyte/Mf and monocytic cell lines. In this respect, we as well as other groups demonstrated that exposure of Mf to bovine Lf results in the production of several cytokines/ chemokines, including IL-6, IL-8, IL-12, tumour necrosis factor (TNF)-a and interferon (IFN)-b [41,44e46]. Interestingly, the bovine Lf stimulated IFN-b expression was responsible for the antiviral effect observed in murine resting PMf [45]. Although exposure of na€ıve macrophages to Lf results in the induction of some cytokines/chemokines, the effects of this molecule on activated Mf (i.e. LPS-treated or pathogen infected) are more complex. In particular, it has been demonstrated that LPS-induced production of IL-6, TNF-a, IL-1-b and IL-8 is inhibited by Lf, both bovine and human or its fragment lactoferricin B, in various human monocytic cell lines [36,47,48]. Furthermore, it has been observed that addition of bovine Lf to spleen Mf and J774A.1 murine cell line stimulated with suboptimal LPS concentrations increased production of IL-12 whereas the secretion of IL-10 was decreased [49]. These observations support the hypothesis that Lf could sustain Mf polarization versus a Th1 immune response. In keeping with this hypothesis, Guillen and colleagues reported an enhanced Th1 response to Staphylococcus aureus infection in huLf-transgenic mice, characterized by an enhanced in vivo production of IFN-g and TNF-a and reduced IL-5 and IL-10 secretion. Similarly, huLf-transgenic mice exhibited enhanced severity of Th1-driven collagen-induced arthritis [50].
Table 1 Immunomodulatory effects of lactoferrin on APCs Function
Effect
Lf origin
Model species
Cell types
Reference
Cytotoxic activity Antibody-dependent cytotoxic activity Pathogen up-take and intracellular killing Expression of surface molecules Cytokine production LPS-induced cytokines/chemokines IFN mediated antiviral state Production of Th1 cytokines Cell migration Viral transmission DC activation
j k j j j k j j k k j
Hu Hu Hu/Bo/P Bo Bo Hu/Bo/F Bo Bo Rec Bo Rec
Hu Hu Hu/Mu/Ra Mu Mu/Ra Hu/mu Mu Mu Mu/Hu Hu Hu
PBM PBM PBM, PMf, BMMf, cell lines PMf, BMMf, cell lines PMf, BMMf, cell lines cell lines PMf spleen Mf, BMMf, cell lines LCs MD-DCs MD-DCs
[37] [37] [38e43] [43,44] [41,44e46] [36,47,48] [45] [49] [78,79] [34] [83]
Summary of the principal experimental observations showing the immunomodulatory effects of Lf of different origins (murine, human, bovine or recombinant) on both primary cells and cell lines of the monocyte/DC lineage. Hu ¼ human; Mu ¼ murine; Ra ¼ rat; Bo ¼ bovine; P ¼ Lf synthetic peptide; F ¼ Lf fragment; Rec ¼ recombinant Lf.
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Overall, these results indicate that Lf can differently affect several Mf functions, ultimately leading to attenuation of excessive inflammation and stimulation of host response against pathogen threats. However, further studies are strongly needed to define molecular and cellular determinants involved in the immunomodulatory activity of this molecule. Structurally, Lf contains a highly basic region close to the N-terminus which binds to a variety of anionic biological molecules. Human Lf binds specifically and with a high affinity to the lipid A region of bacterial LPS [13,51], sCD14 and the sCD14/LPS complex [14]. Two N terminal basic clusters of human Lf, residues 1e5 and 28e34, are responsible for the binding to anionic molecules, such as LPS [51,52], heparin, or cell-surface heparan sulphates [53e55]. The LPS-binding property of Lf play an important role in the immunomodulatory activity of this molecule, as suggested by numerous in vitro and in vivo studies. In particular, the protective effect of exogenous Lf against endotoxin shock in various animal models was extensively reported [29e32] as well as its in vitro capacity to inhibit the LPS-induced IL-8, E-selectin and ICAM-1 expression in human endothelial cells [14,56], and reactive oxygen species production in neutrophils [15,57]. However, Na and co-workers recently reported that when LPS and purified Lf were mixed, and formed a complex, induction of proinflammatory mediators rather than inhibition of LPS challenge was observed in RAW 264.7 cells and PMf harvested from C3H/HeN mice [58]. Comparative studies carried out with LPS responsive and LPS hypo-responsive mice demonstrated a strong dependency of the LfeLPS complex triggered signals on toll-like receptor 4 (TLR4), leading to the conclusion that the immunomodulatory properties of Lf could be due, at least in part, to LPS binding [58]. Lf binds to the lipid A portion of LPS via chargeecharge interaction. The portion of Lf that binds to anionic molecules, including lipid A, is limited to its N-terminus arginine rich domain [52]. Thus, it is likely that bound LPS can still expose the unbound part of lipid A that is recognized by LPS receptors such as TLR4. Such a LfeLPS complex recognition would result in Mf activation [58]. Of note, the lipid A backbone is also the epitope being recognized in the Limulus assay (LAL), the standard method for detection of endotoxin contamination, thus explaining why the LfeLPS complex is found to be LAL positive [58,59]. Collectively, these results suggest that lipid A can be recognized even after LfeLPS complex has been formed, and that this complex retains the capacity to activate Mf through TLR4. However, the intimate relationship between Lf and LPS does not completely account for the different biological activities ascribed to this molecule. In keeping with these results, we have recently reported that the capacity of Lf to induce a type I IFN mediated antiviral state, but not TNF-a production, relies on the function of TLR4 in responding cells [45]. Our results showing that TLR4 is not essential for Lf-induced production of TNF-a by murine PMf, strongly suggest that this molecule induces Mf activation via TLR4dependent and -independent mechanisms. Accordingly, it has been recently reported that Lf-induced IL-6 secretion and CD40 expression in murine PMf was achieved via TLR4-
independent and TLR4-dependent mechanisms, respectively, thus indicating potentially separate pathways for Lf-mediated Mf events in innate immunity [44]. Thus, Lf binding to LPS may represent an important aspect, but does not entirely account for all immunomodulatory effects of this molecule. This aspect could be mostly relevant in those cell types, such as the Mf, in which TLR4 function is of critical importance in the regulation of their activity. Originally described for its antiviral activity, type I IFN has recently been shown to exert important effects on the immune system, including promotion of cellular and humoral responses, by virtue of its adjuvant effects on APCs [60e62]. Early in viral infection, IFNs interfere with viral replication and activate natural killer (NK) cells. Later in the adaptive immune response, these cytokines up-regulate MHC class I antigen expression and promote cross-priming, thus favouring the killing of virally infected cells by cytotoxic lymphocytes [63,64]. The induction of type I IFN by virus-infected cells occurs very rapidly, within a few hours post-infection, and represents the earliest antiviral response in the host [65]. Although every cell or tissue of the body can produce and secrete IFN upon stimulation, a specialized cell type, the blood plasmacytoid DC, has been shown to be the greatest IFN producer on viral challenge [66,67]. IFN production is transcriptionally activated upon recognition of a virus, or its molecular patterns, by TLRs of DCs [68]. In turn, the IFN secreted stimulates DC maturation and subsequent expression of proinflammatory cytokines and co-stimulatory molecules, leading to the transition to an adaptive antiviral immunity [67]. In addition to DCs, Mf are considered important elements in natural resistance against infection, also involved in early IFN production [69]. These cells are intimately related to the IFN system, and the existence of a so-called ‘‘IFN-macrophage alliance’’ has been postulated [69]. Several studies have revealed that low levels of type I IFN, mainly IFN-b, are spontaneously expressed in resting mouse PMf and are responsible for the natural antiviral state of these cells [70,71]. The antiviral activity of Lf has been largely demonstrated for a number of viruses [6]. Several studies indicated that Lf may exert its antiviral effects through inhibition of viral entry rather than stimulation of the immune cells [6]. In this regard, we have recently demonstrated that bovine Lf also exerts indirect effects on viral replication by stimulating the production of antiviral factors, such as type I IFN, which in turn allows to restrict viral replication [45]. Interestingly, a number of studies have previously suggested that Lf could somehow act on the IFN system by promoting its expression. In this regard, it has been demonstrated that oral administration of bovine Lf modulates the expression of immunity related genes in small intestines of mice. Interestingly, a significant up-modulation of transcripts coding for IFN-b was observed in animals that received Lf with respect to control mice [72]. Likewise, oral administration of bovine Lf was reported to increase the expression of IFN-a and IFN-b mRNA in Peyer’s patches and mesenteric lymph nodes, as well as the levels of IL-18 protein in the portal circulation [73]. Of note, administration of liposomal Lf also induced a significant up-modulation of IFN-a levels in healthy human
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Fig. 1. Immunomodulatory activity of Lf on APCs. The plethora of Lf activities relies on its capacity to bind iron but also to interact with molecular and cellular components of both host and pathogens. A schematic representation of the effects of Lf interaction with APC receptors leading to the control of excessive inflammation and enhancement of immune response is shown.
volunteers [74]. It is well known that type I IFN induces NK cell activation by enhancing their cytotoxic activity and IFNg production [75]. Interestingly, in studies describing that oral Lf administration stimulates type I IFN production, an increased NK cell activity was also found [73]. Overall, these studies suggest the importance of Lf-mediated type I IFN induction in at least some of the immunomodulatory effects ascribed to this molecule. Although it remains to be proved that APCs are in vivo targets of Lf activity, their marked capacity to produce type I IFN in response to different stimuli and their intimate relationship with these cytokines, argue in favour of a role of these cells in the Lf-mediated effects on the IFN system. Lastly, these results provide further evidence that some of the immunomodulatory effects ascribed to Lf may be related to its capacity to stimulate IFN production. 4. Effects of Lf on dendritic cell subsets Although the immunomodulatory effects of Lf on monocytes/Mf have been largely investigated, limited information is currently available on the activity of this compound on DCs (Table 1). Some studies have been carried out to evaluate the role of Lf in the regulation of epidermal LCs migration. LCs, a subset of DCs, are considered to play pivotal roles in the induction and regulation of cutaneous immune responses [76]. Consistent with the anti-inflammatory properties of Lf, in a mouse model of cutaneous inflammation, it was found that the intradermal injection of recombinant
15
murine Lf significantly inhibits allergen-induced LC migration and DC accumulation [77]. This inhibitory effect was secondary to the inhibition of TNF-a local production. Finally, immunohistochemical analysis demonstrated that recombinant human Lf was indeed present into the skin, associated primarily with keratinocytes [78]. Additional evidence on the capacity of Lf to influence cutaneous immune and inflammatory processes was provided by subsequent studies assessing the effect of Lf on chemical allergen- and cytokine-induced LC migration in healthy human volunteers [79]. This study demonstrated that Lf, when administered topically, is able to inhibit the subsequent migration of LCs. In keeping with the results obtained in the mouse model, inhibition of LC migration was dependent on the de novo synthesis of TNF-a. Since Lf is expressed constitutively in both mouse and human skin [78,80], these results suggest that moderating inflammatory reactions secondary to regulation of local cytokine production might represent a physiological function of Lf. Subsequent studies examining the contribution of IL-1b to human LC migration provided further evidence on the role of Lf in the regulation of LC migration and inflammation. In particular, it was reported that recombinant IL-1b administered intradermally to healthy human volunteers provides a stimulus for LC migration and stimulates TNF-a production at the site of inoculum [81]. Prior topical application of human recombinant Lf inhibited the IL1b-mediated LC migration and also impaired TNF-a production [81]. Overall, these studies indicate that topical Lf may potentially represent a novel therapeutic in the treatment of skin inflammation where TNF-a is an important mediator. However, a recent study argues against Lf as an anti-inflammatory drug, because of its lack of inhibitory effects on monocytederived DC (MD-DC) maturation [82]. In this report, a panel of anti-inflammatory drugs were assessed for their capacity to revert nickel-induced maturation and cytokine secretion in MD-DCs. The results of these experiments indicated that all tested anti-inflammatory drugs, except talactoferrin, a recombinant human Lf, inhibit DC maturation, as assessed by impaired CD86 up-modulation and CXCL8 production [82]. In addition, Spadaro and colleagues very recently reported that the same preparation of Lf-induced MD-DC maturation and promoted Th1 type immune responses [83]. Another interesting aspect of Lf interaction with DCs is represented by the DC-SIGN, a C-type lectin receptor expressed by DCs that interacts with a variety of pathogens, including HIV-1, hepatitis C virus, Ebola virus, cytomegalovirus, Dengue virus, mycobacterium, Leishmania, and C. albicans [84]. In this regard, it has been reported that bovine Lf prevents MD-DC-mediated HIV-1 transmission by blocking DC-SIGN interaction with the viral surface glycoprotein gp120 [34]. Likewise, the efficacy of Lf to prevent HIV-1 capture by DCs was reported for both R5 and X4 HIV strains [85]. Conversely, Lf-reacting natural antibodies enhanced HIV attachment to DCs by forming complexes with the virus. HIV particles were found to directly interact with Lf following its interaction with Lf-reacting natural antibodies. Thus, these results are consistent with a model in which Lf would appear as
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a double-edge sword that could have beneficial or detrimental effects depending on both cellular and molecular microenvironments [85]. 5. Concluding remarks Lf, a natural compound of mammalian secretions, exerts a plethora of biological activities. Its protective effects range from direct antimicrobial activities against a variety of pathogens, including bacteria, viruses, fungi and parasites, to anti-inflammatory and anti-tumour activities. These multiple functions rely not only on the capacity of Lf to bind iron but also on its marked capacity to interact with molecular and cellular components of both host and pathogens. In this context, Lf interaction with APCs, cells of critical importance in the maintenance of immune system homeostasis, may play a pivotal role (Fig. 1). Understanding the molecular mechanisms underlying the capacity of Lf to reduce excessive inflammation and stimulate host immune responses, as well as identifying cell targets and receptors involved in Lf immunomodulatory activities hold the promise of a better exploitation of Lf therapeutic potential. Acknowledgements This work was supported by a research grant from the Italian Ministry of Health (COSM2) to S.G, PRIN 2004 and 2001 to P.V. References [1] S. Gordon, The macrophage: past, present and future, Eur. J. Immunol. 37 (Suppl. 1) (2007) S9eS17. [2] F.O. Martinez, A. Sica, A. Mantovani, M. Locati, Macrophage activation and polarization, Front. Biosci. 13 (2008) 453e461. [3] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Annu. Rev. Immunol. 18 (2000) 767e811. [4] R.M. Steinman, Some interfaces of dendritic cell biology, Apmis 111 (2003) 675e697. [5] D. Legrand, A. Pierce, E. Elass, M. Carpentier, C. Mariller, J. Mazurier, Lactoferrin structure and functions, Adv. Exp. Med. Biol. 606 (2008) 163e194. [6] P. Valenti, G. Antonini, Lactoferrin: an important host defence against microbial and viral attack, Cell Mol. Life Sci. 62 (2005) 2576e2587. [7] D. Legrand, E. Elass, M. Carpentier, J. Mazurier, Lactoferrin: a modulator of immune and inflammatory responses, Cell Mol. Life Sci. 62 (2005) 2549e2559. [8] P.P. Ward, S. Uribe-Luna, O.M. Conneely, Lactoferrin and host defense, Biochem. Cell Biol. 80 (2002) 95e102. [9] T. Kawamata, I. Tooyama, T. Yamada, D.G. Walker, P.L. McGeer, Lactotransferrin immunocytochemistry in Alzheimer and normal human brain, Am. J. Pathol. 142 (1993) 1574e1585. [10] K. Uchida, R. Matsuse, S. Tomita, K. Sugi, O. Saitoh, S. Ohshiba, Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer, Clin. Biochem. 27 (1994) 259e264. [11] E. Decoteau, A.M. Yurchak, R.E. Partridge, T.B. Tomasi Jr., Lactoferrin in synovial fluid of patients with inflammatory arthritis, Arthritis Rheum. 15 (1972) 324e325.
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