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Alarmins: chemotactic activators of immune responses
Alarmins: chemotactic activators of immune responses Joost J Oppenheim and De Yang The recruitment and activation of antigen-presenting cells are critical early steps in mounting an immune response. Many microbial components and endogenous mediators participate in this process. Recent studies have identified a group of structurally diverse multifunctional host proteins that are rapidly released following pathogen challenge and/or cell death and, most importantly, are able to both recruit and activate antigen-presenting cells. These potent immunostimulants, including defensins, cathelicidin, eosinophil-derived neurotoxin, and high-mobility group box protein 1, serve as early warning signals to activate innate and adaptive immune systems. We propose to highlight these proteins’ unique activities by grouping them under the novel term ‘alarmins’, in recognition of their role in mobilizing the immune system. Addresses Laboratory of Molecular Immunoregulation, Center for Cancer Research, Basic Research Program, Scientific Application and International Cooperation, Inc. (SAIC)-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA
to become mature DCs (mDCs) [2]. The maturation process is characterized by loss of phagocytic capacity, increased expression of MHC molecules and antigenpresenting capacity, expression of co-stimulatory molecules including CD40, CD80 and CD86, the production of proinflammatory cytokines, particularly IL-12, and upregulation of CCR7 and CXCR5 chemokine receptors. Consequently, mDCs develop the capacity to migrate to draining lymphoid tissues and to present antigens to T cells to initiate adaptive immune reactions. Recent studies have identified several structurally diverse endogenous mediators of innate immunity with certain features: firstly, they are rapidly released in response to infection or tissue injury; secondly, they have both chemotactic and activating effects on APCs; and thirdly, they exhibit particularly potent in vivo immunoenhancing activity. This subset of mediators alerts host defenses by augmenting innate and adaptive immune responses to tissue injury and/or infection. On the basis of their unique activities, we propose to name them ‘alarmins’.
Corresponding author: Oppenheim, Joost J ([email protected])
Current Opinion in Immunology 2005, 17:359–365 This review comes from a themed issue on Host–pathogen interactions Edited by Bali Pulendran and Robert A Seder Available online 13th June 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.06.002
Introduction ‘Danger signals’ include exogenous invasive microorganisms, endogenous tissue injury, and the intercellular inflammatory mediators generated to defend the host [1]. Since these mediators are released and/or secreted in response to danger, in reality they act as ‘warning’ signals that alert innate and adaptive immune host defense mechanisms. These warning signals interact with receptors including those that activate antigen-presenting cells (APCs) [1]. The most effective APCs, dendritic cells (DCs), are located in blood and peripheral tissues as resting, immature DCs (iDCs) and have a high capacity for antigen uptake. These iDCs are chemoattracted to sites of tissue damage and infection, take up antigens and are activated www.sciencedirect.com
In this review, we discuss the structure, expression and functional characteristics of alarmins with a particular focus on the receptors responsible for attracting and activating APCs and augmenting the adaptive immune response.
Antimicrobial peptides and proteins with alarmin activity Innate-immune mediators possessing alarmin activity include defensins, cathelicidin, eosinophil-derived neurotoxin (EDN), and high mobility group box protein 1 (HMGB1). These structurally diverse molecules have other well-established functions. Defensins consist of a family of small (3–6 kDa) antimicrobial peptides with a characteristic b-sheet-rich fold and six cysteines forming three intra-chain disulfide bonds, which, primarily on the basis of disulfide connectivity of cysteine residues, are classified into three (a, b, and u) subfamilies [3]. Cathelicidins belong to another family of antimicrobial proteins with a conserved N-terminal prosequence of approximately 100 residues known as the ‘cathelin’ domain, and a C-terminal antimicrobial domain that is highly heterogeneous in terms of size (12–80 amino acids) and structure, which can be either an a-helix, b-hairpin with one or two intra-chain disulfide bonds, or extended polyproline-type folding [4]. Both defensins and cathelicidins have antimicrobial activity against bacteria, fungi, some viruses and parasites [3,4,5,6,7,8]. Current Opinion in Immunology 2005, 17:359–365
360 Host–pathogen interactions
EDN belongs to the RNase family of proteins, which shows a typical RNase V-shaped folding organized into two lobes, each consisting of three anti-parallel b-strands and one a-helix, and with two a-helices located between the two lobes [5]. EDN has antiviral, including antiHIV-1, activity [5]. HMGB1 is one of three non-histone chromosomal structural proteins (HMGB1–3) that contain three distinct domains: two strongly positively charged DNA-binding domains termed A and B box, each of 80 amino acids, arranged in three a-helices, and a highly negatively charged C-terminal domain of 30 repetitive Asp and Glu residues [9]. Because of its bipolar charges, HMGB1 is also known as ‘amphoterin’. HMGB1 stabilizes nucleosomes and allows bending of DNA to facilitate gene transcription. HMGB1 also has antimicrobial activity. These structurally and functionally diverse molecules all have receptor-dependent chemotactic and activating effects on host leukocytes.
Alarmins are rapidly released in response to infection and tissue injury Many alarmins are stored in distinct anatomical compartments (Table 1). Human a-defensins are stored in granules of either neutrophils or small-intestinal Paneth cells. Of the four human b-defensins, HBD1 is constitutively expressed by keratinocytes and epithelial lining cells. Cathelicidin is stored in the granules of neutrophils and mast cells [10] and also expressed by keratinocytes and various epithelial cells. EDN is stored in eosinophil granules. HMGB1 is ubiquitously present in the nuclei of all vertebrate cells. In response to pathogen-associated molecular patterns (PAMPs) or proinflammatory cytokines, there is a rapid release of defensins, cathelicidin, and EDN from storage compartments by local epithelial cells or infiltrating leukocytes [3,4,5], whereas injured cells release HMGB1 [9,11,12]. The expression of many alarmins is also up-regulated by proinflammatory stimuli. HBD2–4, cathelicidin, EDN
(and the mouse ortholog, mEAR2), and HMGB1 can be rapidly induced in local leukocytes and epithelial cells (including keratinocytes) by PAMPs and/or proinflammatory cytokines (Table 1). b-Defensin-2–4 are induced by PAMPs such as LPS and cytokines such as TNF-a, IL-1, IL-17, IL-22, and IFN-g by multiple signal transduction pathways including activation of NF-kB [3,4,5,13,14, 15,16,17]. Cathelicidin is induced by skin infection or injury [6,18]. Macrophages can be induced to produce EDN rapidly by a combination of LPS and TNF-a [19]. The release of HMGB1 from monocytes/macrophages and pituicytes is induced within 3–8 h by LPS or proinflammatory cytokines [9,20,21,22,23]. Thus, alarmins are rapidly released from storage sites or induced by inflammatory and injurious stimuli.
Chemotactic receptors for alarmins All alarmins are multifunctional and have antimicrobial activities. Defensins, cathelicidins, and HMGB1 also promote inflammatory innate immune reactions by induction of proinflammatory mediators such as histamine, prostaglandins, chemokines (e.g. CXCL2/MIP-2, CXCL8/IL-8, CXCL5/ENA-78, CCL2/MCP-1), and TNF-a, suppression of IL-10, and enhancement of phagocytosis as recently reviewed [3,4,5,9]. Defensins, cathelicidins, EDN, and HMGB1 at nanomolar concentrations are chemotactic for distinct subpopulations of leukocytes (Table 2) as well as some nonleukocytes [5,24,25,26,27]. Human neutrophil adefensins are chemotactic for resting naı¨ve CD4 T cells, CD8 T cells, and iDCs [5]. HBD1–3 and mouse bdefensins 2–3 (mBD2–3) are chemotactic for memory T cells and iDCs [28,29]. HBD2 also attracts mast cells [30] and activated neutrophils [31], whereas HBD3–4 are also chemotactic for monocytes/macrophages [15,32]. Very recently, mBD29 has been demonstrated to chemoattract DC precursors and DCs [33]. Cathelicidins are chemotactic for neutrophils, monocytes/macrophages,
Table 1 Regulation of the generation of alarmins. Family
Subfamily
Name
Cell source
Transcription factor
Synthesis
Release mode
Defensin
a
HNP1–4 HD5–6, Cryptidins HBD1, mBD1 HBD2–4, mBD2–4 hLL-37, mCRAMP hLL-37, mCRAMP hEDN, mEAR2 hEDN
PMN PC EpC EpC PMN, Mono, MC, EpC Mono, MC, EpC Eo, placental EpC PMN, Mono/mac All nucleated cells Mono/mac, astrocyte
PU.1, C/EBP PU.1, C/EBP AP-1,2 NF-kB, NF-IL6, IRF C/EBP NF-kB, NF-IL6, IRF AP-1, PU.1, C/EBP, EoTF NF-AT CTF, NF-I, p73a JAK, STAT,
Constitutive Constitutive Constitutive Inducible Constitutive Inducible Constitutive Inducible Constitutive Inducible
Degranulation Degranulation Secretion Secretion Degranulation Secretion Degranulation Secretion Necrosis Secretion
b Cathelicidin EDN HMGB1
Abbreviations: EDN, eosinophil-derived neurotoxin; Eo, eosinophil; EpC, epithelial cell (including keratinocyte); HBD, human b-defensin; HD, human defensin; HMGB1, high-mobility group box 1 protein; HNP, human neutrophil-derived peptide; Mac, macrophage; mBD, mouse b-defensin; MC, mast cell; mCRAMP, mouse cathelin-related antimicrobial peptide; mEAR, mouse eosinophil-associated ribonuclease; Mono, monocyte; PC, Paneth cell; PMN, polymorphonuclear neutrophil.
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Alarmins Oppenheim and Yang 361
Table 2 Leukocyte targets and receptors of alarmins. Target leukocyte/receptor
Neutrophil Mast cell Monocyte/macrophage Dendritic cell T cell Chemotactic receptor Activating receptor
Defensin
Cathelicidin
EDN
HMGB1
+ + +
?
? + + ? GiPCR RAGE, TLR2, TLR4
a
HNP
BD1
BD2
BD3
? + + + GiPCR ?
?
+
+ + CCR6
+ + CCR6 TLR4 for mBD2
? + + + CCR6
+ + FPRL1 P2X7, EGFR
GiPCR TLR2
a
BD3 has an additional receptor (other than CCR6), as it attracts monocytes that have no functional CCR6. ? unknown or unidentified. + and indicate a given subpopulation of leukocytes can or can not migrate to a indicated alarmin, respectively. Abbreviations: BD, b-defensin; CCR, CC chemokine receptor; EDN, eosinophil-derived neurotoxin; EGFR, epidermal growth factor receptor; FPRL1, formyl peptide receptorlike 1; HMGB1, high-mobility group box 1 protein; HNP, human neutrophil-derived peptide; mBD, mouse BD; P2X7, ligand-gated ion channel-type purinergic receptor 7; RAGE, receptor for advanced glycation end-products; TLR, Toll-like receptor.
and T cells [5,34]. EDN and its closely related mouse ortholog, murine eosinophil-associated ribonuclease 2 (mEAR2) exclusively chemoattract DCs [35]. The B box domain of HMGB1 is a chemoattractant for iDCs (Q Chen et al., unpublished) and regulates the migration of monocytes [36]. All alarmins are chemotactic for APCs, including monocytes/macrophages and/or DCs (Table 2). The chemotactic effect of alarmins is suppressed by pretreatment of target cells with pertussis toxin, suggesting that they use Gi-protein-coupled receptors (GiPCRs). The b-defensins share CCR6 with the CC chemokine CCL20/MIP-3a/LARC [28,29,31,33]. In addition, HBD3 and HBD4 presumably use additional GiPCR(s) to chemoattract monocytes/macrophages because these cells do not express functional CCR6 [5]. Cathelicidins such as human LL-37 and mouse CRAMP use human FPRL1 or its mouse ortholog mFPR2 as a receptor to mediate their chemotactic effect on both leukocytes and endothelial cells [5,26,34]. The chemotactic receptors for EDN, a-defensins, and HMGB1 are also probably GiPCRs [5,27,35], but have not yet been identified.
Immunostimulating and APC-activating activities of alarmins Alarmins have direct activating effects on APCs and immunoenhancing adjuvant activities. Human neutrophil-derived a-defensins and b-defensin-1–2 promote humoral and cellular immune responses against soluble antigens [5]. Human a-defensins promote both Th1 and Th2 responses [5]. Of the mouse molecules, mBD2 is a potent inducer of DC maturation and promotes cellmediated antitumor immunity [29,37] whereas mBD3 activates DCs and promotes humoral immunity [29]. Cathelicidins, including human LL-37 and mouse CRAMP, activate MAPK pathways in monocytes/macrophages [34,38]. LL-37 promotes the in vitro differentiation of precursor monocytes into DCs, which exhibit up-regulated phagocytic capacity, elevated expression www.sciencedirect.com
of co-stimulatory molecules, and enhanced production of IL-12 [39]. When DCs differentiated in the presence of LL-37 were used to stimulated allogeneic T cells in vitro, the responder T cells generated more IFN-g than IL-4, suggesting that cathelicidin might polarize the T cell response into a Th1 type [39]. However, coadministration of CRAMP and ovalbumin into mice enhanced ovalbumin-specific serum IgG1 and IgG2a, as well as both IL-4 and IFN-g by ovalbumin-specific T cells, indicating that cathelicidin promotes humoral and cellular immune responses without polarizing into either a Th1 or Th2 type [34]. EDN is capable of inducing DC maturation, activating multiple MAPKs [35], up-regulating surface markers (CD83, CD86, and MHC class II), enhancing the production of many proinflammatory cytokines and chemokines, and enhancing the capacity to stimulate the proliferation of allogeneic T cells in vitro [19]. Co-administration of EDN and a soluble antigen into mice enhanced antigenspecific immune responses, indicating the adjuvant activity of EDN (D Yang et al., unpublished). HMGB1 through its B-box domain induces phenotypic and functional maturation of DCs as evident from an increase in the expression of CD40, CD54, CD58, CD80, CD83, CD86, and MHC class II, a decrease in CD206 expression, stimulation of cytokines such as IL-12, IL-6, IL-1a, TNF-a and chemokines (CXCL8/IL-8 and CCL5/ RANTES), and enhanced capacity to stimulate allogeneic T cells [12,40]. HMGB1 promotes serum antibody response against ovalbumin and protective cellular immune responses against tumor cells, suggesting that HMGB1 induces a polarized Th1 response in vivo [12].
Activating receptors of alarmins The receptors used by alarmins to activate APCs are distinct from GiPCRs (Table 2). The receptor responsible for DC-activating effect of mBD2 was shown to be TLR4 [37]. Although FPRL1 mediates the chemotactic effect of cathelicidins on monocytes/macrophages Current Opinion in Immunology 2005, 17:359–365
362 Host–pathogen interactions
[5,34], interaction with P2X7 is reported to enhance IL-1b processing and release from monocytes by LL-37 [41]. However, LL-37 stimulates IL-8 production by lung epithelial cells by trans-activating EGFR through metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands [42]. Airway epithelial cells were recently found to have two binding sites for LL-37: the low affinity FPRL1 and a high affinity site that awaits identification [43]. Preliminary results suggest that TLR2 accounts for the DC-activating effect of EDN. In transfected cell lines, EDN activated NF-kB in a TLR2-dependent manner. Furthermore, EDN failed to stimulate the production of cytokines (e.g. IL-6) by DCs and macrophages from MyD88 / and TLR2 / mice (D Yang et al., unpublished). The receptor for HMGB1 is reported to be RAGE (receptor for advanced glycation end products), a transmembrane protein belonging to the immunoglobulin superfamily, which is expressed primarily by cells of the central nervous system, endothelial cells, smooth muscle cells, and mononuclear phagocytes [9]. HMGB1 signaling via RAGE activates at least two intracellular signal transduction cascades: the small-GTPase (e.g. Rac and Cdc42) pathway; and the Ras-MAPK pathway. These pathways lead to cytoskeleton reorganization and NF-kB activation [9]. RAGE can therefore contribute to the activating effect of HMGB1 on APCs [12,40]. Although TLR2 and TLR4 are also proposed to mediate the macrophage-activating effect of HMGB1 on the basis
of dominant-negative inhibition experiments [44], interaction with RAGE is most effective in activating mouse primary macrophages on the basis of studies of macrophages derived from IL-1RI / , TLR2 / , and RAGE / mice [45]. Overall, the interaction between alarmins and multiple receptors on APCs promotes adaptive immune response by three major means (Figure 1). One is enhancement of APC recruitment via interaction with GiPCRs as demonstrated in experimental mouse models for some alarmins [33,34,35]. The second is promotion of antigen uptake and processing through chemotactic-receptor-mediated internalization. This is supported by data showing that an mBD2–antigen fusion product preferentially targets antigen into the MHC class II pathway through CCR6mediated internalization [46]. At sites of infection and injury, alarmins bind tightly to the membrane of microorganisms before killing. After killing, alarmins may still bind to fragments of lysed microorganisms to form noncovalently linked ‘alarmin–antigen’ complexes. Finally, interaction between alarmins and various activating receptors on APCs leading to macrophage activation and DC maturation is another crucial means by which alarmins promote adaptive immune responses.
Alarmins are distinct from other endogenous warning signals Although during infection or tissue injury, numerous endogenous soluble signals are generated and/or released,
Figure 1
Ep Leuk
Injury and infection
Alarmins (Defensin, cathelicidin HMGB1, EDN, etc.)
Blood vessel
pre-DC
Differentiation and recruitment ↑
iDC
Antigen uptake and processing ↑ Maturation ↑
Pathogen
mDC
T LN
Antigen presentation
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Schematic illustration of the contributions of alarmins to the initiation of host adaptive immune response. Upon tissue injury and infection, alarmins are generated rapidly due to release or induced production from epithelial cells (Ep), including keratinocytes and infiltrating leukocytes (Leuk). Alarmins enhance APC functions in three ways. Firstly, alarmins enhance DC differentiation from DC precursors (pre-DC) and DC recruitment from peripheral blood and/or surrounding tissue. Secondly, alarmins facilitate antigen uptake and processing by forming ‘defensin–antigen’ complexes and sorting antigen to the MHC class II pathway through alarmin-receptor-mediated internalization. Thirdly, alarmins enhance the maturation of iDCs to mDCs that subsequently migrate to draining lymph nodes (LN) to initiate immune responses. The cumulative consequence is augmentation of antigen uptake, processing and presentation, hence the augmentation of T-cell activation and adaptive immune responses. Current Opinion in Immunology 2005, 17:359–365
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Alarmins Oppenheim and Yang 363
alarmins are unique because, on the basis of their simultaneous APC-attracting and activating capacities, they act as endogenous immunoenhancing adjuvants. Proinflammatory cytokines possess APC-activating effects, but have generally been disappointing in their efficacy as endogenous adjuvants due perhaps to the lack of APCchemoattracting effect. Only GM-CSF, which exhibits limited chemotactic effect and potent activating effect on APCs, has considerable adjuvant activity. Administration of tumor cells transfected to express GM-CSF results in tumor infiltration by DC, macrophages and granulocytes, tumor rejection and subsequent tumor immunity [47]. Consequently, GM-CSF can be considered an alarmin. Chemokines are potent chemoattractants of leukocytes including APCs, and a number of them might also have little or no activating effect on APCs (see Update). These recent observations may account for the fact that certain chemokine– or defensin–antigen fusion products induce greater immnue responses to the fused antigens [29]. Nevertheless, the combination of proinflammatory cytokines capable of activating APCs and APC-attracting chemokines, both of which are often present at sites of infection and injury, can potentially mimic the effects of alarmins. Heat-shock proteins (HSPs) are also likely to be present at the site of infection and tissue injury. On the basis of their peptide-binding capacity, HSPs can facilitate antigen delivery to the endocytic Ag-processing pathway of APCs. Although HSPs also activate APCs, there is no evidence HSPs are chemotactic for APCs [48].
neous receptors on the same cell to initiate distinct and potentially synergistic signal transduction pathways. We therefore consider it a promising approach to identify more of these endogenous alarmins and to evaluate their potency as immune stimulants in an effort to identify lesstoxic and more-effective vaccine adjuvants.
Concluding remarks
Acknowledgements
Probably numerous alarmins remain to be identified. Urokinase and ribosomal protein S19 have recently been shown to be chemotactic by interacting with GiPCRs on mononuclear cells including DCs; however, it is not known whether they can activate DCs and exhibit adjuvant activity (see [19]). Another candidate is the anaphylotoxin C5a, which chemoattracts leukocytes including DCs by interacting with C5aR, a GiPCR. C5a also stimulates DCs to mature in vivo through the induction of TNF-a [49]. However, the capacity of C5a to induce DC maturation is indirect and it is also unclear if it alone acts as an adjuvant. Uric acid has been reported to be a very potent activator of DCs; however, it is not known if it is also chemotactic for APCs [50].
We are grateful to Scott Durum, Andy Hurwitz, Zack Howard and Joshua Farber for critical reading of this manuscript, and to Joshua Farber for suggesting the ‘alarmin’ term. This project has been funded in part by DHHS #NO1-CO-12400.
The concept of alarmins may aid in the identification of less-toxic adjuvants derived from endogenous sources. The plethora of proinflammatory cytokines and chemokines that appear at a site of tissue damage can theoretically have the same effects as an alarmin. However, the data showing that mixtures of antigens with chemokines or defensins have weaker adjuvant activity than antigens fused to these mediators suggest that antigen is processed more effectively by APCs if delivered directly to an appropriate receptor. Alarmins may crosslink heterogewww.sciencedirect.com
Update Several recent reports indicate that a number of chemokines, either by directly activating DCs or by inducing more prolonged interaction of APCs with T cells at the immunological synapse, have immunoactivating or costimulating effects. Molon et al. [51] have demonstrated that CCL5/RANTES derived from APCs can enhance T cell activation during APC–T cell interaction by prolonging the recruitment of CCR5 into the immunological synapse. Marsland et al. [52] have shown that CCL19/ ELC and CCL21/SLC promote T cell proliferation and activation by directly stimulating DC maturation. These three CC chemokines are also chemotactic for DCs and other leukocytes. Consequently, ligand interactions with chemokine receptors, in addition to resulting in chemotaxis, might also have concomitant activating effects. Granulysin, a cationic protein that is produced by activated human NK and cytotoxic T cells and that exhibits a cytolytic effect against microorganisms and tumor cells, has been reported to have both chemotactic and leukocyte-activating effects [53]. These molecules (CCL5/ RANTES, CCL19/ELC, CCL21/SLC, and granulysin) can therefore also be considered alarmins.
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27. Degryse B, Bonaldi T, Scaffidi P, Muller S, Resnati M, Sanvito F, Arrigoni G, Bianchi ME: The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. J Cell Biol 2001, 152:1197-1206. 28. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OMZ et al.: b-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999, 286:525-528. 29. Biragyn A, Surenhu M, Yang D, Ruffini PA, Haines BA, Klyushnenkova E, Oppenheim JJ, Kwak LW: Mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens. J Immunol 2001, 167:6644-6653. 30. Niyonsaba F, Iwabuchi K, Matsuda H, Ogawa H, Nagaoka I: Epithelial cell-derived human b-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway. Int Immunol 2002, 14:421-426. 31. Niyonsaba F, Ogawa H, Nagaoka I: Human b-defensin-2 functions as a chemotactic agent for tumour necrosis factora-treated human neutrophils. Immunology 2004, 111:273-281. 32. Garcia JR, Jaumann F, Schulz S, Krause A, Rodriguez-Jimenez J, Forssmann U, Adermann K, Kluver E, Vogelmeier C, Becker D et al.: Identification of a novel, multifunctional b-defensin (human b-defensin 3) with specific antimicrobial activity: its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res 2001, 306:257-264. 33. Conejo-Garcia JR, Benencia F, Courreges MC, Kang E, Mohamed-Hadley A, Buckanovich RJ, Holtz DO, Jenkins A, Na H, Zhang L et al.: Tumor-infiltrating dendritic cell precursors www.sciencedirect.com
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recruited by a b-defensin contribute to vasculogenesis under the influence of Vegf-A. Nat Med 2004, 10:950-958. This is the first paper demonstrating that b-defensin-29 uses CCR6 to recruit dendritic cell (DC) precursors and DCs, which, under the influence of vascular endothelial growth factor-A in a tumor microenvironment, come to resemble endothelial cells and promote tumor-associated angiogenesis. 34. Kurosaka K, Chen Q, Yarovinsky F, Oppenheim JJ, Yang D: Mouse cathelin-related antimicrobial peptide (CRAMP) chemoattracts leukocytes using FPRL1/mFPR2 as the receptor and acts as an immune adjuvant. J Immunol 2005, 174:6257-6265. Mouse cathelicidin/CRAMP chemoattracted phagocytes using mFPR2 and promoted ovalbumin-specific immune responses, demonstrating for the first time that cathelicidin has immunoenhancing effect in vivo. 35. Yang D, Rosenberg HF, Chen Q, Dyer KD, Kurosaka K, Oppenheim JJ: Eosinophil-derived neurotoxin (EDN), an antimicrobial protein with chemotactic activities for dendritic cells. Blood 2003, 102:3396-3403. This is the initial report, documenting the identification of the chemoattracting activity of eosinophil-derived neurotoxin (EDN) for dendritic cells. 36. Rouhiainen A, Kuja-Panula J, Wilkman E, Pakkanen J, Stenfors J, Tuominen RK, Lepantalo M, Carpen O, Parkkinen J, Rauvala H: Regulation of monocyte migration by amphoterin (HMGB1). Blood 2004, 104:1174-1182. This is the first paper to describe the participation of high mobility group box protein 1 (HMGB1) and receptor for advanced glycation end products (RAGE) in the transendothelial migration of monocytes. 37. Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ et al.: Toll-like receptor 4-dependent activation of dendritic cells by b-defensin 2. Science 2002, 298:1025-1029. 38. Bowdish DM, Davidson DJ, Speert DP, Hancock RE: The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004, 172:3758-3765. 39. Davidson DJ, Currie AJ, Reid GS, Bowdish DM, MacDonald KL, Ma RC, Hancock RE, Speert DP: The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization. J Immunol 2004, 172:1146-1156. The first report demonstrating that human cathelicidin/LL-37 promotes the differentiation of monocytes into dendritic cells with increased endocytic capacity, co-stimulatory molecule expression, and secretion of Th1inducing cytokines in vitro. 40. Messmer D, Yang H, Telusma G, Knoll F, Li J, Messmer B, Tracey KJ, Chiorazzi N: High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization. J Immunol 2004, 173:307-313. See annotation to [12]. 41. Elssner A, Duncan M, Gavrilin M, Wewers MD: A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1b processing and release. J Immunol 2004, 172:4987-4994. 42. Tjabringa GS, Aarbiou J, Ninaber DK, Drijfhout JW, Sorensen OE, Borregaard N, Rabe KF, Hiemstra PS: The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 2003, 171:6690-6696. 43. Lau YE, Rozek A, Scott MG, Goosney DL, Davidson DJ, Hancock RE: Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells. Infect Immun 2005, 73:583-591.
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The authors showed that human lung epithelial cells possess both highand low-affinity binding sites for human cathelicidin/LL-37, providing the first indication that cathelicidin might interact with more than one receptor on epithelial cells. 44. Park JS, Svetkauskaite D, He Q, Kim JY, Strassheim D, Ishizaka A, Abraham E: Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J Biol Chem 2004, 279:7370-7377. 45. Kokkola R, Andersson A, Mullins G, Ostberg T, Treutiger CJ, Arnold B, Nawroth P, Andersson U, Harris RA, Harris HE: RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scand J Immunol 2005, 61:1-9. Using macrophages from receptor for advanced glycation end products (RAGE) / and Toll-like receptor 2 (TLR2) / mice, the authors demonstrated, for the first time, that high mobility group box protein 1 (HMGB1) activation of mouse macrophages is predominantly mediated by RAGE. 46. Biragyn A, Ruffini PA, Coscia M, Harvey LK, Neelapu SS, Baskar S, Wang JM, Kwak LW: Chemokine receptor-mediated delivery directs self-tumor antigen efficiently into the class II processing pathway in vitro and induces protective immunity in vivo. Blood 2004, 104:1961-1969. This paper is the first report to show that mouse b-defensin 2 (mBD2) – target-antigen and mCCL20/MIP3a–target-antigen fusion products promoted the uptake, processing, and presentation of the target antigen to CD4 T cells via a CCR6-mediated sorting of target antigen into the MHC class II pathway, resulting in the augmentation of antigen-specific immune response. 47. Dranoff G: GM-CSF-based cancer vaccines. Immunol Rev 2002, 188:147-154. 48. Manjili MH, Wang XY, MacDonald IJ, Arnouk H, Yang GY, Pritchard MT, Subjeck JR: Cancer immunotherapy and heatshock proteins: promises and challenges. Expert Opin Biol Ther 2004, 4:363-373. 49. Soruri A, Riggert J, Schlott T, Kiafard Z, Dettmer C, Zwirner J: Anaphylatoxin C5a induces monocyte recruitment and differentiation into dendritic cells by TNFa and prostaglandin E2-dependent mechanisms. J Immunol 2003, 171:2631-2636. 50. Shi Y, Evans JE, Rock KL: Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 2003, 425:516-521. 51. Molon B, Gri G, Bettella M, Gomez-Mouton C, Lanzavecchia A, Martinez AC, Manes S, Viola A: T cell costimulation by chemokine receptors. Nat Immunol 2005, 6:465-471. In this paper, the authors reported the recruitment of T cell CCR5 to the immunological synapse during APC–T cell interaction, presumably by APC-derived CCL5/RANTES, which prolongs/stabilizes APC–T cell interaction and enhances subsequent T cell activation. 52. Marsland BJ, Battig P, Bauer M, Ruedl C, Lassing U, Beerli RR, Dietmeier K, Ivanova L, Pfister T, Vogt L et al.: CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells. Immunity 2005, 22:493-505. The authors identified CCL19/ELC as an inducer of T cell proliferation in a DC–T co-culture system using a novel expression cloning technique, and subsequently established that both CCL19/ELC and CCL21/SLC, two chemokines using the same receptor CCR7, act as inducers of DC maturation for the promotion of T cell proliferation and activation. 53. Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM: Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol 2005, 174:5243-5248. This is the first report demonstrating the chemotactic and activating effects of granulysin on diverse subpopulations of human leukocytes.
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to become mature DCs (mDCs) [2]. The maturation process is characterized by loss of phagocytic capacity, increased expression of MHC molecules and antigenpresenting capacity, expression of co-stimulatory molecules including CD40, CD80 and CD86, the production of proinflammatory cytokines, particularly IL-12, and upregulation of CCR7 and CXCR5 chemokine receptors. Consequently, mDCs develop the capacity to migrate to draining lymphoid tissues and to present antigens to T cells to initiate adaptive immune reactions. Recent studies have identified several structurally diverse endogenous mediators of innate immunity with certain features: firstly, they are rapidly released in response to infection or tissue injury; secondly, they have both chemotactic and activating effects on APCs; and thirdly, they exhibit particularly potent in vivo immunoenhancing activity. This subset of mediators alerts host defenses by augmenting innate and adaptive immune responses to tissue injury and/or infection. On the basis of their unique activities, we propose to name them ‘alarmins’.
Corresponding author: Oppenheim, Joost J ([email protected])
Current Opinion in Immunology 2005, 17:359–365 This review comes from a themed issue on Host–pathogen interactions Edited by Bali Pulendran and Robert A Seder Available online 13th June 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.06.002
Introduction ‘Danger signals’ include exogenous invasive microorganisms, endogenous tissue injury, and the intercellular inflammatory mediators generated to defend the host [1]. Since these mediators are released and/or secreted in response to danger, in reality they act as ‘warning’ signals that alert innate and adaptive immune host defense mechanisms. These warning signals interact with receptors including those that activate antigen-presenting cells (APCs) [1]. The most effective APCs, dendritic cells (DCs), are located in blood and peripheral tissues as resting, immature DCs (iDCs) and have a high capacity for antigen uptake. These iDCs are chemoattracted to sites of tissue damage and infection, take up antigens and are activated www.sciencedirect.com
In this review, we discuss the structure, expression and functional characteristics of alarmins with a particular focus on the receptors responsible for attracting and activating APCs and augmenting the adaptive immune response.
Antimicrobial peptides and proteins with alarmin activity Innate-immune mediators possessing alarmin activity include defensins, cathelicidin, eosinophil-derived neurotoxin (EDN), and high mobility group box protein 1 (HMGB1). These structurally diverse molecules have other well-established functions. Defensins consist of a family of small (3–6 kDa) antimicrobial peptides with a characteristic b-sheet-rich fold and six cysteines forming three intra-chain disulfide bonds, which, primarily on the basis of disulfide connectivity of cysteine residues, are classified into three (a, b, and u) subfamilies [3]. Cathelicidins belong to another family of antimicrobial proteins with a conserved N-terminal prosequence of approximately 100 residues known as the ‘cathelin’ domain, and a C-terminal antimicrobial domain that is highly heterogeneous in terms of size (12–80 amino acids) and structure, which can be either an a-helix, b-hairpin with one or two intra-chain disulfide bonds, or extended polyproline-type folding [4]. Both defensins and cathelicidins have antimicrobial activity against bacteria, fungi, some viruses and parasites [3,4,5,6,7,8]. Current Opinion in Immunology 2005, 17:359–365
360 Host–pathogen interactions
EDN belongs to the RNase family of proteins, which shows a typical RNase V-shaped folding organized into two lobes, each consisting of three anti-parallel b-strands and one a-helix, and with two a-helices located between the two lobes [5]. EDN has antiviral, including antiHIV-1, activity [5]. HMGB1 is one of three non-histone chromosomal structural proteins (HMGB1–3) that contain three distinct domains: two strongly positively charged DNA-binding domains termed A and B box, each of 80 amino acids, arranged in three a-helices, and a highly negatively charged C-terminal domain of 30 repetitive Asp and Glu residues [9]. Because of its bipolar charges, HMGB1 is also known as ‘amphoterin’. HMGB1 stabilizes nucleosomes and allows bending of DNA to facilitate gene transcription. HMGB1 also has antimicrobial activity. These structurally and functionally diverse molecules all have receptor-dependent chemotactic and activating effects on host leukocytes.
Alarmins are rapidly released in response to infection and tissue injury Many alarmins are stored in distinct anatomical compartments (Table 1). Human a-defensins are stored in granules of either neutrophils or small-intestinal Paneth cells. Of the four human b-defensins, HBD1 is constitutively expressed by keratinocytes and epithelial lining cells. Cathelicidin is stored in the granules of neutrophils and mast cells [10] and also expressed by keratinocytes and various epithelial cells. EDN is stored in eosinophil granules. HMGB1 is ubiquitously present in the nuclei of all vertebrate cells. In response to pathogen-associated molecular patterns (PAMPs) or proinflammatory cytokines, there is a rapid release of defensins, cathelicidin, and EDN from storage compartments by local epithelial cells or infiltrating leukocytes [3,4,5], whereas injured cells release HMGB1 [9,11,12]. The expression of many alarmins is also up-regulated by proinflammatory stimuli. HBD2–4, cathelicidin, EDN
(and the mouse ortholog, mEAR2), and HMGB1 can be rapidly induced in local leukocytes and epithelial cells (including keratinocytes) by PAMPs and/or proinflammatory cytokines (Table 1). b-Defensin-2–4 are induced by PAMPs such as LPS and cytokines such as TNF-a, IL-1, IL-17, IL-22, and IFN-g by multiple signal transduction pathways including activation of NF-kB [3,4,5,13,14, 15,16,17]. Cathelicidin is induced by skin infection or injury [6,18]. Macrophages can be induced to produce EDN rapidly by a combination of LPS and TNF-a [19]. The release of HMGB1 from monocytes/macrophages and pituicytes is induced within 3–8 h by LPS or proinflammatory cytokines [9,20,21,22,23]. Thus, alarmins are rapidly released from storage sites or induced by inflammatory and injurious stimuli.
Chemotactic receptors for alarmins All alarmins are multifunctional and have antimicrobial activities. Defensins, cathelicidins, and HMGB1 also promote inflammatory innate immune reactions by induction of proinflammatory mediators such as histamine, prostaglandins, chemokines (e.g. CXCL2/MIP-2, CXCL8/IL-8, CXCL5/ENA-78, CCL2/MCP-1), and TNF-a, suppression of IL-10, and enhancement of phagocytosis as recently reviewed [3,4,5,9]. Defensins, cathelicidins, EDN, and HMGB1 at nanomolar concentrations are chemotactic for distinct subpopulations of leukocytes (Table 2) as well as some nonleukocytes [5,24,25,26,27]. Human neutrophil adefensins are chemotactic for resting naı¨ve CD4 T cells, CD8 T cells, and iDCs [5]. HBD1–3 and mouse bdefensins 2–3 (mBD2–3) are chemotactic for memory T cells and iDCs [28,29]. HBD2 also attracts mast cells [30] and activated neutrophils [31], whereas HBD3–4 are also chemotactic for monocytes/macrophages [15,32]. Very recently, mBD29 has been demonstrated to chemoattract DC precursors and DCs [33]. Cathelicidins are chemotactic for neutrophils, monocytes/macrophages,
Table 1 Regulation of the generation of alarmins. Family
Subfamily
Name
Cell source
Transcription factor
Synthesis
Release mode
Defensin
a
HNP1–4 HD5–6, Cryptidins HBD1, mBD1 HBD2–4, mBD2–4 hLL-37, mCRAMP hLL-37, mCRAMP hEDN, mEAR2 hEDN
PMN PC EpC EpC PMN, Mono, MC, EpC Mono, MC, EpC Eo, placental EpC PMN, Mono/mac All nucleated cells Mono/mac, astrocyte
PU.1, C/EBP PU.1, C/EBP AP-1,2 NF-kB, NF-IL6, IRF C/EBP NF-kB, NF-IL6, IRF AP-1, PU.1, C/EBP, EoTF NF-AT CTF, NF-I, p73a JAK, STAT,
Constitutive Constitutive Constitutive Inducible Constitutive Inducible Constitutive Inducible Constitutive Inducible
Degranulation Degranulation Secretion Secretion Degranulation Secretion Degranulation Secretion Necrosis Secretion
b Cathelicidin EDN HMGB1
Abbreviations: EDN, eosinophil-derived neurotoxin; Eo, eosinophil; EpC, epithelial cell (including keratinocyte); HBD, human b-defensin; HD, human defensin; HMGB1, high-mobility group box 1 protein; HNP, human neutrophil-derived peptide; Mac, macrophage; mBD, mouse b-defensin; MC, mast cell; mCRAMP, mouse cathelin-related antimicrobial peptide; mEAR, mouse eosinophil-associated ribonuclease; Mono, monocyte; PC, Paneth cell; PMN, polymorphonuclear neutrophil.
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Table 2 Leukocyte targets and receptors of alarmins. Target leukocyte/receptor
Neutrophil Mast cell Monocyte/macrophage Dendritic cell T cell Chemotactic receptor Activating receptor
Defensin
Cathelicidin
EDN
HMGB1
+ + +
?
? + + ? GiPCR RAGE, TLR2, TLR4
a
HNP
BD1
BD2
BD3
? + + + GiPCR ?
?
+
+ + CCR6
+ + CCR6 TLR4 for mBD2
? + + + CCR6
+ + FPRL1 P2X7, EGFR
GiPCR TLR2
a
BD3 has an additional receptor (other than CCR6), as it attracts monocytes that have no functional CCR6. ? unknown or unidentified. + and indicate a given subpopulation of leukocytes can or can not migrate to a indicated alarmin, respectively. Abbreviations: BD, b-defensin; CCR, CC chemokine receptor; EDN, eosinophil-derived neurotoxin; EGFR, epidermal growth factor receptor; FPRL1, formyl peptide receptorlike 1; HMGB1, high-mobility group box 1 protein; HNP, human neutrophil-derived peptide; mBD, mouse BD; P2X7, ligand-gated ion channel-type purinergic receptor 7; RAGE, receptor for advanced glycation end-products; TLR, Toll-like receptor.
and T cells [5,34]. EDN and its closely related mouse ortholog, murine eosinophil-associated ribonuclease 2 (mEAR2) exclusively chemoattract DCs [35]. The B box domain of HMGB1 is a chemoattractant for iDCs (Q Chen et al., unpublished) and regulates the migration of monocytes [36]. All alarmins are chemotactic for APCs, including monocytes/macrophages and/or DCs (Table 2). The chemotactic effect of alarmins is suppressed by pretreatment of target cells with pertussis toxin, suggesting that they use Gi-protein-coupled receptors (GiPCRs). The b-defensins share CCR6 with the CC chemokine CCL20/MIP-3a/LARC [28,29,31,33]. In addition, HBD3 and HBD4 presumably use additional GiPCR(s) to chemoattract monocytes/macrophages because these cells do not express functional CCR6 [5]. Cathelicidins such as human LL-37 and mouse CRAMP use human FPRL1 or its mouse ortholog mFPR2 as a receptor to mediate their chemotactic effect on both leukocytes and endothelial cells [5,26,34]. The chemotactic receptors for EDN, a-defensins, and HMGB1 are also probably GiPCRs [5,27,35], but have not yet been identified.
Immunostimulating and APC-activating activities of alarmins Alarmins have direct activating effects on APCs and immunoenhancing adjuvant activities. Human neutrophil-derived a-defensins and b-defensin-1–2 promote humoral and cellular immune responses against soluble antigens [5]. Human a-defensins promote both Th1 and Th2 responses [5]. Of the mouse molecules, mBD2 is a potent inducer of DC maturation and promotes cellmediated antitumor immunity [29,37] whereas mBD3 activates DCs and promotes humoral immunity [29]. Cathelicidins, including human LL-37 and mouse CRAMP, activate MAPK pathways in monocytes/macrophages [34,38]. LL-37 promotes the in vitro differentiation of precursor monocytes into DCs, which exhibit up-regulated phagocytic capacity, elevated expression www.sciencedirect.com
of co-stimulatory molecules, and enhanced production of IL-12 [39]. When DCs differentiated in the presence of LL-37 were used to stimulated allogeneic T cells in vitro, the responder T cells generated more IFN-g than IL-4, suggesting that cathelicidin might polarize the T cell response into a Th1 type [39]. However, coadministration of CRAMP and ovalbumin into mice enhanced ovalbumin-specific serum IgG1 and IgG2a, as well as both IL-4 and IFN-g by ovalbumin-specific T cells, indicating that cathelicidin promotes humoral and cellular immune responses without polarizing into either a Th1 or Th2 type [34]. EDN is capable of inducing DC maturation, activating multiple MAPKs [35], up-regulating surface markers (CD83, CD86, and MHC class II), enhancing the production of many proinflammatory cytokines and chemokines, and enhancing the capacity to stimulate the proliferation of allogeneic T cells in vitro [19]. Co-administration of EDN and a soluble antigen into mice enhanced antigenspecific immune responses, indicating the adjuvant activity of EDN (D Yang et al., unpublished). HMGB1 through its B-box domain induces phenotypic and functional maturation of DCs as evident from an increase in the expression of CD40, CD54, CD58, CD80, CD83, CD86, and MHC class II, a decrease in CD206 expression, stimulation of cytokines such as IL-12, IL-6, IL-1a, TNF-a and chemokines (CXCL8/IL-8 and CCL5/ RANTES), and enhanced capacity to stimulate allogeneic T cells [12,40]. HMGB1 promotes serum antibody response against ovalbumin and protective cellular immune responses against tumor cells, suggesting that HMGB1 induces a polarized Th1 response in vivo [12].
Activating receptors of alarmins The receptors used by alarmins to activate APCs are distinct from GiPCRs (Table 2). The receptor responsible for DC-activating effect of mBD2 was shown to be TLR4 [37]. Although FPRL1 mediates the chemotactic effect of cathelicidins on monocytes/macrophages Current Opinion in Immunology 2005, 17:359–365
362 Host–pathogen interactions
[5,34], interaction with P2X7 is reported to enhance IL-1b processing and release from monocytes by LL-37 [41]. However, LL-37 stimulates IL-8 production by lung epithelial cells by trans-activating EGFR through metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands [42]. Airway epithelial cells were recently found to have two binding sites for LL-37: the low affinity FPRL1 and a high affinity site that awaits identification [43]. Preliminary results suggest that TLR2 accounts for the DC-activating effect of EDN. In transfected cell lines, EDN activated NF-kB in a TLR2-dependent manner. Furthermore, EDN failed to stimulate the production of cytokines (e.g. IL-6) by DCs and macrophages from MyD88 / and TLR2 / mice (D Yang et al., unpublished). The receptor for HMGB1 is reported to be RAGE (receptor for advanced glycation end products), a transmembrane protein belonging to the immunoglobulin superfamily, which is expressed primarily by cells of the central nervous system, endothelial cells, smooth muscle cells, and mononuclear phagocytes [9]. HMGB1 signaling via RAGE activates at least two intracellular signal transduction cascades: the small-GTPase (e.g. Rac and Cdc42) pathway; and the Ras-MAPK pathway. These pathways lead to cytoskeleton reorganization and NF-kB activation [9]. RAGE can therefore contribute to the activating effect of HMGB1 on APCs [12,40]. Although TLR2 and TLR4 are also proposed to mediate the macrophage-activating effect of HMGB1 on the basis
of dominant-negative inhibition experiments [44], interaction with RAGE is most effective in activating mouse primary macrophages on the basis of studies of macrophages derived from IL-1RI / , TLR2 / , and RAGE / mice [45]. Overall, the interaction between alarmins and multiple receptors on APCs promotes adaptive immune response by three major means (Figure 1). One is enhancement of APC recruitment via interaction with GiPCRs as demonstrated in experimental mouse models for some alarmins [33,34,35]. The second is promotion of antigen uptake and processing through chemotactic-receptor-mediated internalization. This is supported by data showing that an mBD2–antigen fusion product preferentially targets antigen into the MHC class II pathway through CCR6mediated internalization [46]. At sites of infection and injury, alarmins bind tightly to the membrane of microorganisms before killing. After killing, alarmins may still bind to fragments of lysed microorganisms to form noncovalently linked ‘alarmin–antigen’ complexes. Finally, interaction between alarmins and various activating receptors on APCs leading to macrophage activation and DC maturation is another crucial means by which alarmins promote adaptive immune responses.
Alarmins are distinct from other endogenous warning signals Although during infection or tissue injury, numerous endogenous soluble signals are generated and/or released,
Figure 1
Ep Leuk
Injury and infection
Alarmins (Defensin, cathelicidin HMGB1, EDN, etc.)
Blood vessel
pre-DC
Differentiation and recruitment ↑
iDC
Antigen uptake and processing ↑ Maturation ↑
Pathogen
mDC
T LN
Antigen presentation
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Schematic illustration of the contributions of alarmins to the initiation of host adaptive immune response. Upon tissue injury and infection, alarmins are generated rapidly due to release or induced production from epithelial cells (Ep), including keratinocytes and infiltrating leukocytes (Leuk). Alarmins enhance APC functions in three ways. Firstly, alarmins enhance DC differentiation from DC precursors (pre-DC) and DC recruitment from peripheral blood and/or surrounding tissue. Secondly, alarmins facilitate antigen uptake and processing by forming ‘defensin–antigen’ complexes and sorting antigen to the MHC class II pathway through alarmin-receptor-mediated internalization. Thirdly, alarmins enhance the maturation of iDCs to mDCs that subsequently migrate to draining lymph nodes (LN) to initiate immune responses. The cumulative consequence is augmentation of antigen uptake, processing and presentation, hence the augmentation of T-cell activation and adaptive immune responses. Current Opinion in Immunology 2005, 17:359–365
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Alarmins Oppenheim and Yang 363
alarmins are unique because, on the basis of their simultaneous APC-attracting and activating capacities, they act as endogenous immunoenhancing adjuvants. Proinflammatory cytokines possess APC-activating effects, but have generally been disappointing in their efficacy as endogenous adjuvants due perhaps to the lack of APCchemoattracting effect. Only GM-CSF, which exhibits limited chemotactic effect and potent activating effect on APCs, has considerable adjuvant activity. Administration of tumor cells transfected to express GM-CSF results in tumor infiltration by DC, macrophages and granulocytes, tumor rejection and subsequent tumor immunity [47]. Consequently, GM-CSF can be considered an alarmin. Chemokines are potent chemoattractants of leukocytes including APCs, and a number of them might also have little or no activating effect on APCs (see Update). These recent observations may account for the fact that certain chemokine– or defensin–antigen fusion products induce greater immnue responses to the fused antigens [29]. Nevertheless, the combination of proinflammatory cytokines capable of activating APCs and APC-attracting chemokines, both of which are often present at sites of infection and injury, can potentially mimic the effects of alarmins. Heat-shock proteins (HSPs) are also likely to be present at the site of infection and tissue injury. On the basis of their peptide-binding capacity, HSPs can facilitate antigen delivery to the endocytic Ag-processing pathway of APCs. Although HSPs also activate APCs, there is no evidence HSPs are chemotactic for APCs [48].
neous receptors on the same cell to initiate distinct and potentially synergistic signal transduction pathways. We therefore consider it a promising approach to identify more of these endogenous alarmins and to evaluate their potency as immune stimulants in an effort to identify lesstoxic and more-effective vaccine adjuvants.
Concluding remarks
Acknowledgements
Probably numerous alarmins remain to be identified. Urokinase and ribosomal protein S19 have recently been shown to be chemotactic by interacting with GiPCRs on mononuclear cells including DCs; however, it is not known whether they can activate DCs and exhibit adjuvant activity (see [19]). Another candidate is the anaphylotoxin C5a, which chemoattracts leukocytes including DCs by interacting with C5aR, a GiPCR. C5a also stimulates DCs to mature in vivo through the induction of TNF-a [49]. However, the capacity of C5a to induce DC maturation is indirect and it is also unclear if it alone acts as an adjuvant. Uric acid has been reported to be a very potent activator of DCs; however, it is not known if it is also chemotactic for APCs [50].
We are grateful to Scott Durum, Andy Hurwitz, Zack Howard and Joshua Farber for critical reading of this manuscript, and to Joshua Farber for suggesting the ‘alarmin’ term. This project has been funded in part by DHHS #NO1-CO-12400.
The concept of alarmins may aid in the identification of less-toxic adjuvants derived from endogenous sources. The plethora of proinflammatory cytokines and chemokines that appear at a site of tissue damage can theoretically have the same effects as an alarmin. However, the data showing that mixtures of antigens with chemokines or defensins have weaker adjuvant activity than antigens fused to these mediators suggest that antigen is processed more effectively by APCs if delivered directly to an appropriate receptor. Alarmins may crosslink heterogewww.sciencedirect.com
Update Several recent reports indicate that a number of chemokines, either by directly activating DCs or by inducing more prolonged interaction of APCs with T cells at the immunological synapse, have immunoactivating or costimulating effects. Molon et al. [51] have demonstrated that CCL5/RANTES derived from APCs can enhance T cell activation during APC–T cell interaction by prolonging the recruitment of CCR5 into the immunological synapse. Marsland et al. [52] have shown that CCL19/ ELC and CCL21/SLC promote T cell proliferation and activation by directly stimulating DC maturation. These three CC chemokines are also chemotactic for DCs and other leukocytes. Consequently, ligand interactions with chemokine receptors, in addition to resulting in chemotaxis, might also have concomitant activating effects. Granulysin, a cationic protein that is produced by activated human NK and cytotoxic T cells and that exhibits a cytolytic effect against microorganisms and tumor cells, has been reported to have both chemotactic and leukocyte-activating effects [53]. These molecules (CCL5/ RANTES, CCL19/ELC, CCL21/SLC, and granulysin) can therefore also be considered alarmins.
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