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Originally published online as doi:10.1189/jlb.0808495 on October 27, 2008

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(Journal of Leukocyte Biology. 2009;85:344-351.)
© 2009 by Society for Leukocyte Biology

Neutrophil-derived azurocidin alarms the immune system

Oliver Soehnlein*,{dagger},1 and Lennart Lindbom*

* Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden; and
{dagger} Institute of Molecular Cardiovascular Research (IMCAR), University Hospital, RWTH Aachen University, Aachen, Germany

1 Correspondence: IMCAR, Universitätsklinik Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. E-mail: oliver.sohnlein{at}ki.se or osoehnlein{at}ukaachen.de


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ABSTRACT
 
Azurocidin (heparin-binding protein/cationic antimicrobial protein of 37 kD) is a protein that is mobilized rapidly from emigrating polymorphonuclear leukocytes (PMN). Initially, this inactive serine protease was recognized for its antimicrobial effects. However, it soon became apparent that azurocidin may act to alarm the immune system in different ways and thus serve as an important mediator during the initiation of the immune response. Azurocidin, released from PMN secretory vesicles or primary granules, acts as a chemoattractant and activator of monocyte and macrophages. The functional consequence is enhancement of cytokine release and bacterial phagocytosis, allowing for a more efficient bacterial clearance. Leukocyte activation by azurocidin is mediated via β2-integrins, and azurocidin-induced chemotaxis is dependent on formyl-peptide receptors. In addition, azurocidin activates endothelial cells leading to vascular leakage and edema formation. For these reasons, targeting azurocidin release and its actions may have therapeutic potential in inflammatory disease conditions.

Key Words: granule proteins • inflammation • alarmin • heparin-binding protein • CAP37


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INITIATION OF THE IMMUNE RESPONSE
 
The function of the immune system is to prevent the takeover of the body by genomes other than that encoded in the germline [1 ]. Central to this function is the ability to kill pathogens, which is one of the core competences of the polymorphonuclear leukocyte (PMN), one of the body’s main cellular components for the destruction of microorganisms. Over the last years, the view of the PMN has changed considerably. It was realized that the PMN not only exerts functiones privatae such as bacterial killing by phagocytosis, release of reactive oxygen species, or antimicrobial peptides but also that the PMN carries out plenty of functiones publicae, much of which is mediated by the PMN granule proteins. These are set free at distinct steps during the journey of the PMN from the bloodstream to the site of injury. The functional potential of the PMN granule contents in the inflammatory process was already appreciated by the German Nobel Laureate Paul Ehrlich: "... it is likely that the leukocyte granulations are in fact secretory products, which the cell dissolves and spreads to the environment as needed" [2 ]. Discharge of PMN granule proteins is, however, not only of benefit, as emigration of the PMN and release of proteolytic granule proteins are also associated with cell and tissue damage to the host [3 ]. On the other hand, signals from tissue injury and PMN activation may contribute to ignite the immune response.

To explain the ignition of the immune response, several models have been proposed. Initially Burnet’s [4 ] self-nonself model postulated that lymphocytes are activated by recognition of foreign particles. Over the years, this theory underwent several modifications. Nowadays, the danger theory holds that danger-associated molecular patterns (DAMPs) activate APC, thereby initiating an immune response [5 ]. DAMPs may originate from pathogens, which are then termed pathogen-associated molecular patterns (PAMPs), detected via pattern-recognition receptors [6 ], or they are derived from distressed or injured host cells, which are then termed alarmins [7 ].


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COMMON CHARACTERISTICS OF ALARMINS
 
Alarmins are the equivalent of PAMPs but are endogenous molecules comprising a group of diverse compounds that today include high-mobility group box protein 1, eosinophil-derived neurotoxin, as well as PMN-derived defensins and LL-37 [7 ]. In general, alarmins are released rapidly in response to infection or tissue injury. In addition, they may also be de novo-synthesized. LL-37, for example, is stored in secondary granules of PMN and therefore, discharged rapidly upon extravasation of PMN [8 ]. In addition, LL-37 is induced in monocytes or macrophages by proinflammatory stimuli [9 ] and secreted into the surrounding. Alarmins exert chemotactic and activating effects on APC. For example, human neutrophil peptides (HNPs; {alpha}-defensins) are chemotactic for resting, naïve CD4 T cells, CD8 T cells, and immature dendritic cells (DCs), whereas cathelicidins [LL-37 in the human; mouse cathelin-related antimicrobial peptide (mCRAMP) in the mouse] are chemotactic for PMN, monocytes/macrophages, and T cells [10 , 11 ]. Cathelicidins also activate MAPK pathways in monocytes/macrophages, promoting the in vitro differentiation of precursor monocytes into DCs [12 , 13 ]. Finally, alarmins exhibit potent in vivo-immunoenhancing activity. Coadministration of mCRAMP and OVA into mice enhanced OVA-specific serum IgG1 and IgG2a, as well as IL-4 and IFN-{gamma} by OVA-specific T cells [11 ]. Recent studies about azurocidin indicate that this PMN-derived granule protein may, like LL-37 and HNPs, also satisfy the criteria of an alarmin. This review focuses on the physiological functions of azurocidin with particular emphasis on its potential role as an alarmin.


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AZUROCIDIN IS UNIQUE AMONG THE PMN GRANULE PROTEINS
 
The PMN granule protein azurocidin/cationic antimicrobial protein of 37 kD (CAP37)/heparin-binding protein (HBP) was first identified and isolated by Shafer et al. in 1984 [14 ]. Because of its potent antimicrobial activity, its cationicity, and hydrophobicity, it was considered a component of the oxygen-independent host defense. Its charge and its proposed size gave it the name CAP37. Somewhat later, Gabay et al. [15 ] characterized a PMN-derived bactericidal protein from the azurophilic granules of human PMN, which they named azurocidin. In parallel, Flodgaard et al. [16 ] isolated a protein from human and porcine PMN that displayed strong binding capability for heparin, earning it the name HBP. Complete sequencing has shown that CAP37, azurocidin, and HBP are the same protein. In this review, we will refer to the protein as azurocidin only.

Azurocidin was viewed a member of the family of PMN-derived antimicrobial proteins, such as defensins, lysozyme, and LL-37. However, soon it became evident that azurocidin, like other antimicrobial proteins, not only exerts antimicrobial activity but also modulates immune function in a multifaceted manner. Azurocidin, however, possesses some features that make it unique among the PMN granule proteins: Azurocidin is the only PMN granule protein stored in two different compartments. As a result of its storage in secretory vesicles and primary granules, azurocidin is released at a very early stage of PMN extravasation as well as at a later stage when the PMN has reached the site of inflammation [17 ], thereby allowing it to target cells in the bloodstream, the endothelial lining, and the extravascular environment. The amino acid sequence and the three-dimensional structure of azurocidin have been unveiled and show that azurocidin is a member of the serine protease superfamily. However, as a result of mutations in two of the three essential amino acids in the highly conserved catalytic triad seen in all serine proteases, azurocidin is devoid of protease activity [16 , 18 19 20 ]. Azurocidin is released almost completely after granule mobilization. In contrast to, e.g., HNPs, which are released mainly into the phagolysosome [21 ], 90% of the azurocidin are released upon degranulation [17 , 19 ]. These three distinct properties of azurocidin favor the promiscuous mode of action that this protein displays.


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AZUROCIDIN INDUCES RECRUITMENT OF MONOCYTES
 
Once the PMN senses a signal to extravasate at sites of injury or infection, it becomes activated and adheres to the endothelial lining [22 ]. Upon these initial events of PMN extravasation, the content of rapidly mobilizable, secretory vesicles is discharged in the secluded compartment between the PMN and the endothelial cell (EC). Azurocidin, a major component of secretory vesicles, is strongly, positively charged [20 ] and may thus accumulate on the negatively charged EC surface. In this way, azurocidin becomes immobilized on the endothelium and thereby exposed to cells in the blood flow [23 ] (Fig. 1A ). Interestingly, azurocidin is only deposited by adherent but not rolling PMN, indicating that PMN activation via β2-integrins is an important signal for discharge of secretory vesicles. The accumulation of azurocidin on the endothelium is reduced by treatment with heparinase and chondroitinase, suggesting that negatively charged proteoglycans in the endothelial glycocalyx act as primary binding sites [23 , 24 ]. A specific receptor for azurocidin on EC has not been identified. In line with this, treatment of EC with inflammatory stimuli such as LPS or TNF-{alpha} does not enhance binding of azurocidin to EC. Azurocidin immobilized on the endothelium may interact with inflammatory cells in the bloodstream. In fact, it has been shown that azurocidin with preference binds to monocytes [25 , 26 ]. Once monocytes in flow recognize azurocidin presented on the endothelial surface, a mobilization of intracellular Ca2+ is initiated, which is crucial for the azurocidin-mediated adhesion of monocytes [23 ]. Similar to PMN-derived elastase and proteinase-3 [27 , 28 ] monocyte adhesion stimulated by azurocidin was mediated via β2-intergins (unpublished data). The ability of azurocidin to enhance adhesion depends on a previous capturing of the monocyte from free flow. However, once the monocyte has slowed down, it is able to recognize azurocidin, which thus fulfills the function of a depositable CAM (Fig. 1A) . The clinical relevance of such a mechanism is suggested by the detection of azurocidin on the EC surface of acutely inflamed appendicitis specimens (unpublished observations) and of specimens from chronic inflammatory diseases such as Morbus Alzheimer [29 ] and atherosclerotic plaques [30 ].


Figure 1
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  		Figure 1. Contribution of azurocidin to monocyte recruitment in inflammation. (A) Adherent PMN release azurocidin rapidly from secretory vesicles that bind to proteoglycans of the endothelial glycocalyx. There, azurocidin is presented to cells in the blood flow and activates monocytes and enhances their adhesion via β2-integrins. (B) After its release from PMN, azurocidin is internalized in EC and may contribute to expression of E-selectin, ICAM-1, and VCAM-1, which will mediate the adhesion of inflammatory cells. (C) Azurocidin released from PMN at the inflammatory site is chemotactic for inflammatory monocytes, promoting a directed locomotion of these cells to the site of inflammation. CAMs, Cell adhesion molecules.

An additional mechanism directly affecting the expression of CAMs on the EC surface may contribute to enhanced leukocyte adhesion in response to the release of azurocidin (Fig. 1B) . As shown by Lee et al. [31 ], azurocidin enhances the expression of E-selectin, ICAM-1, and VCAM-1, which is associated with an increased adhesion of PMN and monocytes. Moreover, it has been suggested that azurocidin is produced by EC themselves upon cytokine stimulation [30 ]. However, as azurocidin is only detectable in permeabilized EC, indicative of intracellular accumulation rather than extracellular release, it remains unclear in what way EC-derived azurocidin may contribute to monocyte extravasation.

Adhesion of leukocytes is followed by migration through the vessel wall and directed locomotion to the site of injury. PMN granule proteins released extravascularly may exert a chemotactic effect on following mononuclear cells and thereby, put a causal link between the early PMN tissue infiltration and the second wave of monocyte extravasation. Indeed, studies about patients with specific granule deficiency lacking proteins in their PMN granules indicate that the supernatants from activated PMN contain proteins with chemoattracting effects for monocytes [32 ]. This is even more important, knowing that monocytes are able to herald the acquired immune response. Chertov et al. [33 ] found azurocidin to be strongly chemotactic for monocytes and to a lesser extent, for PMN. In addition, azurocidin was proven to be chemotactic for T cells. Interestingly, these data could be confirmed in a mouse model, where azurocidin induced pronounced leukocyte infiltration. However, binding studies have shown that azurocidin does not bind to lymphocytes, and therefore, indirect mechanisms involving chemokine synthesis may be involved in this response. The monocyte chemotactic activity is 80–100% of that of fMLP, a strong enhancer of monocyte chemotactic migration. Peripheral blood monocytes are, however, a heterogeneous cell population. In the human and the mouse, at least two populations may be distinguished [34 ]. It was demonstrated recently that azurocidin specifically stimulates efflux of inflammatory monocytes (Fig. 1C) [35 ]. Murine inflammatory monocytes were defined as CX3CR1loCCR2+GR1+ [36 ]. This population of murine monocytes shares morphological characteristics and chemokine receptor expression patterns with the classical human CD14hiCD16– monocytes, and murine resident monocytes are thought to correspond to human CD14+CD16+ nonclassical monocytes [34 ]. Human classical monocytes are potent phagocytes [37 ] and produce higher amounts of cytokines such as IL-6 and TNF-{alpha} [38 , 39 ]. In contrast, nonclassical monocytes are potent APC [40 ]. Although these characteristics have not been described for murine inflammatory monocytes, it is likely that the murine homologue functions similarly to the human subtype. Therefore, the specific recruitment of inflammatory monocytes by azurocidin may stand out as an important mechanism in enhancing the immune response. In contrast to HNPs and other alarmins, azurocidin has not yet been shown to attract DCs. However, blood monocytes represent precursors for populations of inflammatory DCs such as TNF/NO-producing DCs. Recent work has shown that these cells are the progeny of Gr1+ monocytes [41 ], and therefore, PMN-derived azurocidin may be an important enhancer of an adaptive immune response. Another intriguing feature of azurocidin-mediated chemotaxis is the relatively low concentration required to carry out this action. Although micromolar concentrations are needed for its antimicrobial activity, nanomolar concentrations are sufficient for its chemotactic activity, a concentration range that is reachable in the tissue [17 ].


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AZUROCIDIN ACTIVATES MONOCYTES AND MACROPHAGES
 
Monocytes and macrophages are multifunctional cells contributing to bacterial clearance by phagocytosis and killing of bacteria. Moreover, mononuclear phagocytes are powerful in the control and fine-tuning of the immune response. They do so by presenting antigens and releasing a wide array of chemokines and cytokines [42 ]. Cytokines are mostly de novo-produced and generally act over short distances and bind to a specific membrane receptor, which then signals via second messengers, often tyrosine kinases, to alter the target cell’s behavior.

Rasmussen et al. [43 ] were the first to describe an enhanced cytokine release from monocytes when treated with azurocidin. Interestingly, azurocidin in itself had no effect on the release of TNF-{alpha} and IL-6. However, in the presence of LPS, azurocidin could enhance the release of these two cytokines several fold. TNF-{alpha} is a multifunctional cytokine that is involved in EC activation, activation of macrophages, and initiation of a local inflammatory response. IL-6 is a cytokine that is mainly involved in T and B cell growth and differentiation, initiating the acquired immune response.

Recently, azurocidin was identified as an activator of human macrophages, as demonstrated not only by intracellular Ca2+ mobilization but also by a change in the phenotype [44 ]. Treatment of macrophages with azurocidin enhanced the expression of HLA II, CD40, and CD86, in agreement with findings from microglial cells treated similarly [45 ]. Expression of these molecules is a sign of the classical macrophage activation being functionally related to enhanced antimicrobial effectiveness and a more powerful activation of the adaptive immune system [46 ]. Furthermore, Fc{gamma}Rs CD64 and CD32 are up-regulated in response to treatment with azurocidin but not CD16 or the complement receptors. This type of activation is typically mediated by a concerted action of the prototypic macrophage activators TNF-{alpha} and IFN-{gamma} [46 ], both of which were found to be released from the macrophage in the presence of azurocidin [44 ]. More importantly, they were not only secreted from the macrophage in response to azurocidin, but they were also found to be responsible for the macrophage activation pattern. The autocrine activation of the macrophage by its own secretion products has been disputed [46 ]. However, the cytokine release in response to azurocidin and the subsequent macrophage activation seem to be an interesting contribution in favor of autocrine activation. In addition, this activation pattern does not only include a structural change but is also the causal link to enhanced phagocytosis in response to stimulation with azurocidin.


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AZUROCIDIN ENHANCES BACTERIAL PHAGOCYTOSIS
 
The process that mediates the recognition and removal of particles is known as phagocytosis and was first described by Metchnikoff in 1882 [47 ]. In mammals, the professional phagocytes of the immune system use phagocytosis to assist in wound repair and removal of tissue debris, apoptotic cells, and most importantly, pathogens. Phagocytosis is much more efficient when the bacteria or other foreign particles are prepared for ingestion—opsonized—which in the body, is achieved by coating the particle with complement-derived proteins and Igs. These complexes are recognized by the complement receptors and the FcRs on phagocytic cells [48 ]. In addition, other opsonins such as fibronectin, LPS-binding protein, thrombospondin, mannose-binding lectin, lung surfactant protein A, and conglutin have been identified [49 ]. Findings by Heinzelmann et al. [50 ] indicate that azurocidin can be added to this list of opsonins, as they found a strong binding of FITC-azurocidin to Staphylococcus aureus, which led to increased phagocytosis in monocytes (Fig. 2A ) and increased superoxide production during oxidative burst. The authors compared the binding of FITC-azurocidin to S. aureus with that of FITC-IgG, which is a well-established opsonin. Interestingly, azurocidin had a stronger tendency to bind S. aureus than IgG. This correlates to enhanced phagocytosis of azurocidin-opsonized bacteria in monocytes but not in PMN. A similar mechanism was found for HNPs, which colocalize with azurocidin in the primary granules of PMN. HNPs also opsonize bacteria, thereby enhancing phagocytosis [51 ].


Figure 2
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  		Figure 2. Contribution of azurocidin to phagocytosis of bacteria by macrophages. (A) Neutrophil-derived azurocidin opsonizes bacteria and enhances phagocytosis. (B) Azurocidin binds to β2-integrins on macrophages and induces expression of TNF-{alpha} and IFN-{gamma}. These cytokines activate macrophages in an autocrine manner, increasing the expression of Fc{gamma}RII/CD32 and Fc{gamma}RI/CD64, which mediates enhancement of phagocytosis of IgG-opsonized bacteria.

Recently, azurocidin has been identified as a main inducer of macrophage phagocytosis by a mechanism that is different from that described above. In this study, PMN-derived azurocidin activates macrophages via β2-integrins, which causes release of TNF-{alpha} and IFN-{gamma} [44 , 52 ]. As a consequence, CD64 and CD32 are up-regulated. Enhanced expression of these receptors increases phagocytosis of IgG-opsonized bacteria (Fig. 2B) but not of complement-opsonized bacteria. IL-10 and IFN-{gamma} were shown to enhance expression of Fc{gamma}Rs [53 , 54 ], both of which are expressed by PMN after stimulation [55 , 56 ]. However, the interesting feature about the recently published study is the activation of macrophages by preformed PMN granule proteins, namely azurocidin and HNPs, which are released instantly. That such a mechanism may be of importance in vivo was demonstrated in a peritonitis model [57 ]. i.p. application of azurocidin increases the survival in a murine fecal peritonitis model, which might relate to the enhanced phagocytosis of bacteria by peritoneal macrophages [57 ].

The antimicrobial activity of azurocidin, as described originally by Shafer et al. [14 ], is directed mainly against gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, and Pseudomonas aeroginosa, which may be a result of the binding of azurocidin to lipid A on the surface of these bacteria, and gram-positive bacteria such as S. aureus are resistant to azurocidin. However, with the two mechanisms described above, azurocidin may circumvent its inability to kill gram-positive bacteria directly by opsonizing bacteria and increase the expression of Fc{gamma}Rs, resulting in increased phagocytosis and bacterial clearance.


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AZUROCIDIN MEDIATES ITS EFFECTS ON LEUKOCYTES VIA β2-INTEGRINS AND FORMYL-PEPTIDE RECEPTORS
 
Soon after the first immunomodulatory effects of azurocidin were discovered, the question of a putative azurocidin receptor on immune cells was raised. Labeling azurocidin with FITC seemed a suitable tool for investigating the interaction of azurocidin with white blood cells. Heinzelmann et al. [25 ] and Påhlman et al. [26 ] found that azurocidin binds predominantly to monocytes and to a minor extent to PMN. Binding to lymphocytes could not be detected. As azurocidin enhanced the LPS-mediated TNF-{alpha} release from monocytes, CD14 seemed a likely receptor, but experiments with blocking antibodies to CD14 could rule out this receptor as the primary binding site [25 ]. Using EDTA and fucoidan, Heinzelmann et al. [58 ] could establish that the binding of azurocidin to monocytes was partially dependent on divalent cations and that fucoidan could prevent the azurocidin-monocyte interaction. This result drew attention to P- and L-selectin as well as scavenger receptors. Because of binding of azurocidin preferentially to monocytes, P- and L-selectin were considered less likely. Unfortunately, the possibility of an interaction between azurocidin and scavenger receptors has not been followed up until today.

In another approach, intracellular Ca2+ mobilization assays were applied to investigate the activation of monocytes in response to azurocidin. Following treatment with azurocidin, a strong and transient enhancement of intracellular Ca2+ was observed shortly, indicating a receptor-signaling mechanism to be involved in this event [23 , 26 ]. Early findings by Cai and Wright [28 ] pointed at a possible interaction between azurocidin and β2-integrins (CD11/CD18). In accordance, the use of a blocking antibody to CD18 abolished the azurocidin-mediated Ca2+ increase completely [26 ], and, e.g., antibodies to L-selectin were without effect (unpublished data). As discussed earlier, immobilized azurocidin enhances the adhesion of monocytes to EC, a response that could be blocked as well with an antibody to CD18 (unpublished observation). Similarly, release of TNF-{alpha} and IFN-{gamma} from macrophages and enhanced Fc{gamma}R expression on macrophages in response to azurocidin can be inhibited by treatment with a CD18 antibody [44 ]. In addition, Ca2+ mobilization in macrophages was inhibited by herbimycin, a tyrosine kinase inhibitor, underlining the β2-integrins to be of major importance in the azurocidin-monocyte interaction [44 ].

Besides β2-integrins mediating monocyte activation, including cytokine release, yet another receptor for azurocidin has been proposed in experiments studying the chemotactic effect of the protein. There, the chemotactic effect of azurocidin on monocytes vanished when the cells were pretreated with pertussis toxin (PTx), indicative of the involvement of a Gi-protein-coupled receptor (GiPCR) [33 ]. Azurocidin shares strong homology with the serine proteases elastase, proteinase-3, and cathepsin G [20 ]. The latter has been shown to be chemotactic for monocytes in a PTx-sensitive manner. A member of the formyl peptide receptor (FPR) family has been identified recently as the receptor for cathepsin G on leukocytes mediating its chemotactic effect [59 ]. In line with this, it was demonstrated recently that FPR antagonists efficiently block the chemotactic response of inflammatory monocytes to azurocidin in vivo, suggesting that a member of this family is important in mediating azurocidin-dependent chemotaxis [35 ].

The fact that two or more receptors are involved in mediating effects of of azurocidin on monocyte function may seem surprising. Such a pattern, however, has been demonstrated earlier for several members of the alarmin family. Interestingly, activation of APC and chemotaxis is mediated frequently via different receptors. Although chemotaxis is mediated in a PTx-dependent manner, APC activation is regulated via adenosine receptors, members of the TLR family, or the epidermal growth factor receptor (EGFR). In this respect, cathelicidins exert their chemotactic activity via FPR-like 1 [11 ], and they activate APC via P2X7 [60 ] or EGFR [61 ]. Eosinophil-derived neurotoxin exerts its chemotactic activity via GiPCR [62 ], whereas APC are activated via TLR2 [63 ].


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IMMUNOENHANCING ACTIVITIES OF AZUROCIDIN
 
Members of the alarmin family have been shown to exhibit potent in vivo immunoenhancing activity. The coadministration of mCRAMP and OVA into mice, for example, enhanced OVA-specific serum IgG1 and IgG2a, as well as IL-4 and IFN-{gamma} by OVA-specific T cells [11 ]. However, until today, such experiments have not been done for azurocidin, and it therefore remains elusive whether azurocidin possesses properties of an immunadjuvant. In addition, alarmins have been shown to attract and activate DCs repeatedly [7 ]. Research about azurocidin, however, so far, has focused on the activation and mobilization of monocytes rather than DCs. The recent finding that azurocidin exerts a chemotactic effect on Gr1+ monocytes, which may differentiate into DC subsets [41 ], suggests that azurocidin may also activate DCs.


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AZUROCIDIN ACTIVATES EC
 
Besides monocytes, other cell types are also activated by azurocidin. In fact, EC are the first target of PMN-derived azurocidin released from secretory vesicles. As described above, this may induce endothelial CAM expression and result in a more pronounced adhesion of immune cells. In addition, derangement of the endothelial barrier function, leading to plasma leakage and edema formation, is a characteristic feature of the inflammatory reaction. Previous studies clearly indicate that emigration of PMN is accompanied by efflux of plasma from the vasculature and that these cells are in a position to trigger permeability changes themselves [64 , 65 ]. Of critical importance in a PMN-evoked permeability increase is the PMN adhesion and activation via β2-integrins [66 ]. Adhesion of the PMN to the EC induces rapid intracellular Ca2+ mobilization in both cell types, leading to granule exocytosis in the PMN and rearrangement of the EC cytoskeleton. Blockage of β2-integrin function abrogated these responses completely [66 ]. More recently, it was shown that azurocidin is released upon β2-integrin ligation and that this protein has a central role in the PMN-evoked permeability change. Its location in rapidly mobilized secretory vesicles allows a rapid discharge upon PMN adhesion and activation. PMN-derived azurocidin could be demonstrated to provoke a rapid rise in cytosolic-free Ca2+ in adjacent EC, formation of actin stress fibers, and increased paracellular permeability [67 ]. The responses to azurocidin stimulation are identical to those achieved by chemoattractant stimulation of PMN, and immunoneutralization of azurocidin in PMN-derived secretion inhibits the activity completely, substantiating the critical role of this protein in PMN-evoked alterations in vascular permeability. Besides the importance of the localization of azurocidin in secretory vesicles, which allows an almost instant permeability change upon PMN adhesion, another feature of azurocidin is at least equally important in this process. Azurocidin carries a large number of positively charged amino acid residues concentrated on one side of the protein, creating a strong dipole moment [18 ]. It is likely that the basic patch of azurocidin interacts with negatively charged proteoglycans on the EC surface by which EC conformational changes are induced. Yet, the exact mechanisms by which azurocidin activates signaling pathways in EC and stimulates reorganization of cytoskeletal and junctional complexes remain elusive.

That release of azurocidin and activation of EC are of clinical importance has also been demonstrated in a model of septic acute lung injury. Streptococcus pyogenes infections may lead to the streptococcal toxic shock syndrome, which is characterized by hypotension, multiple organ failure, and lung edema. In the course of the infection, S. pyogenes shed M1 protein, which forms complexes with fibrinogen [68 ]. These activate PMN to degranulate in the circulation [69 ], releasing proteins from all granule subsets, including azurocidin [68 ]. Degranulation of PMN was found to be causative of the subsequent lung damage and edema formation [69 ]. Interestingly, injection of antibodies to azurocidin abrogated the lung injury (unpublished observation), pointing at the central position of this protein in the pathogenesis of M1 protein-induced lung damage.


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ACTIVATION OF OTHER CELL TYPES
 
Although EC, monocytes, and macrophages seem the main targets for azurocidin, additional cell types are also activated (Table 1 ). Indeed, one of the earliest studies about pleiotropic effects of azurocidin reports the contraction of fibroblasts in response to azurocidin [74 ]. More recently, Pereira and colleagues [72 ] demonstrated that azurocidin induces migration of corneal epithelial cells, which is an important step during healing after bacterial cornea injury. Interestingly, in this model, azurocidin was detected before infiltration of PMN, indicating a local expression of this protein [75 ]. Healing mechanisms in the cornea may also be supported by azurocidin-induced corneal epithelial cell proliferation and corneal epithelial cell adhesion molecule expression [72 ]. Similar results were also found with regard to the effects of azurocidin on smooth muscle cells. There, Lee et al. [30 ] demonstrate the presence of azurocidin in smooth muscle cells of atherosclerotic vessels. Functionally, azurocidin was found to stimulate proliferation, migration, and expression of E-selectin and ICAM-1 in aortic smooth muscle cells [73 ].


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  		Table 1. Biological Actions Mediated by Extracellular Azurocidin


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CONCLUDING REMARKS
 
Azurocidin exhibits certain characteristics of an alarmin. Following tissue injury or infection, it is released from PMN granules upon cell activation during the PMN extravasation cascade. Once released, azurocidin not only exerts direct antimicrobial activity but also activates APC. Specifically, azurocidin contributes to monocyte recruitment and activation, resulting in cytokine release and enhanced phagocytosis. However, until today, data about immunoenhancing properties of azurocidin are not at hand, and therefore, azurocidin does not fulfill all criteria of an alarmin. In addition, β2-integrins and FPRs have been shown to be important in transmitting azurocidin-dependent cell activation. However, receptor-binding studies, confirming that azurocidin acts through these receptors, have not been performed yet.

As an alarmin, azurocidin can play a pivotal role in host defense but also in the pathogenesis of a wide variety of inflammatory conditions and may thus constitute a novel target for therapeutic interventions in acute and chronic inflammatory diseases. Further studies are needed to define the role of azurocidin in clinical disorders. However, observations from preclinical animal models point at the potential importance of neutralizing azurocidin to prevent edema formation and limit the inflammatory response. On the contrary, as a result of the immunostimulating properties of the protein, recombinant forms of azurocidin may be beneficial in treating bacterial infections.


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ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (SO876/1-1), the Swedish Research Council, the Swedish Heart-Lung Foundation, the Lars Hierta Memorial Fund, and the Karolinska Institute.

Received August 25, 2008; revised October 6, 2008; accepted October 9, 2008.


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