Proteolytic processing of chemokines: Implications in physiological and pathological conditions

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The International Journal of Biochemistry & Cell Biology 40 (2008) 1185–1198

Review

Proteolytic processing of chemokines: Implications in physiological and pathological conditions Marlene Wolf ∗ , Stefan Albrecht, Christa M¨arki Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3012 Bern, Switzerland Available online 28 December 2007

Abstract Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and finetunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing. © 2007 Elsevier Ltd. All rights reserved. Keywords: Chemokine; Protease; Inflammation; Cancer; Leukocyte; Haematopoiesis

Contents 1. 2. 3. 4.

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∗

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation of chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemokine modifying proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological implications of chemokine processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Haematopoiesis and trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +41 31 631 4150; fax: +41 31 631 5377. E-mail address: [email protected] (M. Wolf).

1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.12.009

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1. Introduction Chemokines are the main regulators of leukocyte traffic under inflammatory conditions as well as in the process of cell homing during homeostasis. In addition, they control the migration of other cell types and contribute to the regulation of various cellular functions such as angiogenesis, haematopoiesis, lymphatic organogenesis, embryonic development, and tumour growth. As precise regulation of these events is essential, it is of central importance that chemokine activity is under strict control. Chemokines are found in all vertebrates, and chemokine homologues can also be encoded by viruses. Most chemokines are secreted proteins consisting of 67–127 amino acids and are characterized by four conserved cysteine residues which form two disulfide bridges (Moser, Wolf, Walz, & Loetscher, 2004; Zlotnik, Yoshie, & Nomiyama, 2006). The chemokine family contains nearly 50 members which are divided into four subgroups. The two major subfamilies are distinguished by the arrangement of the two NH2 -terminal cysteine residues, which are either adjacent (CC) or separated by a single amino acid (CXC). Chemokines of the two minor subfamilies have only a single cysteine residue (XC) or the cysteine residues are separated by three amino acids (CX3C). The chemokines act via heterotrimeric G protein coupled surface receptors. Most chemokines are known by their original name, nonetheless, a systematic nomenclature for chemokine and chemokine receptors was introduced in 2000. Receptors were designated as CXC, CC, XC and CX3C followed by “R” and a number, and the chemokines were assigned the same acronyms followed by “L” (Murphy et al., 2000; Zlotnik & Yoshie, 2000). Members of the chemokine family are also classified according to their function and are described as either inflammatory or homeostatic chemokines (Moser et al., 2004). The function of inflammatory chemokines is to recruit leukocytes for host defence. Their expression is rapidly induced in leukocytes and other cells types including endothelial cells during an inflammatory response or after tissue injury. Additionally, inflammatory chemokines can be induced in tumourtransformed cells. This class of chemokines includes the ELR-chemokines, a subgroup of the CXC chemokines having the tri-peptide motif Glu-Leu-Arg (ELR motif) preceding the first cysteine and members of the CCLchemokines that activate CCR1, CCR2, CCR3, and CCR5 expressed on immune cells. In contrast, homeostatic chemokines are constitutively expressed. They direct the migration of lymphocytes and dendritic cells to specialized areas of

secondary lymphoid organs for differentiation and maturation and are involved in haematopoiesis in the bone marrow and in immune surveillance of peripheral tissues. Examples are the CCR7-ligands CCL19 and CCL21, which are important for the homing of dendritic cells, na¨ıve and central memory T cells and B cells through high-endothelial venules into peripheral lymph nodes. The distinction between inflammatory and homeostatic chemokines is not strict, several chemokines belong to both categories and are defined as dual-function chemokines (Allen, Crown, & Handel, 2007; Moser et al., 2004). Dual function chemokines are upregulated under inflammatory conditions and also target nonclassical effector cells at sites of leukocyte development and immune surveillance. CCL20 is an example for a dual-function chemokine, it is upregulated in skin in response to inflammatory stimuli and it targets immature dendritic cells. There is high redundancy among chemokines with regard to target cell selectivity and in many cases, it is difficult to ascribe a distinct role to a single chemokine. Chemokine activity is regulated at different levels: (i) at the level of gene expression and protein secretion; chemokines may be stored in granules of leukocytes (Catalfamo et al., 2004), platelets (von Hundelshausen, Petersen, & Brandt, 2007) or endothelial cells (Oynebraten et al., 2005) or exported as inactive membrane-bound forms (Hundhausen et al., 2007), (ii) at the level of interaction with glycosaminoglycans; chemokines are retained on extracellular matrices or cell surfaces by binding to glycosaminoglycans which leads to increased local concentrations and potential protection from proteolytic processing (Proudfoot, 2006), (iii) at the level of binding to non-signalling receptors (interceptors); these receptors include DARC which binds inflammatory CC and CXC chemokines, D6 which binds inflammatory CC chemokines, and CCX–CKR which has a preference for constitutively expressed CC chemokines (Comerford, Litchfield, Harata-Lee, Nibbs, & McColl, 2007) and (iv) at the level of modulating active chemokine concentrations by means of proteolytic processing. In this review, emphasis is placed on the control of chemokine activity at the protein level, in particular, the impact of proteolytic processing on the function of secreted chemokines by different proteases. Numerous studies have appeared during the last 10 years addressing the issue of proteolytic chemokine processing. Early work focused on NH2 -terminal processing, mainly because different NH2 -terminally truncated forms of a given chemokine have been isolated from natural sources (Proost, Struyf, & Van Damme, 2006) and because it

M. Wolf et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1185–1198

is the NH2 -terminal region of a chemokine which is critical for receptor-binding and activity. Though many chemokines have been described as substrates for proteases in vitro, the physiological relevance of chemokine processing is not straightforward. It is technically demanding and time consuming to detect unambiguously truncated forms generated by a specific protease in tissue. Furthermore, in in vitro experiments, many chemokines are completely degraded by a single protease. This may not be the case in a physiological environment where fine-tuning of protease activity and of chemokine availability is more tightly regulated than in a test tube. Hence, in vitro results do not adequately predict the potential impact of chemokine proteolysis in vivo in conditions such as infection and tumour progression. 2. Activation of chemokines Most chemokines are synthesized as proteins with signal sequences of 20–25 amino acids. The secreted mature forms of some chemokines are processed further to yield-truncated forms with various levels of biological activity. Because the NH2 -terminal sequence preceding the first cysteine is exposed and conformationally disordered, many chemokines are processed easily at the NH2 -terminus, and in the case of CXC inflammatory chemokines, such cleavage often results in an amplification of their activity (Baggiolini, Dewald, & Moser, 1994; Proost et al., 2006). Only a few studies have reported chemokine processing at the COOH-terminus or at internal sites, but generally, removal of up to six COOH-terminal amino acids has little or no consequences on activity. The subgroup of CXC chemokines with the ELR motif has a high occurrence of naturally truncated forms (Proost et al., 2006). Proteolytic removal of NH2 -terminal amino acids up to the ELR motif generally has no major biological consequence; however, enhanced potency resulting in a positive feedback-loop was reported for truncated forms of CXCL8/IL8, CXCL5/ENA-78, CXCL1/Gro␣ and CXCL3/Gro␥ (Nufer, Corbett, & Walz, 1999; Padrines, Wolf, Walz, & Baggiolini, 1994; Wuyts et al., 1999). A unique case is CXCL7/NAP-2; this chemokine is derived from the precursor of platelet basic protein (PBP) which is secreted by platelets as a 128 amino acid molecule. Precursor PBP is NH2 -terminally processed to PBP and further cleaved to the 81 amino acid connective tissue-activating peptide III (CTAP-III). After removal of an additional seven amino acids by chymotrypsin or cathepsin G, the chemotactically active CXCL7 is formed (Car, Baggiolini, &

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Walz, 1991; Schiemann et al., 2006; Walz, Dewald, von Tscharner, & Baggiolini, 1989). Additional forms of CXCL7 with diverging NH2 -termini were also identified (Walz & Baggiolini, 1990). Despite the fact that PBP and CTAP-III are chemotactically inactive, they may be involved in other biological functions like modulating processes related to atherosclerosis and angiogenesis (von Hundelshausen et al., 2007). CCL14/HCC-1 and CCL15/HCC-2 are two chemokines, which are abundant in human plasma, but they are rather inactive in their unprocessed forms (Detheux et al., 2000; Richter et al., 2005). CCL14 is expressed constitutively in many tissues and released into plasma where it is cleaved by urokinase-type plasminogen activator (uPA) and plasmin (Vakili et al., 2001). Full size CCL14 activates CCR1 receptors with an IC50 of 93 nM and is inactive on CCR5, whereas the NH2 -terminally processed form which has lost eight amino acids (CCL14(9-74)) is a much stronger agonist for CCR1 (IC50 of 2.8 nM) and in addition triggers CCR5-mediated cell migration (IC50 4.8 nM). Similarly, NH2 -terminal processing of CCL15 is required to retain full activity. Cathepsin G was identified as the responsible protease (Richter et al., 2005). The conversion of highly abundant inactive precursors into active chemokines enables the rapid recruitment of leukocytes without the need for transcriptional activation. In the case of CCL14 and CCL15, it reveals a mechanistic link to the activation of the uPA–plasmin system as well as the degranulation of neutrophils to the activation of monocytes and other cells bearing inflammatory CC-receptors. Two members of the minor chemokine families, CX3CL1/fractalkine and CXCL16 are unique as they are synthesized as transmembrane molecules consisting of a NH2 -terminal chemokine domain, followed by a mucin-like stalk, a transmembrane ␣-helix and a short cytoplasmic tail (Ludwig & Weber, 2007). Upon limited proteolysis by disintegrin and metalloproteinases ADAM10 and ADAM17, soluble CX3CL1 and CXCL16 are generated (Garton et al., 2001; Hundhausen et al., 2003, 2007). The soluble form is comprised of the chemokine domain and most of the extracellular mucin-like stalk. The membrane-bound form, which is expressed on numerous cell types, functions as adhesion molecule and promotes shear-resistant adhesion of CX3CR1-bearing leukocytes. The chemoattractive function is attributed to the soluble form. It is believed that ADAM10 is responsible for constitutive shedding and ADAM17 activity mediates enhanced cleavage in response to cell activation by phorbolesters. In a new study, Hundhausen et al. reported that the calcium-

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ionophore ionomycin acts as an efficient inducer of ADAM10 activity linking protease activity to calcium signalling (Hundhausen et al., 2007). CX3CL1 and CXCL16 are considered as inflammatory chemokines, but may also exhibit constitutive functions (Ludwig & Weber, 2007). Chemerin is a chemokine-related protein, which structurally belongs to the cathelicidin/cystatin family of proteins. It has chemoattractant properties and high levels are found in plasma. Chemerin circulates as an inactive precursor and requires proteolytic processing by serine proteases for activation, mediated by several proteases of the inflammatory or coagulation cascades (Wittamer et al., 2005; Zabel et al., 2005). Five to 10 COOH-terminal amino acids are removed to produce the active chemerin, which selectively attracts antigen-presenting cells such as immature plasmacytoid dendritic cells and macrophages, which express the receptor chemerinR. Interestingly, recent work by Kulig et al. demonstrated that Staphylococcus aureus-derived cysteine protease staphopain B is a potent activator of chemerin by cleaving off six COOHterminal amino acids (Kulig et al., 2007). The presence of active chemerin in plasma may result in the accumulation of immature plasmacytoid dendritic cells and macrophages, leading to an inflammatory environment. This could explain the chronic inflammation often seen after S. aureus infection. 3. Chemokine modifying proteases A wide range of different proteases is able to process chemokines at the NH2 -terminus, at the COOH-terminus, as well as the core regions. Table 1 (NH2 -terminal processing) and Table 2 (COOH-terminal and core processing) summarize the hitherto known reports on proteolytical processing of chemokines. A protease considered relevant for chemokine processing is CD26/dipeptidyl-peptidase IV (DPPIV). CD26/DPPIV is a membrane-bound serine protease which is expressed on many cells, particular on activated T cells, but also found as soluble form in normal plasma (Struyf, Proost, & Van Damme, 2003). CD26/DPPIV is highly selective for substrates containing a proline or alanine residue at position two of the NH2 -terminus. About one-third of human chemokines has an X-Pro NH2 -terminal dipeptide motif and may thus be candidate substrates. However, the NH2 -termini of several X-Pro-dipeptide containing chemokines are modified by pyroglutamate and therefore not available for cleavage by CD26/DPPIV. Surprisingly, CD26/DPPIV removes two dipeptides from CCL22/MDC, first the expected

Gly1-Pro2 dipeptide and subsequently the Tyr3-Gly4 dipeptide (Proost et al., 1999). Several chemokines are described as being processed by members of the family of matrix metalloproteinases (MMPs). MMPs are expressed by stromal cells and leukocytes where some of them are highly upregulated during inflammation or in cancer. They have important functions in extracellular matrix degradation and as such are involved in angiogenesis and tumour progression (Overall & Kleifeld, 2006). Particular chemokine substrates for MMPs are the monocyte chemoattractant proteins (MCPs) CCL2/MCP-1, CCL7/MCP-3, CCL8/MCP-2 and CCL13/MCP-4 (McQuibban et al., 2002), and CXCL8 as well as CXCL12/SDF-1 (McQuibban et al., 2001; Vergote et al., 2006; Zhang et al., 2003). Cathepsin G, elastase and proteinase-3 are released from neutrophil and monocyte granules upon cell activation and as a result, chemokines of both the CXC and CC family might be proteolytically cleaved be these proteases. Depending on the chemokine, the agonistic properties of the products are either increased or decreased. Moreover, NH2 -terminal proteolysis of chemokines was also shown to be mediated by enzymes present in plasma like CD13/aminopeptidase N (CD13/APN), uPA, plasmin and thrombin (Table 1). More recent reports describe COOH-terminal processing (Davis et al., 2005) or processing in the core of the chemokines (Hasan et al., 2006; Wolf et al., 2003) (Table 2). CCL20/LARC and CCL21/SLC were identified as substrates for cathepsin B and D and were cleaved in the core region resulting in their inactivation. The respective truncated products have not been identified in tissues, but interestingly, both proteases responsible for this processing are upregulated in cancer (Berdowska, 2004; Mohamed & Sloane, 2006). It is an attractive hypothesis that cancer-related proteases might help to define the route of metastasis by selectively cleaving and inactivating chemokines. The regulatory interplay between chemokines and proteases occurs in both directions; chemokines are also known to be modulators of protease expression. CCL-2, -4 and -5 stimulate MMP-9 expression in macrophages (Balkwill, 2004). CCL11/eotaxin and CXCL12 induce the release of MMP-2 in arterial smooth muscle cells (Kodali et al., 2006), CCL2 and CXCL8 regulate MT1MMP (Galvez et al., 2005) and CCL23 induces MMP-2 expression in vascular endothelial cells (Son, Hwang, Kwon, & Kim, 2006). Furthermore, proteases may also interfere with the function of chemokines by shedding cell- or extracellular matrix-bound glycosaminoglycans.

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Table 1 NH2 -terminal processing of chemokines Protease

Chemokine

Product(s)

Biological effects of product(s) and reference

MMP-1

CXCL5 CXCL8 CXCL12 CCL2 CCL7

10-78 7-77 5-67 5-76 5-76

CCL13

4-75; 5-75; 8-75

No information (Tester et al., 2007) No information (Tester et al., 2007) No activity (McQuibban et al., 2001) Reduced cell migration in an in vivo inflammatory model (McQuibban et al., 2002) Reduced cell migration in an in vivo inflammatory model (McQuibban et al., 2000, 2002) Reduced cell migration in an in vivo inflammatory model CCR2 and CCR3 antagonist (McQuibban et al., 2002)

MMP-2

CXCL12 CCL7

5-67 5-76

CXCR3-mediated neurotoxicity (Vergote et al., 2006; Zhang et al., 2003) See MMP-1

MMP-3

CXCL12 CCL2 CCL7 CCL8 CCL13

5-67 5-76 5-76 5-76 4-75; 5-75

See MMP-1 See MMP-1 See MMP-1 CCR2 antagonist (McQuibban et al., 2002) See MMP-1

MMP-8

CXCL5 CXCL6 CXCL8 CCL2

8-78 5-77; 6-77 6-77 5-76

Increased activity (Tester et al., 2007) No changes (Van Den Steen et al., 2003b) Increased activity (Tester et al., 2007) See MMP-1

MMP-9

CXCL5 CXCL6 CXCL8 CXCL12

6-78; 7-78: 8-78 5-77; 6-77; 7-77 7-77 5-67

Increased activity (Nufer et al., 1999; Van Den Steen et al., 2003b; Wuyts et al., 1999) No changes (Van Den Steen et al., 2003b) See MMP-1 See MMP-1

MMP-13

CXCL8 CXCL12 CCL7

6-77 5-67 5-76

See MMP-8 See MMP-1 See MMP-1

MMP-14

CXCL8 CXCL12 CCL7

6-77 5-67 5-76

See MMP-8 See MMP-1 See MMP-1

CD26/DPP IV

CXCL6 CXCL9 CXCL10 CXCL11 CXCL12

3-77 3-103 3-77 3-73 3-68

CCL3L1

3-70

CCL5

3-68

CCL11

3-74

CCL22

3-69

No changes (Proost et al., 1998a) Impaired activity (Proost et al., 2001) Impaired activity, CXCR3 antagonist (Proost et al., 2001) Impaired activity, CXCR3 antagonist (Ludwig et al., 2002; Proost et al., 2001) Impaired activity, CXCR4 antagonist (Christopherson et al., 2002; Ohtsuki et al., 1998; Proost et al., 1998b; Shioda et al., 1998) Increased activity for monocytes, CCR1+ and CCR5+ cells (Proost et al., 2000); decreased activity for CCR3+ cells; anti-HIV activity (Struyf et al., 2001) Reduced activity for CCR1+ and CCR3+ cells; increased activity for CCR5+ cells, increased anti-HIV activity (Oravecz et al., 1997; Schols et al., 1998) Reduced chemotactic activity for CCR3+ cells, unchanged anti-HIV activity (Struyf et al., 1999) Impaired binding to CCR4; reduced activity for lymphocytes, but not for monocytes; (Struyf et al., 1998) Impaired binding to CCR4, reduced activity for lymphocytes, but not for monocytes (Proost et al., 1999)

5-69 CD13/APNa

CXCL11

3-73; 5-73; 7-73

Reduced activity for lymphocytes and CXCR3+ cells, antagonistic effects (Proost et al., 2007)

Cathepsin G

CXCL5 CXCL12 CCL5 CCL15

9-78 6-67 4-68 24-92; 27-92 29-92 27-99

Increased activity for neutrophils (Nufer et al., 1999) No activity (Delgado et al., 2001) Reduced activity (Lim, Burns, Lu, & DeVico, 2005; Lim, Lu, Hartley, & DeVico, 2006) Increased activity for monocytes and CCR1+ cells (Richter et al., 2005) Increased activity for CCR1+ cells (Berahovich et al., 2005) Increased activity for CCR1+ cells (Berahovich et al., 2005)

CCL23

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Table 1 ( Continued ) Protease

Chemokine

Product(s)

Biological effects of product(s) and reference

Elastase

CXCL12 CCL15 CCL23

4-67 22-92 30-99

No activity (Valenzuela-Fernandez et al., 2002) Increased activity for CCR1+ cells (Berahovich et al., 2005; Richter et al., 2005) Increased activity for CCR1+ cells (Berahovich et al., 2005)

Proteinase-3 uPAb

CXCL8 CCL14

8-77 9-74

Increased activity (Padrines et al., 1994) Increased activity for CCR1+ , CCR3+ , and CCR5+ cells, anti-HIV activity (Detheux et al., 2000; Vakili et al., 2001)

Chymase

CCL15 CCL23

29-92 27-99

Increased activity for CCR1+ cells (Berahovich et al., 2005) Increased activity for CCR1+ cells (Berahovich et al., 2005)

Plasmin

CXCL8 CCL14

6-77; 9-77 9-74

Increased activity for neutrophils (Nakagawa et al., 1991) CCL14(9-74) has increased activity, but plasmin further degrades this active product (Vakili et al., 2001)

Thrombin

CXCL8

6-77

Increased activity for neutrophils (Hebert et al., 1990)

Cathepsin L

CXCL8

6-77

Increased activity for neutrophils (Ohashi, Naruto, Nakaki, & Sano, 2003)

a b

CD13/aminopeptidase N. uPA: urokinase plasminogen activator.

Table 2 COOH-terminal processing of chemokines Protease

Chemokine

Product(s)

Biological effect of product(s) and references

MMP-8 MMP-9

CXCL10 CXCL9 CXCL10

1-71; 1-73 1-90; 1-93; 1-94 1-68

No information (Van den Steen, Husson, Proost, Van Damme, & Opdenakker, 2003a) No information (Van den Steen et al., 2003a) No information (Van den Steen et al., 2003a)

Cathepsin B Cathepsin D

CCL20 CCL20

CCL21

1-66 1-19 1-52; 1-55 59-70 1-58; 59-111

No changes (Hasan et al., 2006) No information (Hasan et al., 2006) No activity (Hasan et al., 2006) Antimicrobial activity (Hasan et al., 2006) No activity (Wolf et al., 2003)

CXCL12(1-68) Met-CXCL10

1-67 1-76

Reduced activity (Davis et al., 2005; De La Luz Sierra et al., 2004) No change (Hensbergen et al., 2004)

CPNa Furin a

Carboxypeptidase N.

Chemokine–glycosaminoglycan interactions are thought to facilitate the retention of chemokines on cell surfaces, and disruption of this interaction may therefore destroy high local chemokine concentrations, and prevent their action (Proudfoot, 2006). 4. Physiological implications of chemokine processing 4.1. Inflammation Controlling chemokine activity at sites of inflammation is important, because excess and accumulation of leukocytes contributes to local tissue damage. Upon detection of pathogens, numerous types of tissue cells and leukocytes rapidly secrete different pro-inflammatory chemokines. Their chemotactic specificities determine the cellular infiltrates at the site

of pathogen entry. A therapeutically relevant question in the pathogenesis of inflammatory diseases is how chemokine signals are annihilated to reduce new cell recruitment and to promote termination of the inflammatory response. Chemokines with the ELR motif bind to the neutrophil receptors CXCR1 and CXCR2 and attract these cells to sites of inflammation while the inflammatory CC chemokines recruit monocytes, basophils and eosinophils. These infiltrating cells induce effector functions, e.g. by the release of granule contents like proteases, histamine and cytotoxic proteins (Baggiolini, 2001). The release of proteases at inflammatory sites might have significant consequences for the fate of local chemoattractants. Cathepsin G, elastase and mast cell chymase have been reported to modify chemokines and thereby changing their activity (Proost et al., 2006; Zabel et al., 2006). Depending on the outcome of such

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protease–chemokine interactions (activation or inactivation), proteolytic processes might amplify or dampen immune responses. The human chemokines CCL15 and CCL23 and the mouse chemokines CCL6 and CCL9 possess an extended NH2 -terminal domain and contain six cysteine residues that appear to form a third disulfide bond. Little is known about the biology of these four CC chemokines, but since all are ligands for CCR1, they were suggested to be involved in local inflammatory responses to pathogens (Berahovich et al., 2005). The study of Berahovich et al. demonstrated that proteases released by infiltrating neutrophils and mast cells cleave the NH2 -terminal domain of these chemokines yielding truncated forms with higher potency to induce CCR1-mediated calcium mobilization and cell migration. This cleavage was confirmed using recombinant mast cell chymase or purified neutrophil cathepsin G and elastase. Proteolytic conversion of relatively inactive ligands into potent chemoattractants might be significant under inflammatory conditions and may play a role in rheumatoid arthritis patients where sufficient amounts of NH2 -terminally truncated CCL15 and CCL23 were found in synovial fluids to be responsible for recruiting CCR1-bearing leukocytes (Berahovich et al., 2005). MMP-9 and MMP-8 released by activated neutrophils efficiently process the ELR-CXC chemokines CXCL5, CXCL6, and CXCL8. The truncation products CXCL8(6-77), CXCL8(7-77) and CXCL5(8-78) display several fold enhanced biological activity. Regulation of CXCL8 and CXCL5 activation by MMP-9 and MMP-8 therefore seems to exercise a positive feedback loop in neutrophil recruitment. In contrast, CTAPIII and CXCL1, two other ELR-chemokines are completely degraded by MMP-9. (Tester et al., 2007; Van den Steen, Proost, Wuyts, Van Damme, & Opdenakker, 2000). MMPs may also mediate the function of chemokines by releasing chemokines, which are retained on the cell surface or extracellular matrix through their binding to GAGs. For example, MMP-7 is important to promote shedding of syndecan-1, a heparan sulfate proteoglycan that binds KC, the mouse homologue of CXCL8. (Li, Park, Wilson, & Parks, 2002) have shown that MMP-7 generates a gradient of KC by shedding of syndecan-1 thereby inducing the influx of neutrophils in bleomycininjured lungs. In MMP-7 null mice the neutrophils did not migrate into the alveolar space, these mice were protected against the lethal effects of bleomycininduced injury. Similarly, neutrophil influx into the liver is impaired in mice lacking functional MMP-8 (Van Lint, Wielockx, Puimege, Noel, Lopez-Otin, & Libert, 2005).

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Conversion of CXCL5 by cathepsin G to CXCL5(978) results in two to threefold increased potency. The fact that CXCL5 is predominantly expressed by epithelial cells, monocytes and macrophages and is transformed into a stable active product by cathepsin G, strongly suggests that this chemokine is part of the amplification loop in cell recruitment during inflammatory processes in vivo. In pathological conditions, such as rheumatoid arthritis, where both, cathepsin G and CXCL5, are present in high levels in synovial fluids (Nordstrom et al., 1996), proteolytic processing might therefore modulate the degree of the inflammatory response (Nufer et al., 1999). On the other hand, several studies describe inactivation of inflammatory chemokines after proteolytic processing. One example with reduced activity after proteolysis is CXCL11. The product of CD26/DPPIVprocessing, CXCL11(3-73), displays reduced receptor binding, and completely abrogated chemotactic activity. As an antagonist, CXCL11(3-73) may therefore desensitize T cells towards the intact CXCR3 ligands CXCL9, CXCL11 and CXCL10. These results imply that CD26/DPPIV, which is highly expressed on activated T cells may be a regulator of T cell recruitment (Ludwig, Schiemann, Mentlein, Lindner, & Brandt, 2002). Likewise, removal of four to seven NH2 -terminal amino acid residues of the MCPs by different MMPs results in reduced activity and simultaneous conversion to CCR1, -2, -3 receptor antagonists (Table 1). In an in vivo rat paw model, where oedema formation was measured after injection of the pro-inflammatory substance carrageenan, MMP-cleaved MCPs were found to reduce the paw volume suggesting a role as inhibitors by blocking the infiltration of inflammatory cells (McQuibban et al., 2002). The strongest anti-inflammatory effects were revealed by cleaved CCL7, CCL7(5-76). Allergic reactions are characterized by the accumulation of eosinophils at the site of irritation. Truncated CCL11 missing two NH2 -terminal amino acids, shows reduced chemotactic activity for eosinophils and impaired binding and signalling toward CCR3. Furthermore, it displays antagonistic activity and desensitized calcium signalling and chemotaxis toward intact CCL11. CCL11(3-74) might affect the response of eosinophils to other CCR3 ligands as well and it is likely that allergic reactions mediated by CCL7, CCL13 and CCL5/RANTES through CCR3 might also be downregulated (Struyf et al., 1999). Proteolytic processing does not only modulate the activity of chemokines, but also alters receptor selectivity. CCL5 for example, when converted to CCL5(3-68)

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by CD26/DPPIV, shows reduced signalling through CCR1 and CCR3, but is a more potent ligand for CCR5 (Oravecz et al., 1997; Schols et al., 1998). Similarly, CD26/DPPIV processing of CCL22 leads to reduced biological activity on lymphocytes and dendritic cells, but not on monocytes and thus, brings up the possibility that proteases are competent to direct an immune response in a particular direction (Proost et al., 1999; Struyf et al., 1998). In summary, all these examples of proteolytic processing of chemokines during inflammatory conditions underline that there is no consistency in the functional outcome of a protease–chemokine interaction. A single protease may increase the activity of one chemokine while reducing it of another.

tion towards CXCL12, as the chemokine is cleaved and inactivated by CD26/DPPIV. This abrogated migration could be overcome by inhibiting the activity of CD26/DPPIV. Hence, the authors propose that pharmacological inhibitors of CD26/DPPIV would be useful to increase homing of haematopoietic stem and progenitor cells to the bone marrow during cord blood transplantation. Davis et al. identified carboxypeptidase N in human serum as the enzyme responsible for the removal of COOH terminal lysine from CXCL12(1-68) (Davis et al., 2005). The conversion to CXCL12(1-67) reduces the chemokine‘s biological activity as a pre-B-cell growth factor and chemoattractant and the study identifies another protease as candidate to regulate cell migration within tissues and from the blood stream to tissues.

4.2. Haematopoiesis and trafficking 4.3. Cancer CXCL12 is expressed in the bone marrow by stromal, endothelial cells and osteoblasts and is a potent attractant of immature and mature haematopoietic cells (Kucia et al., 2004; Ma, Jones, & Springer, 1999). It is therefore a key regulator of the migration of cells of the haematopoietic cell lineage, which express CXCR4, the best-characterized receptor for CXCL12 (Lapidot & Petit, 2002; Valenzuela-Fernandez et al., 2002). Endogenous CXCL12 provides a retention signal for haematopoietic stem and progenitor cells and degradation of CXCL12 by proteolytic enzymes has been suggested to affect mobilization of these cells. During G-CSF-stimulated cell mobilization, elastase is released from neutrophils and may be responsible for CXCL12 processing (Petit et al., 2002). Other studies confirmed that the neutrophil proteases cathepsin G and elastase cleave CXCL12 and remove five or three NH2 -terminal residues, respectively (Delgado et al., 2001; Valenzuela-Fernandez et al., 2002). Both truncated forms of CXCL12 lose their agonistic abilities leading to the disruption of the CXCL12–CXCR4 axis (Levesque, Hendy, Takamatsu, Simmons, & Bendall, 2003) and enabling mobilization of haematopoietic stem cells from the bone marrow to the peripheral circulation. In addition, several metalloproteinases as well as CD26/DPPIV can process CXCL12 at the NH2 -terminus (McQuibban et al., 2001; Proost et al., 1998b). However, it is still not established how important these proteases are in processing and clearing of CXCL12 in vivo. In favour of a physiological role of CD26/DPPIV is the study of Christopherson et al., which shows that a subpopulation of CD34+ cord blood cells expresses CD26/DPPIV on the surface (Christopherson, Hangoc, & Broxmeyer, 2002). These cells reveal reduced migra-

Most tumours produce chemokines and cause infiltration of immune cells to the tumour (Balkwill, 2004). The macrophage and lymphocyte infiltrates contribute to tumour growth and progression by producing growth factors, angiogenic factors, immunosuppressive cytokines and proteases. Abundant expression of certain MMPs and members of the cathepsin family is often associated with a more aggressive tumour phenotype (Gocheva & Joyce, 2007). Proteases influence the tumour microenvironment in different ways. They may cleave various structural components of the extracellular matrix, release and activate matrix bound growth factors and cytokines (Sternlicht & Werb, 2001), or may alter the function of chemokines by specifically processing them (Egeblad & Werb, 2002). Furthermore, many cancer cells express chemokine receptors, most commonly CXCR4 (Burger & Kipps, 2006). Expression of CXCR3, CXCR4 and CCR7 is implicated in the metastasis of breast and colon cancer cells to organs where the respective chemokine ligands are produced (Kawada et al., 2007; Kim et al., 2005; Muller et al., 2001). S´ezary syndrome illustrates how chemokinemediated migration of tumour cells could be affected by proteolytic processing (Narducci et al., 2006). S´ezary patients are characterized by an uncontrolled accumulation of CXCR4+ T lymphoma cells (S´ezary cells) in the skin expressing abundant CXCL12. The accumulation is believed to be due to the impaired expression of CD26/DPPIV on these cells and reduced CD26/DPPIV activity in the plasma of the patients. Therefore, CXCL12 would remain intact and be responsible for CXCR4-mediated metastasis of S´ezary cells to the skin. This concept is supported by an in

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vitro transmigration assay, which demonstrated that preincubation of CXCL12 with soluble CD26/DPPIV results in decreased numbers of transmigrating CXCR4+ S´ezary cells. Such altered migration behaviour due to downregulation of chemokine-modulating proteases could as well be relevant for other tumour cells bearing functional chemokine receptors. Some chemokines have angiogenic activities while others are angiostatic (Strieter et al., 2006). The beststudied angiogenic chemokines CXCL1, CXCL5 and CXCL8 belong to the group of ELR-CXC chemokines. They aid in blood vessel formation by attracting CXCR2+ endothelial cells to the tumour microenvironment (Addison et al., 2000). Several angiogenic chemokines are substrates for proteases such as MMP9 (Van den Steen et al., 2000), cathepsin G (Nufer et al., 1999), or plasmin (Nakagawa, Hatakeyama, Ikesue, & Miyai, 1991). Interestingly, CXCL8 processed by MMP-9 (CXCL8(7-77)) has higher affinity for CXCR1 and CXCR2 than the full-length form and could therefore have an elevated angiogenic potential which would be beneficial for tumour development. Most non-ELR CXC chemokines, on the other hand, are angiostatic. The CXCR3 ligands CXCL9, CXCL10 and CXCL11 are implicated in anti-tumour immunity by attracting Th1 cells to the tumour microenvironment, but also block endothelial cell proliferation by binding with high affinity to CXCR3+ microvascular endothelial cells. This angiostatic effect could be inhibited with an anti-CXCR3 antibody (Romagnani et al., 2001). CXCL9, CXCL10 and CXCL11 are all substrates for CD26/DPPIV (Aquaro et al., 2001), and the processed chemokines lose a high proportion of their binding capacity and chemotatic activity for CXCR3 (Table 1). Surprisingly, the truncated chemokines were as effective in inhibiting angiogenesis as their intact counterpart in a rabbit model. When pellets containing either intact or CD26/DPPIV-truncated CXCL9 or CXCL10 were implanted into corneal micropockets on rabbit eyes, no significant difference between the antiangiogenic activity of the intact and truncated chemokines was observed, indicating that the angiostatic effect of the CXCR3 ligands is not exclusively dependent on CXCR3-mediated signalling (Proost et al., 2001; Strieter et al., 2006). The accumulation of specific lymphocyte subsets in the tumour microenvironment is a phenomenon that has been observed in many tumours. A role for T regulatory cells (Tregs) in suppressing autoreactive T cells without killing them has been discovered some time ago, but there still remain questions concerning the impact of Tregs in tumour development, where they could be responsible for tumour driven immune eva-

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sion (Curiel, 2007). Specific recruitment of CCR4+ Tregs into ovarian cancer (Curiel et al., 2004) or into the proximity of Hodgkin lymphoma (Ishida et al., 2006) has been described. CCL22 produced by tumour cells and/or tumour associated cells was involved in both studies. CCL22 is a substrate for CD26/DPPIV (Proost et al., 1999) and truncated CCL22 has reduced chemotactic activity for lymphocytes, which may result in a decreased migration of Tregs to the tumour microenvironment probably leading to an unfavourable effect for the tumour. This raises the question whether chemokine cleavage is a defence mechanism of tumours or a byproduct of protease over expression at tumour sites. But since it is not yet clear if chemokine cleavage by specific proteases like CD26/DPPIV or MMP is taking place in the tumour microenvironment and due to experimental difficulties in detecting truncated chemokines in in vivo models, it is currently not possible to predict the overall contribution of chemokine proteolysis in tumour biology. 4.4. HIV Chemokines and chemokine receptors play a crucial role in HIV infection. Entry of the HIV-1 strains into CD4-expressing target cells is dependent on binding to co-receptors, where CCR5 and CXCR4 are being considered the major co-receptors used by M-tropic (R5) and T-tropic (X4) HIV-1 strains, respectively (reviewed in (Loetscher, Moser, & Baggiolini, 2000)). Correspondingly, the CCR5 ligands CCL5, CCL3 and CCL4, are recognized as potent inhibitors of M-tropic HIV-1 infection, mediated through their binding to CCR5 (Alkhatib et al., 1996). Proteolytic processing of chemokines was shown to influence not only the chemotactic function, but also the antiviral capacity against HIV-1 strains. Several studies have demonstrated that CCL5(3-68), generated by CD26/DPPIV-mediated proteolysis, shows decreased binding to CCR3 and CCR1, but is a more potent ligand for CCR5 compared to the intact form and concurrently also a more potent inhibitor of infections by HIV-1 strains (Oravecz et al., 1997; Proost et al., 1998a; Schols et al., 1998). Furthermore, processing of CCL3L1/LD78␤ by CD26/DPPIV results also in increased CCR5 mediated chemotactic activity and the cleavage product, CCL3L1(3-70), is considered the most potent CCR5 ligand in inhibiting HIV-1 infection (Proost et al., 2000). These studies demonstrate that chemokine ligands for CCR5 are turned into more potent activators of chemotaxis and inhibitors of HIV infection upon processing by CD26/DPPIV and they underline the significance of chemokine–protease interactions in

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the fine-tuning of specific chemokine actions (Proost et al., 2006). Several other chemokine receptors, including CCR3 and CCR4, were proposed to be “minor co-receptors” (Agrawal, Vanhorn-Ali, & Alkhatib, 2002; Struyf et al., 1999), however, their contribution to HIV disease in vivo remains controversial. CD26/DPPIV-truncated CCL11, which shows reduced chemotactic activity through CCR3, was found to have unaltered anti-HIV activity (Struyf et al., 1999). Similar properties were demonstrated for the truncated form of the CCR4 ligand CCL22 (Struyf et al., 1998). CD26/DPPIV-truncated CXCL12 in contrast, loses the ability to bind to its cognate receptor CXCR4 and consequently also its anti-HIV properties for X4-tropic HIV-1 strains. Not only CD26/DPPIV, but also MMP-2 efficiently converts CXCL12 from a potent chemoattractant and HIV inhibitor to a CXCR4-unresponsive protein (CXCL12(5-67) (McQuibban et al., 2001). HIV infection stimulates MMP-2 release from macrophages, and increased activity of this protease is associated with the development of neurodegeneration (Conant et al., 1999). Two interesting recent studies demonstrate that CXCL12(5-67) is highly neurotoxic and that this neurotoxicity is mediated by CXCR3 revealing that CXCL12 cleavage leads to a change in receptor specificity and transformation into a cytotoxic mediator (Vergote et al., 2006; Zhang et al., 2003). Based on the importance of the NH2 -terminus for anti-HIV activity,

several studies analyzed a number of NH2 -terminally modified chemokine variants like Met-RANTES or AOP-RANTES as antiviral agents (Zaitseva, Peden, & Golding, 2003). Low-molecular weight antagonists such as the AMD3100 variants and CXCR4 antagonists, however, seem to be more promising antiviral drugs. In conclusion, proteolytic processing represents an important regulatory mechanism during antiviral responses. 5. Conclusions Regulation of chemokine activity by proteolytic processing is very complex and numerous factors influence, regulate, and fine-tune the precise outcome of protease–chemokine interactions. Differences in the local concentrations of chemokines or proteases, processing kinetics, environmental conditions e.g., pH, co-factors or endogenous inhibitors and substrate availability influence the level of active chemokines. Furthermore, binding of chemokines to GAG or heterodimerization (Guan, Wang, & Norcross, 2001) may also affect their ability to exert their function. Eventually, the ratio of intact and antagonistically active cleaved forms of chemokines is decisive for regulating cell migration and the extent of inflammation caused by cellular infiltrates (Fig. 1). The impact of proteolytic processing of chemokines in the pathogenesis of inflammatory diseases and

Fig. 1. Modification of chemokine activity by proteolytic processing. Proteolytic processing of chemokines is influenced by numerous factors. Modified chemokines may have enhanced or decreased activity and the altered function can influence the outcome of the biological response.

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tumours is difficult to predict. In several diseases such as infections and tumours, chemokines can have beneficial effects by recruiting effector cells to the site of pathogen entry, however, an excess of leukocyte recruitment can also be harmful and may lead to chronic disorders. Furthermore, the redundancy of chemokines renders it more difficult to ascribe a role for a single chemokine to a specific pathological situation. Nevertheless, dysfunctions or alterations of chemokine activities may affect the immune response profoundly and failures in the regulation of chemokine–protease interactions may result in severe outcomes. Up to now, most data of protease–chemokine interactions are derived from in vitro experiments, but studies demonstrating the impact of truncated chemokines in vivo are also available. McQuibban et al. showed in different mouse models that injection of truncated CCL7 could reduce the infiltration of inflammatory cells (McQuibban et al., 2000). Proost et al. used a rabbit cornea micropocket model to investigate neovascularisation (Proost et al., 2001). Furthermore, the neurotoxic effect of the product of CXCL12 processing by MMP-2, CXCL12(5-67), was confirmed by injecting CXCL12(567) into the striatum of mice (Zhang et al., 2003). Although some effects of cleaved chemokines could be confirmed in various in vivo models, the question as to if and to what extent chemokine cleavage occurs in vivo and affects in vivo functions has not been solved so far. Due to the profound implications which might result from proteolytic processing of chemokines and in order to get further insights into the natural regulation of the chemokine system, it is of great importance to develop models where it is possible to analyze the interactions of chemokines and proteases under normal and pathological conditions. A better knowledge in this field could prove helpful and provide a basis for therapeutic interventions. Acknowledgements We thank Bernhard Moser and Deborah Stroka for critical reading of the manuscript. This work was supported by grant no. 03.0441-2 from the Staatssekretariat fuer Bildung und Forschung, and by the Bernische Krebsliga. References Addison, C. L., Daniel, T. O., Burdick, M. D., Liu, H., Ehlert, J. E., Xue, Y. Y., et al. (2000). The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity. J Immunol, 165(9), 5269–5277.

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