CD13/Aminopeptidase N Is a Potential Therapeutic Target for Inﬂammatory Disorders Chenyang Lu,*,† Mohammad A

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CD13/Aminopeptidase N Is a Potential Therapeutic Target for Inflammatory Disorders Chenyang Lu,*,† Mohammad A. Amin,* and David A. Fox* CD13/aminopeptidase N is a widely expressed ectoen- and processing of inflammatory mediators, which are im- zyme with multiple functions. As an enzyme, CD13 portant features of immune responses. In this study, we regulates activities of numerous cytokines by cleaving review evidence concerning the involvement of CD13, includ- their N-terminals and is involved in Ag processing by ing a soluble form of the molecule in inflammation and its trimming the peptides bound to MHC class II. Inde- potential as a therapeutic target in inflammatory disorders. pendent of its enzymatic activity, cell membrane CD13 functions by cross-linking–induced signal transduc- Mechanisms of CD13 functions tion, regulation of receptor recycling, enhancement of CD13 was named aminopeptidase N because of its prefer- FcgR-mediated phagocytosis, and acting as a receptor ence for binding neutral amino acids and because it removes for cytokines. Moreover, soluble CD13 has multiple N-terminal amino acids from unsubstituted oligopeptides, proinflammatory roles mediated by binding to G-pro- with the exception of peptides with proline in the penulti- tein–coupled receptors. CD13 not only modulates de- mate position (11). By cleaving N termini, CD13 regulates velopment and activities of immune-related cells, but activity of numerous hormones,cytokines,andchemokines also regulates functions of inflammatory mediators. that participate in inflammation. Moreover, CD13 is coex- Therefore, CD13 is important in the pathogenesis of pressed by MHC class II (MHC II)–bearing APCs (12), and is various inflammatory disorders. Inhibitors of CD13 involved in the trimming of MHC II–associated peptides on the have shown impressive anti-inflammatory effects, but surface of APCs (5, 13, 14) (Fig. 1). none of them has yet been used for clinical therapy of SomeCD13functionsintheimmunesystemareinde- human inflammatory diseases. We reevaluate CD13’s pendent of its enzymatic activity. These mechanisms include regulatory role in inflammation and suggest that CD13 cross-linking–induced signal transduction (15), enhancement of FcgR-mediated phagocytosis (16), acting as a receptor could be a potential therapeutic target for inflamma- (17, 18), and binding of soluble CD13 (sCD13) to G-protein– tory disorders. The Journal of Immunology, 2020, by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. coupled receptors (GPCRs) (19) (Fig. 1). 204: 3–11. Various cellular responses, including homotypic aggregation, cell–cell adhesion, and migration, have been observed D13, also known as aminopeptidase N or membrane after cross-linking CD13 with mAbs. Interestingly, CD13, alanyl aminopeptidase, is a type II membrane, 150- which includes a 7-aa cytoplasmic tail that was previously as- C kDa metalloprotease with an extracellular-oriented sumed to be inert, is itself phosphorylated in a Src-dependent catalytic domain. It is a seahorse-shaped molecule and manner (20). mAb cross-linking of CD13 induces clustering usually forms a head-to-head homodimer by means of and phosphorylation of tyrosine 6 in the cytoplasmic domain hydrophobic interactions (1, 2). Each monomeric molecule of CD13, activation of Src, FAK, ERK, and potentially other http://classic.jimmunol.org of CD13 possesses a seven-domain organization, which is components of the Ras/MAPK (JNK and p38) and PI3K characteristic of M1 metallopeptidases (1, 3). CD13 has pathways. Initiation of a Ca2+ flux by mAb against CD13 results been termed a “moonlighting enzyme” because of its in increased adhesion of monocytes (MNs) to endothelial cells multiple functions (4). Increasing evidence points to cru- (ECs) or to a monolayer of CD13 and upregulation of cyto- cial regulatory functions for CD13 during normal and kines including IL-8 (15, 20). Anti-CD13 mAbs also induce pathologic immune responses. CD13 has been suggested to integrin-independent homotypic aggregation of monocytic Downloaded from play a role in Ag processing (5, 6), cell trafficking (7–10) U937 cells independent of their effects on CD13’s enzymatic

*Division of Rheumatology, Clinical Autoimmunity Center of Excellence, University of Abbreviations used in this article: a.k.a, also known as; ARF6, adenosine diphosphate Michigan Medical School, Ann Arbor, MI 48109; and †Department of Rheumatology ribosylation factor 6; BK, bradykinin; DP IV, dipeptidyl peptidase IV; EAE, experimen- and Immunology, West China Hospital, Sichuan University, Chengdu 610041, tal autoimmune encephalomyelitis; EC, endothelial cell; FLS, ﬁbroblast-like synoviocyte; Sichuan, China GPCR, G-protein–coupled receptor; HCMV, human CMV; IBD, inﬂammatory bowel disease; ILD, interstitial lung disease; IL-1RA, IL-1R antagonist; IQGAP1, IQ motif Received for publication July 23, 2019. Accepted for publication October 1, 2019. containing GTPase activating protein 1; MHC II, MHC class II; MMP, matrix metal- This work was supported by a National Institute of Allergy and Infectious Diseases loproteinase; MN, monocyte; MS, multiple sclerosis; OA, osteoarthritis; RA, rheuma- Clinical Autoimmunity Center of Excellence Award. toid arthritis; sCD13, soluble CD13; Treg, regulatory T.

Address correspondence and reprint requests to Dr. David A. Fox, Department of Ó Internal Medicine/Division of Rheumatology, University of Michigan Medical School, Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 3918 Taubman Center, 1500 E. Medical Center Drive, SPC 5358, 7C27 North Ingalls Building, Ann Arbor, MI 48109-5422. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900868 4 BRIEF REVIEWS: CD13 IS A THERAPEUTIC TARGET FOR INFLAMMATORY DISORDERS

FIGURE 1. Mechanisms of CD13 functions. CD13 acts by enzyme-dependent mechanisms and enzyme-independent mechanisms. CD13 can cleave the N terminus of numerous cytokines, hormones, and chemokines and trim peptides that bind to MHC II. Ab cross-linking of CD13 induces clustering of CD13, tyrosine phosphorylation of CD13, and activation of Src kinase, FAK and ERK kinases, a calcium ﬂux, and potentially other components of the Ras/MAPK and PI3K pathways, resulting in increased adhesion of MNs to endothelial or monolayer CD13. CD13 also tethers the IQGAP1–ARF6–EFA6 complex on the plasma membrane to promote ARF6 activation, b1 integrin recycling, and cell migration. CD13 functionally interacts with FcgRs and enhances phagocytosis by increasing the level and duration of Syk phosphorylation. CD13 is a cell surface receptor for some cytokines, such as 14-3-3 proteins, signaling via p38 MAPK and JNK. CD13 can be shed from the cell membrane and sCD13 functions through engagement of GPCRs. ECM, extracellular matrix; ER, endoplasmic reticulum. by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd.

activity (7). Such CD13-induced cell–cell adhesion is also by increasing the level and duration of Syk phosphorylation dependent on the carbohydrate-binding protein galectin-3. A induced by FcgRs themselves (16). There is a possibility that possible mechanism could be that Ab cross-linking of CD13 CD13 positively modulates FcgR-mediated signals and triggers a signaling cascade leading to release of galectin-3, functions when CD13 and FcgR bind to their ligands, as which then binds to another receptor and triggers adhesion CD13 is a well-known receptor for viral proteins and other or, in oligomeric form, binds to glycoconjugates on neigh- pathogen-derived molecules. Moreover, CD13 was reported boring cells (21). CD13 can be constitutively phosphorylated to be a cell-surface receptor for some cytokines, including 14- http://classic.jimmunol.org at Tyr6 (10, 20) to interact with IQ motif containing GTPase 3-3e and 14-3-3s (also known as [a.k.a] stratifin) (17, 18). By activating protein 1 (IQGAP1), a scaffold protein that interacts binding to CD13 on cells such as fibroblasts and chon- with over 90 other proteins to mediate biological functions drocytes, 14-3-3e regulates production of matrix-degrading (22). CD13 serves to properly position IQGAP1 to perform enzymes such as matrix metalloproteinases (MMP)–3 and its functions; thus, it was expected that CD13 participates –13 through p38 MAPKs and JNK-signaling cascades, whereas in numerous functions mediated by IQGAP1, including cell 14-3-3s induces expression of MMP-1 through p38 MAPK Downloaded from motility, endocytosis, filopodia formation, angiogenesis, and activation (17, 18). Phosphorylation of the ectodomains of phagocytosis (10). A recent study demonstrated that CD13 can CD13 augments the binding affinity of 14-3-3 proteins to tether IQGAP1 with active adenosine diphosphate ribosylation CD13, regulating the downstream signaling cascade. It has factor 6 (ARF6) and b1 integrin at the plasma membrane to been proposed that enzymatic activity and binding to extra- allow b1 integrin recycling and cell migration (10). However, cellular matrix proteins through Asn–Gly–Arg motifs is me- mAbs binding to distinct epitopes of CD13 mediate dif- diated through the catalytic site involving domains IV–VII; ferent consequences for cell adhesion. For example, cross- cell–cell adhesion is mediated by the C-terminal part, in- linking CD13 by some mAbs inhibits cell adhesion, whereas volving half of domain VI and VII, whereas signal transduc- mAb 452 induces it (23). tion depends on the transmembrane and cytoplasmic domains CD13 colocalizes with FcgRs, including FcgRII/CD32 and (I and II) (8, 23). FcgRI/CD64 after Fc receptor ligation with specific Abs (24). CD13 can also be shed from the cell membrane by MMP-14 During FcgR-mediated phagocytosis, CD13 redistributes to (25), and the resultant sCD13 functions by engaging GPCRs the phagocytic cup and is simultaneously internalized into on cytokine-activated lymphocytes (19), MNs, ECs, and fi- phagosomes with FcgRs, rendering ingestion more efficient broblasts (26). However, the identity of such GPCRs has not The Journal of Immunology 5

2 2 yet been reported. With binding to GPCRs, sCD13 induces (8). In CD13 / mice, lack of monocytic CD13 ablates phosphorylation of Src, ERK1/2, and, to some extent, NF-kB, anti-CD13–dependent MN adhesion to ECs; yet, CD13 activating cell migration, angiogenesis, and other responses (26). appears to be dispensable for phagocytosis, proliferation, Pertussis toxin, a GPCR inhibitor, significantly reduced chemo- and Ag presentation of macrophages (36). Meanwhile, assessment tactic responses of cytokine-activated T cells (19) or monocytic of four inflammatory disease models (thioglycollate-induced cells as well as endothelial capillary morphogenesis in response to peritonitis, collagen Ab-induced arthritis, DSS-induced sCD13 (26). colitis, and croton oil-induced contact hypersensitivity) showed that lack of CD13 had little effect on disease onset CD13 modulates development and function of immune-related cells or progression (36). These models are, however, relatively CD13 is widely expressed. Within the hematopoietic system, independent of autoimmune T cell responses. CD13 is predominantly expressed on myelomonocytic lineage cells, and it has long been used as a lineage marker in the Dendritic cells characterization and typing of leukemia or lymphoma cells CD13 is involved in the development of dendritic cells from (27). However, CD13 is not just a cellular marker, but also CD34+ hematopoietic progenitor cells (37). CD13 is highly a molecule modulating development and function of immune- expressed on dendritic cells and participates in cell-surface Ag related cells (Table I). processing through trimming of MHC II– or MHC I–bound MNs/macrophages peptides on APCs (12, 13, 38). Furthermore, CD13 is highly and specifically expressed on the CD8+ splenic dendritic cells, CD13 has been implicated in MN/macrophage activation and which are responsible for cross-presentation (39). It negatively differentiation (28). It is expressed on the surface of blood regulates receptor-mediated, dynamin-dependent endocytosis MNs and is upregulated by IFN-g, LPSs, C5a, IL-4, and of Ags to control T cell activation in adaptive immunity (39). TGF-b but downregulated by IL-10 (28–31). There is also an CD13 restricts TLR4 endocytic signal transduction, balancing increase of CD13 expression during the differentiation from the innate response by maintaining the inflammatory equilibrium MNs to macrophages (28). Meanwhile, blocking CD13 by critical to innate immune regulation (40). However, develop- mAb or by binding of human CMV (HCMV) inhibits ment, maturation, Ag processing, and presentation of dendritic 2 2 macrophage differentiation (32). cells are normal in CD13 / mice (39). CD13 mediates MN activation either by cross-linking of cell membrane CD13 or by binding of sCD13 to a GPCR. Lymphocytes Cross-linking membrane CD13 by mAbs activates signaling Lymphocytes, including T cells and B cells, are important in pathways involving MAPK and PI3K and a Ca2+ flux, resulting inflammation and autoimmunity. However, the expression in homotypic aggregation, cytokine secretion, and increased and function of CD13 on lymphocytes remain controversial. adhesive capacity (7, 15, 20, 21). SCD13 binding to a GPCR Generally, it is accepted that CD13 is absent on resting increases phosphorylation of multiple downstream signaling lymphocytes but may be inducible by stimulation or contact

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. molecules and activates MNs (26). Moreover, CD13 interacts with some CD13-positive cells. CD13 expression is observed with FcgRs on the monocytic cell membrane and regulates on hematopoietic stem cells and at the earliest stages of B or FcgR-mediated phagocytosis (16, 33). CD13 also regulates T cell differentiation but is then lost upon maturation (41, 42). MN migration and contributes to inflammation. The ex- Consistent with this, some malignant B cells were CD13 pression of CD13 is enriched specifically on the proin- positive (43, 44). Human T cell lines HuT78 and H9 con- flammatory subset of MNs, and blocking CD13 by Ab or tained both Alanine-b-naphthylamide–hydrolyzing activity knockdown of CD13 significantly decreases MN infiltration, and CD13 mRNA; however, CD13 protein was undetectable suggesting that CD13 may regulate trafficking and function (45). Mature B and resting T cells lack CD13 expression of these cells (8). In addition, CD13 expression by ECs is also detectable by standard flow cytometry (46). In contrast, http://classic.jimmunol.org essential for MN infiltration (8, 34). CD13 is upregulated other studies suggested that CD13 mRNA is consistently on activated ECs (35) and mediates MN/EC adhesion by detected in resting T cells, and its surface expression is mark- homotypic interactions that involve the C-terminal domain edly upregulated in response to T cell activation (47–51). For

Table I. Regulation of immune-related cells and inﬂammatory mediators by CD13 Downloaded from

Subjects Major Functions of CD13 Immune cells MN/macrophage Differentiation, adhesion, migration, and activation Dendritic cell Regulates differentiation and Ag presentation and restricts TLR4 signal transduction Lymphocyte T cell proliferation, migration, and secretion of cytokines including IL-2, IL-17, IFN-g, TNF-a, TGF-b1, and IL-1RA Neutrophil TNF-a induced apoptosis; aggregation Mast cell Negative regulator of production of IL-6 and TNF-a Inflammation-related nonimmune cells EC Induces tube formation; angiogenesis; invasion Fibroblast Augments migration; proliferation; cytokine secretion Inflammatory mediators Kinins Both kallidin and its derivative Lys–des-Arg9–BK are substrates of CD13. CD13 also facilitates BK’s biologic functions by participating in membrane protein organization. Tuftsin CD13 can cleave tuftsin, generating an antagonist for receptor binding. Cytokines Truncates CXCL11, leading to a reduction of lymphocyte infiltration and a reduced inhibition of EC migration; cleaves synthetic oligopeptides corresponding to the N-terminal of human IL-1b, IL-2, TNF-b, and IL-6. Contradictory results were found for IL-8. 6 BRIEF REVIEWS: CD13 IS A THERAPEUTIC TARGET FOR INFLAMMATORY DISORDERS

instance, CD13 expression on T cells can be upregulated by actinonin or bestatin significantly enhanced TNF-a–induced Con A (50) or other mitogens such as PHA and PMA (49). apoptosis of neutrophils (67). A possible mechanism was that However, induction of CD13 in lymphocytes was not domi- CD13 inhibition interfered with shedding of TNFR1, resulting nantly dependent on de novo protein biosynthesis or glyco- in augmented TNF-a–induced apoptosis, cell polarization, and sylation, as inhibitors of protein biosynthesis and glycosylation respiratory burst (67). only partly prevented mitogen-induced CD13 expression (49). CD13+ T cells have also been found outside the peripheral Mast cells blood, for instance, in the synovial fluid of patients with ar- Evidence of CD13’s role in mast cells is limited. One study thritis (52), in the pericardial fluid of patients undergoing claimed that CD13 is a negative regulator of mast cell activa- cardiac surgery (53), or in tumor microenvironment (54). The tion, because Ag stimulation of CD13-deficient bone marrow– upregulation of CD13 in lymphocytes was mediated by ad- derived mast cells increased degranulation and proinflammatory hesion to CD13+ cells, including fibroblast-like synoviocytes cytokine production (68). This study also identified a functional (FLS), MNs/macrophages, and ECs (55), which was attributed interaction between FceRI and CD13 on mast cells as Ab- to increased CD13 promoter activity in lymphocytes through mediated cross-linking of CD13 caused IL-6 production cell–cell contact (56). However, the possibility that lymphocytes in an FceRI-dependent manner (68). However, the path- increase CD13 on the cell surface by trogocytosis from ophysiological or physical connection between CD13 cross- nonlymphoid cells has not been excluded. linking and FceRI activation remains uncertain. The CD13 inhibitors, actinonin and probestin, suppress inflammation through anti-proliferation and reduced production Endothelial cells of proinflammatory cytokines such as IL-1b and IL-2, accom- Angiogenesis is a critical step for tumor growth as well as panied by augmented release of anti-inflammatory cytokines metastasis and an integral component of the pathologic including TGF-b1 and IL-1R antagonist (IL-1RA) (46, 57). inflammatory response in arthritis. CD13 was exclusively Although actinonin has no effect on chemotaxis or unstimulated expressed by angiogenic or activated ECs, but not resting ECs cell migration, it selectively suppresses lymphocyte functions, (35, 69). Hypoxia, angiogenic growth factors, and signals including proliferation and production of IFN-g,IL-17,and mediating angiogenesis potently induce CD13 transcription TNF-a, and ameliorates autoimmunity in vivo (58). Addi- in primary ECs, whereas CD13 inhibitors interfered with tionally, CD13 inhibitors could preserve and promote immu- tube formation, but not EC proliferation, suggesting that + + nosuppressive functions of CD4 CD25 regulatory T (Treg) CD13 is a crucial regulator of endothelial morphogenesis + + cells. Inhibition of CD13 on CD4 CD25 Treg cells by during angiogenesis (35, 70). Otherwise, addition of ex- phebestin-enhanced expression of Foxp3 and TGF-b1andal- ogenous CD13 is sufficient to restore arrested endothelial leviated acute colitis in mice (46, 59). Simultaneous application migration and morphogenesis resulting from inhibition of 2 2 of dipeptidyl peptidase IV (DP IV; a.k.a DPP-4 or CD26) Ras/MAPK or PI3K signaling (71). In vivo, CD13 / and CD13 inhibitors significantly suppressed DNA synthesis mice showed a severely impaired angiogenic response un- by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. and increased TGF-b1 production in mitogen- or anti-CD3– der pathological conditions (72). One possible mechanism stimulated human T cells in vitro (60, 61). In response to is that CD13 mediates galectin-3–induced angiogenesis CD13 inhibitors, there is a marked increase of expression and through carbohydrate-recognition interactions (73). Alterna- activity of p42/ERK2 (62); meanwhile, a reduction of ex- tively, CD13, through its peptidase activity, can regulate pression and phosphorylation of glycogen synthase kinase–3b endothelial invasion and filopodia formation by facilitating (GSK-3b) occurs (63). However, another study showed that the bradykinin (BK)–BK receptor B2 (B2R) internalization and + + hi CD13 CD4 CD25 Treg subpopulation exhibits stronger sup- plasma membrane protein organization (74). Pharmacological + hi pressive function among CD4 CD25 Treg cells by expressing a inhibitors of CD13 and anti-CD13 mAbs blocked angio- http://classic.jimmunol.org higher level of Foxp3, CTLA-4, membrane-bound TGF-b1, and genesis and endothelial invasion (74). Moreover, recent work B7H1 (64). A CD13 cross-linking Ab, WM15, suppresses demonstrated that sCD13 can induce angiogenesis through expression of Foxp3, CTLA-4, B7H1, the secretion of GPCRs (26). TGF-b1 and IL-10 by CD13+CD4+CD25hi Treg cells, and their ability to suppress CD25 expression and proliferation of Fibroblasts + 2 CD4 CD25 T cells (64). Synovial fibroblasts (a.k.a FLS) reside in the synovial lining of Downloaded from joints and act as key contributors to arthritis (75). In rheu- Neutrophils matoid arthritis (RA), activated FLS are a major source of CD13 is present on neutrophils and can be upregulated by inflammatory cytokines and catabolic enzymes that promote proinflammatory agonists including fMLF, IL-8, and TNF- joint damage (75). CD13 was found to be highly expressed on a (65). Anti-CD13 mAbs, WM15 and MY7, impair IL-8– RA FLS (19, 76) and upregulated by proinflammatory cyto- mediated neutrophil migration in type I collagen gels and kines, including IL-17, IFN-g, and TNF-a (25). CD13 is induce significant homotypic aggregation of neutrophils, more abundant in synovial fluids of RA compared with os- which is dependent on CD13 cross-linking (65). Neutro- teoarthritis (OA) (19). Moreover, CD13 is present as both a phil apoptosis is critical to resolution of acute inflamma- soluble molecule and on extracellular vesicles in biological tion and prevention of granulocyte-mediated tissue injury fluids including plasma, synovial fluid, and FLS culture su- (66). TNF-a was found as a priming agonist to enhance pernatant (25). SCD13 arises from the shedding of mem- the rate of neutrophil apoptosis, whereas CD13 could regulate brane CD13 on the FLS surface by MMPs such as MMP-14 TNF-a–induced apoptosis in human neutrophils (67). Inhi- (25). CD13 is localized predominantly in caveolae or lipid bition of cell-surface CD13 using CD13 inhibitors such as rafts on RA FLS, which are hotspot regions for functional The Journal of Immunology 7

cell–cell interactions (77). Inhibition of CD13 by either in- The role of CD13 in the pathogenesis of inflammatory disorders hibitors of enzymatic activity or mAb resulted in decreased Rheumatoid arthritis. RA is a chronic inflammatory disease proliferation and diminished migration of RA FLS (25). characterized by persistent synovitis, systemic inflammation, Similarly, there was elevated expression of CD13 in dermal and autoantibodies, causing cartilage and bone damage as well fibroblasts at wound sites (78). Blocking CD13 with mAbs as disability (89). CD13 is strongly expressed in RA synovial (WM15, 3D8, and H300) reduced the migration activity of tissue (28, 90), specifically, it is highly expressed by RA FLS, dermal fibroblast in a dose-dependent manner without any synovial MNs, and T cells and is also present in synovial fluid antiproliferative or cytotoxic effect (78). Recent work has (19, 52, 76). In addition, CD13 is also found in serum and found that sCD13 increases expression of some proin- FLS culture supernatant, which contains sCD13 cleaved from flammatory cytokines, including IL-6 and MCP-1, in RA FLS by MMP-14 or secreted in extracellular vesicles (25). FLS (26). Moreover, sCD13 activated signaling pathways in However, measurement of sCD13 in RA sera did not show RA FLS through GPCRs and pertussis toxin blocked this higher levels than in normal sera (19). cDNA microarray effect (26). analysis showed that the gene encoding laeverin (an enzyme CD13 regulates functions of inflammatory mediators with sequence homology to CD13) was found to be the most significantly overexpressed gene in RA twins’ lymphoblastoid Biological activities of inflammatory mediators can change B cell lines compared with those from healthy twins (91). robustly following short deletions or mutations at the N CD13 facilitates cell trafficking and cytokine secretion of terminus. By cleaving N-terminal amino acids, CD13 can immune-related cells that play an important role in the patho- regulate the activity of numerous hormones, cytokines, and genesis of RA. SCD13 induced chemotaxis of lymphocytes, es- chemokines that participate in inflammation (Table I). Kinins pecially cytokine-activated T cells, a T cell population similar to are naturally occurring vasoactive peptide hormones, influ- that found in RA synovium (19, 76). This chemotactic activity encing inflammation, nociception, and cardiovascular ho- was mediated through GPCRs which can be inhibited by per- meostasis. The kinin family contains BK, kallidin (Lys–BK), 9 9 tussis toxin (19). Immunodepleting CD13 from RA synovial and their des-Arg derivatives des-Arg –BK and Lys–des-Arg – fluid removed a substantial fraction of the total cytokine-activated BK, respectively) (79), and both kallidin and its derivative 9 T cell (19) and MN (26) chemotactic activity from that fluid. Lys–des-Arg –BK are CD13 substrates. N-terminal Lys resi- Inhibitors of CD13 enzymatic activity and anti-CD13 Abs also due of Lys–des-Arg9–BK can be cleaved by CD13, yielding 9 9 decreasedgrowthandmigration of FLS (25). Neutralizing Abs the much less potent des-Arg –BK (80). Des-Arg –BK or BK against CD13 inhibited secretion of proinflammatory cytokines is not as susceptible to CD13 degradation, because the pep- such as IL-6, IL-8, and MCP-1 from FLS (92) and RA synovial tide bond preceding a proline is resistant to this enzyme tissue ex vivo (26). Remarkably, sCD13 also increased MN (27, 81). Nevertheless, CD13 controls EC invasion in re- migration, angiogenesis, and cytokine expression by RA sponse to BK by facilitating signal transduction at plasma FLS, which are key processes for joint inflammation (26).

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. membrane after BK binding, but prior to ligand–receptor Injection of sCD13 into mouse knees caused severe joint internalization; inhibition of CD13 enzymatic activity inflammation in 24 h while upregulating cytokines such as abrogates B2R internalization, leading to the attenuation IL-1b, IL-6, and MCP-1 and augmenting MN/macrophage of downstream events such as BK-induced activation of infiltration (26). Cdc42 and filopodia formation, thus affecting EC motility Psoriasis. Psoriasis is an immune-mediated disease manifesting (74). Interestingly, inhibitors of two BK receptors directly in skin or joints or both (93). The skin plaques are characterized inhibitedCD13aswell(82,83). by epidermal hyperplasia with hyperproliferation, epidermal Tuftsin, a natural tetrapeptide located in the Fc domain of differentiation impairment, angiogenesis, and a brisk infiltration the IgG H chain, can enhance phagocytosis, immune re-

http://classic.jimmunol.org of MNs/macrophages, dendritic cells, and activated T cells in the sponses, bactericidal, and antitumor activities. It is hydrolyzed dermis and the epidermis (94). CD13 has been identified as an by CD13, generating an antagonist that competes for receptor binding and regulates tuftsin functions (81). activation marker in immune cells (27, 57) and keratinocytes (95, CXCL11 selectively recruits immune cells at sites of in- 96). Moreover, it is overexpressed in psoriatic skin and skin flammation and inhibits angiogenesis upon binding to its fibroblasts compared with skin of healthy control (97). receptors CXCR3 and CXCR7 (84). In combination with DP Calcitonin gene–related peptide and IL-4 were implicated Downloaded from IV, CD13 rapidly processed CXCL11 to generate truncated in the regulation of CD13 expression and activity (97). forms, which may bind and desensitize CXCR3 as well as Inhibiting CD13 with actinonin, bestatin, or PAC-22 CXCR7, leading to a reduction of lymphocyte infiltration (a cytosol alanyl aminopeptidase–specific inhibitor) led and an increase of EC migration (85). In vitro, CD13 cleaves to dose-dependent suppression of DNA synthesis in synthetic oligopeptides corresponding to the N terminus of keratinocytes, correlating well with the simultaneous human IL-1b, IL-2, TNF-b (lymphotoxin), and murine IL-6, decrease in enzyme activity (96). In a mouse tail model although natural IL-1a, IL-1b, IL-2, and G-CSF are resistant of psoriasis, actinonin dose-dependently restored the stratum to CD13 (86). This suggests that proteolysis of cytokines by granulosum and ameliorated the impaired keratinocyte cellular exopeptidases may require one or more preceding differentiation (96). In clinical situations, epidermal CD13 steps. CD13 degraded IL-8 and inactivated its chemotactic was especially upregulated in psoriatic plaques, and successful activity (87), whereas othergroupsfoundthatIL-8was treatments were associated with a reduction of CD13 resistant to degradation by CD13 (85, 88). More studies are expression (98). needed to clarify knowledge about inflammatory mediators Multiple sclerosis. Multiple sclerosis (MS) is the most prevalent processed by CD13. chronic inflammatory disease of the CNS, and it is currently 8 BRIEF REVIEWS: CD13 IS A THERAPEUTIC TARGET FOR INFLAMMATORY DISORDERS

incurable (99). A complex immune process, involving T cells, Therefore, 14-3-3e–CD13 interaction could be a new thera- B cells, Abs, and cells of the innate immune system, such as peutic target in OA. bloodborne macrophages and microglia, is found in lesions of Others. Significantly higher aminopeptidase activity was MS (99). Increased expression of CD13 on lymphocytes and detected in bronchoalveolar lavage fluid from patients with PBMC of MS patients was found during acute exacerbation interstitial lung diseases (ILD) because of RA, polymyositis/ and chronic progression, compared with MS remission or dermatomyositis, systemic sclerosis, and Sjogren syndrome other neurologic diseases, suggesting that CD13 can be a than from normal volunteers or control patients who were cell activation and migration marker in MS (100–102). free of ILD, and increased aminopeptidase activity and Compared with use of a single ectopeptidase inhibitor, increased expression of CD13 were found in alveolar combined application of DP IV and CD13 inhibitors macrophages from patients with ILD (114). CD13 may increased suppression of DNA synthesis as well as increased also be a target for acne. DP IV and CD13 inhibitors TGF-b1 production in human PBMC and isolated T cells upregulate IL-1RA in sebocytes and keratinocyte cell line, (61). In experimental autoimmune encephalomyelitis (EAE), suppress Propionibacterium acnes–stimulated T cell proliferation an animal model of MS, combined DP IV and CD13 and IL-2 production, and enhance TGF-b1 expression (115). inhibition, dramatically alleviated clinical severity of EAE CD13+CD33+ B cells were more numerous in Behcet disease compared with the response to each of the inhibitors alone andsepsiscomparedwithhealthycontrolsandRAorsystemic (61). Therefore, a dual inhibitor of DP IV and CD13, lupus erythematosus patients (116), but the authors failed to PETIR-001, exhibits a therapeutic effect in EAE (103). furtherexplorefunctionsofthissubsetofBcells.Recently, Although inhibitors of DP IV and CD13 have no effect another research group identified a distinct CD13+CD33+ on migration of pathogenic effector T cells into target population of leukemic cells contributing to a proinflammatory tissues, they induce release of TGF-b by T cells at the site microenvironment that may be detrimental to long-term of inflammation and suppress lymphocyte proliferation and normal hematopoiesis within acute lymphoblastic leukemia production of IFN-g,IL-17,andTNF-a (58). The amount bone marrow (117). With the capacity to secrete many + + of IL-17 and the number of CD13 IL-17R myeloid cells in proinflammatory cytokines, such as IL-1a,IL-1b,TNF-a, peripheral blood were steadily decreased in MS patients and GM-CSF, the CD13+ B cells may contribute to autoimmune with effective treatment (104). diseases such as Behcet disease. Activated immune cells such Inflammatory bowel diseases. Inflammatory bowel diseases (IBD), as MNs are important factors in the systemic inflammatory comprising Crohn disease and ulcerative colitis, result from response syndrome following trauma or sepsis. Increased an inappropriate inflammatory response to intestinal mi- number of tissue factor+CD13+ microparticles were detected crobes (105). In a mouse model of colitis, simultaneously in peripheral blood from patients in the acute phase of trauma inhibiting DP IV and CD13 reduced colitis activity significantly and severe sepsis, which correlated significantly with severity, (106). Endogenous opioid peptides such as Met- and Leu- suggesting that tissue factor+CD13+ microparticles are important enkephalin have beneficial impact on the function of the in the pathogenesis of early systemic inflammatory response by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. digestive system and exert anti-inflammatory effects through syndrome following trauma or sepsis (118). A role for CD13 direct influence on immune cells. However, those peptides are has been suggested in systemic lupus erythematosus, but the rapidly degraded by endogenous enkephalinases, including data are still preliminary (114, 119–121). CD13 is also a neutral endopeptidase (NEP) and CD13. Blocking NEP receptor for a strain of human coronavirus. A broad spectrum and CD13 with Pal-KKQHNPR (an analogue of natural of variations in the CD13 domain were found critical for enkephalin) or sialorphin (a natural blocker) attenuated coronavirus binding (122). Some studies pointed out that colitis in mice (107, 108). single-site polymorphisms and alternative splicing of human It has been noticed that HCMV infection induces pro- CD13 are characteristic for acute myeloid leukemia, implying http://classic.jimmunol.org duction of CD13-specific autoantibodies, which may promote that CD13 could be a disease marker and target for therapy inflammation and tissue damage (109–111). Evidence suggests (123–125). However, to our knowledge, there are no data an association between HCMV infection and IBD, but the concerning CD13 polymorphisms in autoimmune diseases/ mechanism is unclear. However, cytotoxic CD13–specific inflammatory diseases. autoantibodies were identified in 66% of the sera obtained from HCMV-IgG–positive patients with ulcerative colitis and CD13 inhibitors Downloaded from in 58% of the sera obtained from HCMV-IgG–positive pa- Almost100CD13inhibitorshavebeen reported and tested (126, tients with Crohn disease, but not in control individuals 127). Mina-Osorio (4) divided them into seven categories: (112). These cytotoxic autoantibodies may interfere with cell natural inhibitors (betatin and amastatin), synthetic peptidomi- functions and could thereby contribute to chronic inflam- metic inhibitors, synthetic nonpeptide inhibitors, tumor-homing mation in patients with IBD. peptides Asn–Gly–Arg, cholesterol-lowering drugs (ezetimibe), Osteoarthritis. OA, once viewed as mechanical cartilage deg- rAbs (single-chain, fragment-variable Ab fragments), and mAbs radation, is now known to be a complex condition affecting the (WM15 and mAb 452). Most of the clinical studies with CD13 entire joint, in which matrix proteases, synovium, and systemic enzymatic inhibitors, such as bestatin and tosedostat, were per- inflammation have key roles (113). OA shares some features formed in cancers including leukemia, lymphomas, and carci- with RA, and CD13 has also been detected in OA synovial nomas of lung, bladder, esophagus, and stomach. Inhibition of tissue (19, 90). CD13 is expressed on articular chondrocytes, aminopeptidase yielded a higher efficacy in some cancer types and a proinflammatory cytokine 14-3-3e might directly bind (128). Several other compounds inhibiting CD13 enzymatic to CD13, which transmits a signal in chondrocytes to induce activity are under preclinical development, for instance, LYP, a a catabolic phenotype similar to that observed in OA (17). bestatin dimethylaminoethyl ester (129). Curcumin, a phenolic The Journal of Immunology 9

natural product, has been described as an irreversible inhibitor 11. Hooper, N. M. 1994. Families of zinc metalloproteases. FEBS Lett. 354: 1–6. 12. Hansen, A. S., O. Noreń, H. Sjo¨stro¨m, and O. Werdelin. 1993. A mouse ami- of CD13 and is currently being investigated for its effects on nopeptidase N is a marker for antigen-presenting cells and appears to be co- patients with cancer (130) as well as patients with inflammation expressed with major histocompatibility complex class II molecules. Eur. J. Immunol. 23: 2358–2364. (available from ClinicalTrials.gov: https://clinicaltrials.gov/ct2/ 13. Larsen, S. L., L. O. Pedersen, S. Buus, and A. Stryhn. 1996. T cell responses af- show/NCT04032132?term=Curcumin&draw=2&rank=14; fected by aminopeptidase N (CD13)-mediated trimming of major histocompati- https://clinicaltrials.gov/ct2/show/NCT03122613?term=Curcumin& bility complex class II-bound peptides. J. Exp. Med. 184: 183–189. 14. Dong, X., B. An, L. Salvucci Kierstead, W. J. Storkus, A. A. Amoscato, and draw=2&rank=12). However, there are no available data con- R. D. Salter. 2000. Modification of the amino terminus of a class II epitope confers cerning the clinical use of CD13 mAbs. To our knowledge, no resistance to degradation by CD13 on dendritic cells and enhances presentation to T cells. J. Immunol. 164: 129–135. clinical trial has been completed to test CD13 inhibitors in 15. Santos, A. N., J. Langner, M. Herrmann, and D. Riemann. 2000. Aminopeptidase inflammatory diseases, except one ongoing phase II trial on N/CD13 is directly linked to signal transduction pathways in monocytes. Cell. psoriasis studying IP10.C8, a combined DP IV and CD13 Immunol. 201: 22–32. 16. Mina-Osorio, P., and E. Ortega. 2005. Aminopeptidase N (CD13) functionally inhibitor. The possible reason for this could be the complexity interacts with FcgammaRs in human monocytes. J. Leukoc. Biol. 77: 1008–1017. and extensiveness of CD13 functions. CD13 is essential for 17. Nefla, M., L. Sudre, G. Denat, S. Priam, G. Andre-Leroux, F. Berenbaum, and C. Jacques. 2015. The pro-inflammatory cytokine 14-3-3e is a ligand of CD13 in proper trafficking of inflammatory cells, which is necessary to cartilage. J. Cell Sci. 128: 3250–3262. prime and sustain the reparative responses (9). Therefore, the 18. Ghaffari, A., Y. Li, R. T. Kilani, and A. Ghahary. 2010. 14-3-3 sigma associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1. role of CD13 as well as the potential use of CD13 inhibitors in J. Cell Sci. 123: 2996–3005. inflammatory diseases requires further investigation. 19. Morgan, R., J. Endres, N. Behbahani-Nejad, K. Phillips, J. H. Ruth, S. C. Friday, G. Edhayan, T. Lanigan, A. Urquhart, K. C. Chung, and D. A. Fox. 2015. Ex- pression and function of aminopeptidase N/CD13 produced by fibroblast-like Conclusions synoviocytes in rheumatoid arthritis: role of CD13 in chemotaxis of cytokine- In summary, CD13 functions through enzyme-dependent activated T cells independent of enzymatic activity. Arthritis Rheumatol. 67: 74–85. 20. Subramani, J., M. Ghosh, M. M. Rahman, L. A. Caromile, C. Gerber, K. Rezaul, mechanisms and enzyme-independent mechanisms. It not D. K. Han, and L. H. Shapiro. 2013. Tyrosine phosphorylation of CD13 regulates only can modulate development and activities of immune- inflammatory cell-cell adhesion and monocyte trafficking. J. Immunol. 191: 3905– related cells but also can regulate functions of inflammatory 3912. 21. Mina-Osorio, P., I. Soto-Cruz, and E. Ortega. 2007. A role for galectin-3 in mediators. Furthermore, CD13 plays an important role in CD13-mediated homotypic aggregation of monocytes. Biochem. Biophys. Res. various inflammatory disorders, including RA, psoriasis, MS, Commun. 353: 605–610. 22. White, C. D., H. H. Erdemir, and D. B. Sacks. 2012. IQGAP1 and its binding and IBD. Many inhibitors against CD13 have been found and proteins control diverse biological functions. Cell. Signal. 24: 826–834. tested; yet, none of them has been used for the clinical treatment 23. Garay-Canales, C. A., I. Licona-Limoń, and E. Ortega. 2018. Distinct epitopes on CD13 mediate opposite consequences for cell adhesion. Biomed. Res. Int. 2018: of human inflammatory disorders. The contribution of CD13 to 4093435. various diseases and its significance as a therapeutic target need 24. Riemann, D., A. Tcherkes, G. H. Hansen, J. Wulfaenger, T. Blosz, and further study. E. M. Danielsen. 2005. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc gamma receptors CD32 and CD64. Biochem. Biophys. Res. Commun. 331: 1408–1412. 25. Morgan, R. L., N. Behbahani-Nejad, J. Endres, M. A. Amin, N. J. Lepore, Y. Du, Acknowledgments A. Urquhart, K. C. Chung, and D. A. Fox. 2016. Localization, shedding, regu- We thank Jonatan Hervoso and Mikel Gurrea from the University of Michigan lation and function of aminopeptidase N/CD13 on fibroblast like synoviocytes. by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. for generous help in reviewing the manuscript. Special thanks are due to the PLoS One 11: e0162008. University of Michigan librarians for assistance with access to literature. 26. Du, Y., C. Lu, R. L. Morgan, W. A. Stinson, P. L. Campbell, E. Cealey, W. Fu, N. J. Lepore, J. L. Hervoso, H. Cui, et al. 2019. Angiogenic and arthritogenic properties of the soluble form of CD13. J. Immunol. 203: 360–369. 27. Riemann, D., A. Kehlen, and J. Langner. 1999. CD13--not just a marker in Disclosures leukemia typing. Immunol. Today 20: 83–88. The authors have no financial conflicts of interest. 28. Koch, A. E., J. C. Burrows, A. Skoutelis, R. Marder, P. H. Domer, B. Anderson, and S. J. Leibovich. 1991. Monoclonal antibodies detect monocyte/macrophage activation and differentiation antigens and identify functionally distinct subpop- References ulations of human rheumatoid synovial tissue macrophages. Am. J. Pathol. 138: 1. Wong, A. H., D. Zhou, and J. M. Rini. 2012. The X-ray crystal structure of 165–173. 29. Kehlen, A., M. Geisler, J. Olsen, A. Sablotzki, J. Langner, and D. Riemann. 2004.

http://classic.jimmunol.org human aminopeptidase N reveals a novel dimer and the basis for peptide processing. J. Biol. Chem. 287: 36804–36813. IL-10 and TGF-beta differ in their regulation of aminopeptidase N/CD13 ex- 2. Chen, L., Y. L. Lin, G. Peng, and F. Li. 2012. Structural basis for multifunctional pression in monocytes. Int. J. Mol. Med. 13: 877–882. roles of mammalian aminopeptidase N. Proc. Natl. Acad. Sci. USA 109: 17966– 30. van Hal, P. T., J. P. Hopstaken-Broos, A. Prins, E. J. Favaloro, R. J. Huijbens, 17971. C. Hilvering, C. G. Figdor, and H. C. Hoogsteden. 1994. Potential indirect anti- 3. Sjo¨stro¨m, H., O. Noreń, and J. Olsen. 2000. Structure and function of amino- inflammatory effects of IL-4. Stimulation of human monocytes, macrophages, and peptidase N. Adv. Exp. Med. Biol. 477: 25–34. endothelial cells by IL-4 increases aminopeptidase-N activity (CD13; EC 4. Mina-Osorio, P. 2008. The moonlighting enzyme CD13: old and new functions 3.4.11.2). J. Immunol. 153: 2718–2728. to target. Trends Mol. Med. 14: 361–371. 31. Werfel, T., G. Sonntag, M. H. Weber, and O. Go¨tze. 1991. Rapid increases in the Downloaded from 5. Mouritsen, S., M. Meldal, O. Werdelin, A. S. Hansen, and S. Buus. 1992. MHC membrane expression of neutral endopeptidase (CD10), aminopeptidase N molecules protect T cell epitopes against proteolytic destruction. J. Immunol. 149: (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stim- 1987–1993. ulation of human peripheral leukocytes with human C5a. J. Immunol. 147: 3909– 6. Falk, K., O. Ro¨tzschke, S. Stevanovic´, G. Jung, and H. G. Rammensee. 1994. Pool 3914. sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide 32. Gredmark, S., W. B. Britt, X. Xie, L. Lindbom, and C. So¨derberg-Naucle´r. 2004. motifs, constraints of processing, and general rules. Immunogenetics 39: 230–242. Human cytomegalovirus induces inhibition of macrophage differentiation by 7. Mina-Osorio, P., L. H. Shapiro, and E. Ortega. 2006. CD13 in cell adhesion: binding to human aminopeptidase N/CD13. J. Immunol. 173: 4897–4907. aminopeptidase N (CD13) mediates homotypic aggregation of monocytic cells. J. 33. MacIntyre, E. A., P. J. Roberts, M. Jones, C. E. Van der Schoot, E. J. Favalaro, Leukoc. Biol. 79: 719–730. N. Tidman, and D. C. Linch. 1989. Activation of human monocytes occurs on 8. Ghosh, M., C. Gerber, M. M. Rahman, K. M. Vernier, F. E. Pereira, cross-linking monocytic antigens to an Fc receptor. J. Immunol. 142: 2377–2383. J. Subramani, L. A. Caromile, and L. H. Shapiro. 2014. Molecular mechanisms 34. Mina-Osorio, P., B. Winnicka, C. O’Conor, C. L. Grant, L. K. Vogel, regulating CD13-mediated adhesion. Immunology 142: 636–647. D. Rodriguez-Pinto, K. V. Holmes, E. Ortega, and L. H. Shapiro. 2008. CD13 is a 9. Pereira, F. E., C. Cronin, M. Ghosh, S. Y. Zhou, M. Agosto, J. Subramani, novel mediator of monocytic/endothelial cell adhesion. J. Leukoc. Biol. 84: 448–459. R. Wang, J. B. Shen, W. Schacke, B. Liang, et al. 2013. CD13 is essential for 35. Bhagwat, S. V., J. Lahdenranta, R. Giordano, W. Arap, R. Pasqualini, and inflammatory trafficking and infarct healing following permanent coronary artery L. H. Shapiro. 2001. CD13/APN is activated by angiogenic signals and is essential occlusion in mice. Cardiovasc. Res. 100: 74–83. for capillary tube formation. Blood 97: 652–659. 10. Ghosh, M., R. Lo, I. Ivic, B. Aguilera, V. Qendro, C. Devarakonda, and 36. Winnicka, B., C. O’Conor, W. Schacke, K. Vernier, C. L. Grant, F. H. Fenteany, L. H. Shapiro. 2019. CD13 tethers the IQGAP1-ARF6-EFA6 complex to the F. E. Pereira, B. Liang, A. Kaur, R. Zhao, et al. 2010. CD13 is dispensable for plasma membrane to promote ARF6 activation, b1 integrin recycling, and cell normal hematopoiesis and myeloid cell functions in the mouse. J. Leukoc. Biol. 88: migration. Sci. Signal. 12: eaav5938. 347–359. 10 BRIEF REVIEWS: CD13 IS A THERAPEUTIC TARGET FOR INFLAMMATORY DISORDERS

37. Rosenzwajg, M., L. Tailleux, and J. C. Gluckman. 2000. CD13/N- 63. Lendeckel, U., B. Scholz, M. Arndt, K. Frank, A. Spiess, H. Chen, B. P. Roques, aminopeptidase is involved in the development of dendritic cells and macro- and S. Ansorge. 2000. Inhibition of alanyl-aminopeptidase suppresses the phages from cord blood CD34(+) cells. Blood 95: 453–460. activation-dependent induction of glycogen synthase kinase-3beta (GSK-3beta) in 38. Amoscato, A. A., D. A. Prenovitz, and M. T. Lotze. 1998. Rapid extracellular human T cells. Biochem. Biophys. Res. Commun. 273: 62–65. degradation of synthetic class I peptides by human dendritic cells. J. Immunol. 161: 64. Ju, S., H. Qiu, X. Zhou, B. Zhu, X. Lv, X. Huang, J. Li, Y. Zhang, L. Liu, Y. Ge, 4023–4032. et al. 2009. CD13+CD4+CD25hi regulatory T cells exhibit higher suppressive 39. Ghosh, M., B. McAuliffe, J. Subramani, S. Basu, and L. H. Shapiro. 2012. CD13 function and increase with tumor stage in non-small cell lung cancer patients. Cell regulates dendritic cell cross-presentation and T cell responses by inhibiting Cycle 8: 2578–2585. receptor-mediated antigen uptake. J. Immunol. 188: 5489–5499. 65. Fiddler, C. A., H. Parfrey, A. S. Cowburn, D. Luo, G. B. Nash, G. Murphy, and 40. Ghosh, M., J. Subramani, M. M. Rahman, and L. H. Shapiro. 2015. CD13 re- E. R. Chilvers. 2016. The aminopeptidase CD13 induces homotypic aggregation stricts TLR4 endocytic signal transduction in inflammation. J. Immunol. 194: in neutrophils and impairs collagen invasion. PLoS One 11: e0160108. 4466–4476. 66. Fox, S., A. E. Leitch, R. Duffin, C. Haslett, and A. G. Rossi. 2010. Neutrophil 41. Spits, H., L. L. Lanier, and J. H. Phillips. 1995. Development of human T and apoptosis: relevance to the innate immune response and inflammatory disease. J. natural killer cells. Blood 85: 2654–2670. Innate Immun. 2: 216–227. 42. Syrja¨la¨, M., T. Ruutu, and S. E. Jansson. 1994. A flow cytometric assay of CD34- 67. Cowburn, A. S., A. Sobolewski, B. J. Reed, J. Deighton, J. Murray, positive cell populations in the bone marrow. Br. J. Haematol. 88: 679–684. K. A. Cadwallader, J. R. Bradley, and E. R. Chilvers. 2006. Aminopeptidase N 43. Pinto, A., L. Del Vecchio, A. Carbone, M. Roncadin, R. Volpe, D. Serraino, (CD13) regulates tumor necrosis factor-alpha-induced apoptosis in human neu- S. Monfardini, A. Colombatti, and V. Zagonel. 1991. Expression of myelomo- trophils. J. Biol. Chem. 281: 12458–12467. nocytic antigens is associated with unfavourable clinicoprognostic factors in B-cell 68. Zotz, J. S., F. Wo¨lbing, C. Lassnig, M. Kauffmann, U. Schulte, A. Kolb, chronic lymphocytic leukaemia. Ann. Oncol. 2(Suppl. 2): 107–113. B. Whitelaw, M. Muller,€ T. Biedermann, and M. Huber. 2016. CD13/ 44. Drexler, H. G., E. Thiel, and W. D. Ludwig. 1991. Review of the incidence and aminopeptidase N is a negative regulator of mast cell activation. FASEB J. 30: clinical relevance of myeloid antigen-positive acute lymphoblastic leukemia. Leu- 2225–2235. kemia 5: 637–645. 69. Fukasawa, K., H. Fujii, Y. Saitoh, K. Koizumi, Y. Aozuka, K. Sekine, M. Yamada, 45. Lendeckel, U., T. Wex, T. Ka¨hne, K. Frank, D. Reinhold, and S. Ansorge. 1994. I. Saiki, and K. Nishikawa. 2006. Aminopeptidase N (APN/CD13) is selectively Expression of the aminopeptidase N (CD13) gene in the human T cell lines expressed in vascular endothelial cells and plays multiple roles in angiogenesis. HuT78 and H9. Cell. Immunol. 153: 214–226. Cancer Lett. 243: 135–143. 46. Lendeckel, U., A. Bukowska, J. La¨ttig, and W. Brandt. 2004. Alanyl- 70. Shim, J. S., J. H. Kim, H. Y. Cho, Y. N. Yum, S. H. Kim, H.-J. Park, B. S. Shim, aminopeptidases in Human T Cells. In Aminopeptidases in Biology and Disease. S. H. Choi, and H. J. Kwon. 2003. Irreversible inhibition of CD13/ Proteases in Biology and Disease, Vol 2. N. M. Hooper, and U. Lendeckel, eds. aminopeptidase N by the antiangiogenic agent curcumin. Chem. Biol. 10: 695– Springer, Boston, MA, p. 201–227. 704. 47. Lendeckel, U., T. Wex, D. Reinhold, T. Ka¨hne, K. Frank, J. Faust, K. Neubert, 71. Bhagwat, S. V., N. Petrovic, Y. Okamoto, and L. H. Shapiro. 2003. The angio- and S. Ansorge. 1996. Induction of the membrane alanyl aminopeptidase gene and genic regulator CD13/APN is a transcriptional target of Ras signaling pathways in surface expression in human T-cells by mitogenic activation. Biochem. J. 319: 817– endothelial morphogenesis. Blood 101: 1818–1826. 821. 72. Rangel, R., Y. Sun, L. Guzman-Rojas, M. G. Ozawa, J. Sun, R. J. Giordano, 48. Lendeckel, U., T. Wex, D. Reinhold, M. Arndt, A. Ittenson, K. Frank, and C. S. Van Pelt, P. T. Tinkey, R. R. Behringer, R. L. Sidman, et al. 2007. Impaired S. Ansorge. 1997. Activation-dependent induction of T cell alanyl aminopeptidase angiogenesis in aminopeptidase N-null mice. Proc. Natl. Acad. Sci. USA 104: and its possible involvement in T cell growth. Adv. Exp. Med. Biol. 421: 59–66. 4588–4593. 49. Lendeckel, U., T. Wex, A. Ittenson, M. Arndt, K. Frank, O. Mayboroda, 73. Yang, E., J. S. Shim, H. J. Woo, K. W. Kim, and H. J. Kwon. 2007. Amino- W. Schubert, and S. Ansorge. 1997. Rapid mitogen-induced aminopeptidase N peptidase N/CD13 induces angiogenesis through interaction with a pro-angiogenic surface expression in human T cells is dominated by mechanisms independent of protein, galectin-3. Biochem. Biophys. Res. Commun. 363: 336–341. de novo protein biosynthesis. Immunobiology 197: 55–69. 74. Petrovic, N., W. Schacke, J. R. Gahagan, C. A. O’Conor, B. Winnicka, 50. Kunz, D., F. Buhling,€ H. J. Hutter,€ T. Aoyagi, and S. Ansorge. 1993. Amino- R. E. Conway, P. Mina-Osorio, and L. H. Shapiro. 2007. CD13/APN regulates peptidase N (CD13, EC 3.3.4.11.2) occurs on the surface of resting and conca- endothelial invasion and filopodia formation. Blood 110: 142–150. navalin A-stimulated lymphocytes. Biol. Chem. Hoppe Seyler 374: 291–296. 75. Neumann, E., S. Lefe`vre, B. Zimmermann, S. Gay, and U. Muller-Ladner.€ 2010. 51. Bukowska, A., J. Tadje, M. Arndt, C. Wolke, T. Ka¨hne, J. Bartsch, J. Faust, Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends K. Neubert, Y. Hashimoto, and U. Lendeckel. 2003. Transcriptional regulation of Mol. Med. 16: 458–468. cytosol and membrane alanyl-aminopeptidase in human T cell subsets. Biol. Chem. 76. Shimizu, T., K. Tani, K. Hase, H. Ogawa, L. Huang, F. Shinomiya, and S. Sone.

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. 384: 657–665. 2002. CD13/aminopeptidase N-induced lymphocyte involvement in inflamed 52. Riemann, D., A. Schwachula, M. Hentschel, and J. Langner. 1993. Demonstration joints of patients with rheumatoid arthritis. Arthritis Rheum. 46: 2330–2338. of CD13/aminopeptidase N on synovial fluid T cells from patients with different 77. Riemann, D., G. H. Hansen, L. Niels-Christiansen, E. Thorsen, L. Immerdal, forms of joint effusions. Immunobiology 187: 24–35. A. N. Santos, A. Kehlen, J. Langner, and E. M. Danielsen. 2001. Caveolae/lipid 53. Riemann, D., H. G. Wollert, J. Menschikowski, S. Mittenzwei, and J. Langner. rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains. 1994. Immunophenotype of lymphocytes in pericardial fluid from patients with Biochem. J. 354: 47–55. different forms of heart disease. Int. Arch. Allergy Immunol. 104: 48–56. 78. Lai, A., A. Ghaffari, and A. Ghahary. 2010. Inhibitory effect of anti- 54. Riemann, D., B. Go¨hring, and J. Langner. 1994. Expression of aminopeptidase N/ aminopeptidase N/CD13 antibodies on fibroblast migration. Mol. Cell. Biochem. CD13 in tumour-infiltrating lymphocytes from human renal cell carcinoma. 343: 191–199. Immunol. Lett. 42: 19–23. 79. Leeb-Lundberg, L. M., F. Marceau, W. Muller-Esterl,€ D. J. Pettibone, and 55. Riemann, D., A. Kehlen, K. Thiele, M. Lo¨hn, and J. Langner. 1997. Induction of B. L. Zuraw. 2005. International union of pharmacology. XLV. Classification of aminopeptidase N/CD13 on human lymphocytes after adhesion to fibroblast-like the kinin receptor family: from molecular mechanisms to pathophysiological

http://classic.jimmunol.org synoviocytes, endothelial cells, epithelial cells, and monocytes/macrophages. J. consequences. Pharmacol. Rev. 57: 27–77. Immunol. 158: 3425–3432. 80. Fortin, J. P., L. Gera, J. Bouthillier, J. M. Stewart, A. Adam, and F. Marceau. 56. Kehlen, A., J. Olsen, J. Langner, and D. Riemann. 2000. Increased lymphocytic 2005. Endogenous aminopeptidase N decreases the potency of peptide agonists aminopeptidase N/CD13 promoter activity after cell-cell contact. J. Cell. Biochem. and antagonists of the kinin B1 receptors in the rabbit aorta. J. Pharmacol. Exp. 80: 115–123. Ther. 314: 1169–1176. 57. Lendeckel, U., M. Arndt, K. Frank, T. Wex, and S. Ansorge. 1999. Role of alanyl 81. Xu, Y., D. Wellner, and D. A. Scheinberg. 1995. Substance P and bradykinin are aminopeptidase in growth and function of human T cells (review). Int. J. Mol. natural inhibitors of CD13/aminopeptidase N. Biochem. Biophys. Res. Commun. Med. 4: 17–27. 208: 664–674. 58. Biton, A., S. Ansorge, U. Bank, M. Ta¨ger, D. Reinhold, and S. Brocke. 2011. 82. Bawolak, M. T., J. P. Fortin, L. K. Vogel, A. Adam, and F. Marceau. 2006. The Downloaded from Divergent actions by inhibitors of DP IV and APN family enzymes on CD4+ Teff bradykinin B2 receptor antagonist icatibant (Hoe 140) blocks aminopeptidase N at cell motility and functions. Immunobiology 216: 1295–1301. micromolar concentrations: off-target alterations of signaling mediated by the 59. Bank, U., J. Tadje, M. Ta¨ger, C. Wolke, A. Bukowska, A. Ittenson, D. Reinhold, bradykinin B1 and angiotensin receptors. Eur. J. Pharmacol. 551: 108–111. M. Helmuth, S. Ansorge, A. Shakespeare, et al. 2007. Inhibition of alanyl- 83. Gera, L., J. P. Fortin, A. Adam, J. M. Stewart, and F. Marceau. 2006. Discovery of aminopeptidase on CD4+CD25+ regulatory T-cells enhances expression of a dual-function peptide that combines aminopeptidase N inhibition and kinin B1 FoxP3 and TGF-beta1 and ameliorates acute colitis in mice. Int. J. Mol. Med. 20: receptor antagonism. J. Pharmacol. Exp. Ther. 317: 300–308. 483–492. 84. Lacotte, S., S. Brun, S. Muller, and H. Dumortier. 2009. CXCR3, inflammation, 60. Reinhold, D., A. Biton, A. Goihl, S. Pieper, U. Lendeckel, J. Faust, K. Neubert, and autoimmune diseases. Ann. N. Y. Acad. Sci. 1173: 310–317. U. Bank, M. Ta¨ger, S. Ansorge, and S. Brocke. 2007. Dual inhibition of dipeptidyl 85. Proost, P., A. Mortier, T. Loos, J. Vandercappellen, M. Gouwy, I. Ronsse, peptidase IV and aminopeptidase N suppresses inflammatory immune responses. E. Schutyser, W. Put, M. Parmentier, S. Struyf, and J. Van Damme. 2007. Pro- Ann. N. Y. Acad. Sci. 1110: 402–409. teolytic processing of CXCL11 by CD13/aminopeptidase N impairs CXCR3 and 61. Reinhold, D., A. Biton, S. Pieper, U. Lendeckel, J. Faust, K. Neubert, U. Bank, CXCR7 binding and signaling and reduces lymphocyte and endothelial cell mi- M. Ta¨ger, S. Ansorge, and S. Brocke. 2006. Dipeptidyl peptidase IV (DP IV, gration. Blood 110: 37–44. CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and 86. Hoffmann, T., J. Faust, K. Neubert, and S. Ansorge. 1993. Dipeptidyl peptidase targets of immunotherapy in CNS inflammation. Int. Immunopharmacol. 6: 1935– IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrolysis of cytokines and 1942. peptides with N-terminal cytokine sequences. FEBS Lett. 336: 61–64. 62. Lendeckel, U., T. Ka¨hne, M. Arndt, K. Frank, and S. Ansorge. 1998. Inhibition of 87. Kanayama, N., Y. Kajiwara, J. Goto, E. el Maradny, K. Maehara, K. Andou, and alanyl aminopeptidase induces MAP-kinase p42/ERK2 in the human T cell line T. Terao. 1995. Inactivation of interleukin-8 by aminopeptidase N (CD13). J. KARPAS-299. Biochem. Biophys. Res. Commun. 252: 5–9. Leukoc. Biol. 57: 129–134. The Journal of Immunology 11

88. Kehlen, A., I. Egbert, K. Thiele, K. Fischer, D. Riemann, and J. Langner. 2001. 109. Soderberg, C., S. Sumitran-Karuppan, P. Ljungman, and E. Moller. 1996. CD13- Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: specific autoimmunity in cytomegalovirus-infected immunocompromised patients. interleukin-8 is resistant to degradation by aminopeptidase N/CD13. Eur. Cyto- Transplantation 61: 594–600. kine Netw. 12: 316–324. 110. Soderberg, C., S. Larsson, B. L. Rozell, S. Sumitran-Karuppan, P. Ljungman, and 89. Smolen, J. S., D. Aletaha, and I. B. McInnes. 2016. Rheumatoid arthritis. Lancet E. Moller. 1996. Cytomegalovirus-induced CD13-specific autoimmunity--a pos- 388: 2023–2038. sible cause of chronic graft-vs-host disease. Transplantation 61: 600–609. 90. Haringman, J. J., T. J. Smeets, P. Reinders-Blankert, and P. P. Tak. 2006. Che- 111. Kasman, L. M. 2005. CD13/aminopeptidase N and murine cytomegalovirus in- mokine and chemokine receptor expression in paired peripheral blood mononu- fection. Virology 334: 1–9. clear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, 112. Rahbar, A., L. Bostro¨m, and C. So¨derberg-Naucler. 2006. Detection of cytotoxic and reactive arthritis. Ann. Rheum. Dis. 65: 294–300. CD13-specific autoantibodies in sera from patients with ulcerative colitis and 91. Haas, C. S., C. J. Creighton, X. Pi, I. Maine, A. E. Koch, G. K. Haines, S. Ling, Crohn’s disease. J. Autoimmun. 26: 155–164. A. M. Chinnaiyan, and J. Holoshitz. 2006. Identification of genes modulated in 113. Glyn-Jones, S., A. J. Palmer, R. Agricola, A. J. Price, T. L. Vincent, H. Weinans, rheumatoid arthritis using complementary DNA microarray analysis of lympho- and A. J. Carr. 2015. Osteoarthritis. Lancet 386: 376–387. blastoid B cell lines from disease-discordant monozygotic twins. Arthritis Rheum. 114. Dan, H., K. Tani, K. Hase, T. Shimizu, H. Tamiya, Y. Biraa, L. Huang, 54: 2047–2060. H. Yanagawa, and S. Sone. 2003. CD13/aminopeptidase N in collagen vascular 92. Chomarat, P., M. C. Rissoan, J. J. Pin, J. Banchereau, and P. Miossec. 1995. diseases. Rheumatol. Int. 23: 271–276. Contribution of IL-1, CD14, and CD13 in the increased IL-6 production induced 115. Thielitz, A., D. Reinhold, R. Vetter, U. Bank, M. Helmuth, R. Hartig, by in vitro monocyte-synoviocyte interactions. J. Immunol. 155: 3645–3652. S. Wrenger, I. Wiswedel, U. Lendeckel, T. Ka¨hne, et al. 2007. Inhibitors of 93. Boehncke, W. H., and M. P. Scho¨n. 2015. Psoriasis. Lancet 386: 983–994. dipeptidyl peptidase IV and aminopeptidase N target major pathogenetic steps in 94. Perera, G. K., P. Di Meglio, and F. O. Nestle. 2012. Psoriasis. Annu. Rev. Pathol. acne initiation. J. Invest. Dermatol. 127: 1042–1051. 7: 385–422. 116. Eksxioglu-Demiralp, E., A. Kibaroglu, H. Direskeneli, S. Yavuz, F. Karsli, 95. Hunyadi, J., M. Simon, Jr., A. S. Kenderessy, and A. Dobozy. 1993. Expression of S. Yurdakul, H. Yazici, and T. Akoglu. 1999. Phenotypic characteristics of B cells monocyte/macrophage markers (CD13, CD14, CD68) on human keratinocytes in in Behçet’s disease: increased activity in B cell subsets. J. Rheumatol. 26: 826–832. healthy and diseased skin. J. Dermatol. 20: 341–345. 117. Vilchis-Ordon˜ez, A., A. Contreras-Quiroz, E. Vadillo, E. Dorantes-Acosta, 96. Thielitz, A., A. Bukowska, C. Wolke, R. Vetter, U. Lendeckel, S. Wrenger, A. Reyes-Lo´pez, H. M. Quintela-Nun˜ez del Prado, J. Venegas-Va´zquez, Y. Hashimoto, S. Ansorge, H. Gollnick, and D. Reinhold. 2004. Identification of H. Mayani, V. Ortiz-Navarrete, B. Lo´pez-Martıńez, and R. Pelayo. 2015. Bone extra- and intracellular alanyl aminopeptidases as new targets to modulate kerati- marrow cells in acute lymphoblastic leukemia create a proinflammatory micro- nocyte growth and differentiation. Biochem. Biophys. Res. Commun. 321: 795–801. environment influencing normal hematopoietic differentiation fates. Biomed. Res. 97. Gerbaud, P., J. Guibourdenche, R. Jarray, M. Conti, P. Palmic, S. Leclerc- Int. 2015: 386165. Mercier, J. Bruneau, O. Hermine, Y. Lepelletier, and F. Raynaud. 2018. APN/ 118. Matsumoto, H., K. Yamakawa, H. Ogura, T. Koh, N. Matsumoto, and CD13 is over-expressed by Psoriatic fibroblasts and is modulated by CGRP and T. Shimazu. 2017. Clinical significance of tissue factor and CD13 double-positive IL-4 but not by retinoic acid treatment. J. Cell. Physiol. 233: 958–967. microparticles in Sirs patients with trauma and severe sepsis. Shock 47: 409–415. 98. Bonnekoh, B., A. J. Pommer, R. Bo¨ckelmann, H. Hofmeister, L. Philipsen, and 119. Tsokos, G. C. 2011. Systemic lupus erythematosus. N. Engl. J. Med. 365: 2110– H. Gollnick. 2007. Topo-proteomic in situ analysis of psoriatic plaque under 2121. efalizumab treatment. Skin Pharmacol. Physiol. 20: 237–252. 120. Mitic, B., G. Lazarevic, P. Vlahovic, M. Rajic, and V. Stefanovic. 2008. Diagnostic 99. Reich, D. S., C. F. Lucchinetti, and P. A. Calabresi. 2018. Multiple sclerosis. N. value of the aminopeptidase N, N-acetyl-beta-D-glucosaminidase and dipepti- Engl. J. Med. 378: 169–180. dylpeptidase IV in evaluating tubular dysfunction in patients with glomer- 100. Ziaber, J., H. Tcho´rzewski, H. Chmielewski, Z. Baj, and J. Paśnik. 1999. [In- ulopathies. Ren. Fail. 30: 896–903. creased expression of aminopeptidase N on lymphocytes in multiple sclerosis]. 121. Behzadi, M., A. Ahmadzadeh, M. Valizadeh, M. Haji Molla Hoseini, and Neurol. Neurochir. Pol. 33: 1025–1032. F. Yeganeh. 2017. CD13/aminopeptidase N mRNA expression and enzyme ac- 101. Ziaber, J., Z. Baj, J. Paśnik, H. Chmielewski, and H. Tcho´rzewski. 2000. In- tivity in Systemic Lupus Erythematosus. Acta Reumatol. Port. 42: 162–167. creased expression of neutral endopeptidase (NEP) and aminopeptidase N (APN) 122. Vijgen, L., E. Keyaerts, K. Zlateva, and M. Van Ranst. 2004. Identification of six on peripheral blood mononuclear cells in patients with multiple sclerosis. Immu- new polymorphisms in the human coronavirus 229E receptor gene (aminopepti- nol. Lett. 71: 127–129. dase N/CD13). Int. J. Infect. Dis. 8: 217–222. 102. Ziaber, J., Z. Baj, J. Paśnik, H. Chmielewski, and H. Tcho´rzewski. 2000. Ex- 123. Alfalah, M., M. P. Krahn, G. Wetzel, S. von Ho¨rsten, C. Wolke, N. Hooper, pression of aminopeptidase N (APN) on peripheral blood mononuclear cells’ T. Kalinski, S. Krueger, H. Y. Naim, and U. Lendeckel. 2006. A mutation in surface as a marker of these cells’ transendothelial migration properties in the aminopeptidase N (CD13) isolated from a patient suffering from leukemia leads to

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. course of multiple sclerosis. Mediators Inflamm. 9: 45–48. an arrest in the endoplasmic reticulum. J. Biol. Chem. 281: 11894–11900. 103. Reinhold, D., U. Bank, D. Entz, A. Goihl, D. Stoye, S. Wrenger, S. Brocke, 124. Dybkaer, K., J. S. Kristensen, and F. S. Pedersen. 2001. Single site polymorphisms A. Thielitz, S. Stefin, K. Nordhoff, et al. 2011. PETIR-001, a dual inhibitor of and alternative splicing of the human CD13 gene--different splicing frequencies dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates ex- among patients with acute myeloid leukaemia and healthy individuals. Br. J. perimental autoimmune encephalomyelitis in SJL/J mice. Biol. Chem. 392: 233– Haematol. 112: 691–696. 237. 125. Adamia, S., B. Haibe-Kains, P. M. Pilarski, M. Bar-Natan, S. Pevzner, H. Avet- 104. Esendagli, G., A. T. Kurne, G. Sayat, A. K. Kilic, D. Guc, and R. Karabudak. Loiseau, L. Lode, S. Verselis, E. A. Fox, J. Burke, et al. 2014. A genome-wide 2013. Evaluation of Th17-related cytokines and receptors in multiple sclerosis aberrant RNA splicing in patients with acute myeloid leukemia identifies novel patients under interferon b-1 therapy. J. Neuroimmunol. 255: 81–84. potential disease markers and therapeutic targets. Clin. Cancer Res. 20: 1135–1145. 105. Abraham, C., and J. H. Cho. 2009. Inflammatory bowel disease. N. Engl. J. Med. 126. Bauvois, B., and D. Dauzonne. 2006. Aminopeptidase-N/CD13 (EC 3.4.11.2) 361: 2066–2078. inhibitors: chemistry, biological evaluations, and therapeutic prospects. Med. Res. 106. Bank, U., A. Heimburg, M. Helmuth, S. Stefin, U. Lendeckel, D. Reinhold, Rev. 26: 88–130.

http://classic.jimmunol.org J. Faust, P. Fuchs, B. Sens, K. Neubert, et al. 2006. Triggering endogenous im- 127. Su, L., H. Fang, and W. Xu. 2011. Aminopeptidase N (EC 3.4.11.2) inhibitors munosuppressive mechanisms by combined targeting of dipeptidyl peptidase IV (2006 - 2010): a patent review. Expert Opin. Ther. Pat. 21: 1241–1265. (DPIV/CD26) and aminopeptidase N (APN/ CD13)--a novel approach for the 128. Wickstro¨m, M., R. Larsson, P. Nygren, and J. Gullbo. 2011. Aminopeptidase N treatment of inﬂammatory bowel disease. Int. Immunopharmacol. 6: 1925–1934. (CD13) as a target for cancer chemotherapy. Cancer Sci. 102: 501–508. 107. Salaga, M., A. Mokrowiecka, D. Jacenik, A. I. Cygankiewicz, E. Malecka-Panas, 129. Gao, J. J., Z. H. Gao, C. R. Zhao, Y. Yuan, S. X. Cui, X. F. Zhang, Y. N. Cheng, R. Kordek, W. M. Krajewska, M. K. Sobocinska, E. Kamysz, and J. Fichna. 2017. W. F. Xu, W. Tang, and X. J. Qu. 2011. LYP, a novel bestatin derivative, inhibits Systemic administration of sialorphin attenuates experimental colitis in mice via cell growth and suppresses APN/CD13 activity in human ovarian carcinoma cells interaction with Mu and Kappa opioid receptors. J. Crohn’s Colitis 11: 988–998. more potently than bestatin. Invest. New Drugs 29: 574–582. 108. Kamysz, E., M. Sałaga, M. Sobocinska, A. Giełdon, and J. Fichna. 2016. Anti- 130. Allegra, A., V. Innao, S. Russo, D. Gerace, A. Alonci, and C. Musolino. 2017. Downloaded from inﬂammatory effect of novel analogs of natural enkephalinase inhibitors in a mouse Anticancer activity of curcumin and its analogues: preclinical and clinical studies. model of experimental colitis. Future Med. Chem. 8: 2231–2243. Cancer Invest. 35: 1–22.