Formylpeptide Receptor N Antagonist at The

The Endogenous Opioid Spinorphin Blocks fMet-Leu-Phe-Induced Neutrophil Chemotaxis by Acting as a Specific Antagonist at the N-Formylpeptide Receptor This information is current as Subtype FPR of September 27, 2021. Thomas S. Liang, Ji-Liang Gao, Omid Fatemi, Mark Lavigne, Thomas L. Leto and Philip M. Murphy J Immunol 2001; 167:6609-6614; ; doi: 10.4049/jimmunol.167.11.6609 Downloaded from http://www.jimmunol.org/content/167/11/6609

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Endogenous Opioid Spinorphin Blocks fMet-Leu-Phe-Induced Neutrophil Chemotaxis by Acting as a Speciﬁc Antagonist at the N-Formylpeptide Receptor Subtype FPR

Thomas S. Liang, Ji-Liang Gao, Omid Fatemi, Mark Lavigne, Thomas L. Leto, and Philip M. Murphy1

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), ﬁrst isolated from bovine spinal cord, whose sequence matches a conserved region of ␤-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine

(fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of- Downloaded from function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by con- http://www.jimmunol.org/ centrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR. The Journal of Immunology, 2001, 167: 6609Ð6614.

he generation of opioid peptides from endogenous pro- peptidase (18). Spinorphin has been identified in human cerebro- teins has been characterized in milk protein, mitochon- spinal fluid and bovine spinal cord (10, 18). Concentrations in vivo T drial cytochrome b, and hemoglobin (1–3). Hemorphins are not known, but high levels (115–300 nM) of the related he- are nonclassical opioid peptides found in human pituitary gland, morphin LVV-hemorphin-7 have been measured in cerebrospinal cerebrospinal fluid, adrenal gland, blood, and bronchoalveolar la- fluid from a patient with cerebrovascular bleeding (9). Spinorphin by guest on September 27, 2021 vage fluid as well as bovine brain that have amino acid sequences has been shown to have analgesic activity for morphine-resistant identical with a conserved region of the ␤-chain of bovine and pain pathways (19). human hemoglobin (4–10). Peritoneal macrophages (11) and pro- Expression of the spinorphin targets NEP and aminopeptidase N teolytic enzymes such as aspartic endopeptidase (12) and cathepsin (CD13) on blood phagocytes (20–24) has suggested a potential D (13) have been implicated in hemorphin processing and produc- role for spinorphin in modulating inflammatory responses. NEP tion. Hemorphins have been reported to function as agonists at degrades and inactivates the potent phagocyte chemoattractant AT4 angiotensin receptors in brain and as antagonists at enkeph- fMLF (25), suggesting that spinorphin might act to potentiate re- alinases and angiotensin-converting enzyme (14–17), suggesting sponses to this peptide. In fact, the opposite is the case; spinorphin potential modulatory roles in memory, pain, and blood pressure inhibits fMLF-induced calcium flux, chemotaxis, exocytosis, and control. superoxide production in human neutrophils (26). The mechanism Spinorphin, or LVV-hemorphin-4, has the amino acid sequence appears to involve competition for fMLF binding to surface re- leucyl-valyl-valyl-tyrosyl-prolyl-tryptophyl-threonine, which matches ceptors. Spinorphin has also been shown to block carrageenan- positions 32–38 of human ␤-hemoglobin (5). Like other hemor- induced polymorphonuclear neutrophil accumulation in mouse air phins, spinorphin is an angiotensin-converting enzyme antagonist, pouches (27). and inhibits enkephalin-degrading enzymes such as neutral endo- Here we address the molecular basis of spinorphin action by peptidase (NEP2/CD10), aminopeptidase, and dipeptidyl amino- testing its specificity for known fMLF receptors. Two human neutrophil fMLF receptor subtypes have been cloned, N-formylpep- Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, tide receptor (FPR) and FPR-like 1 receptor (FPRL1R) (28–32). National Institutes of Health, Bethesda, MD 20892 These receptors are members of the G protein-coupled receptor Received for publication December 8, 2000. Accepted for publication September superfamily and have 69% amino acid identity. Compared with 24, 2001. FPR, FPRL1R binds fMLF with lower affinity and has additional The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance agonists, including lipoxin A4, and serum amyloid A (33, 34). In with 18 U.S.C. Section 1734 solely to indicate this fact. the mouse the FPR homologue has been clearly identified (35), but 1 Address correspondence and reprint requests to Dr. Philip M. Murphy, National two other related receptors have been cloned that are equally ho- Institutes of Health, Building 10, Room 11N113, Bethesda, MD 20892. E-mail ad- mologous to human FPRL1R (36). To date one has been charac- dress: [email protected] terized as a lipoxin A4 receptor and is known as LXA4R (37), 2 Abbreviations used in this paper: NEP, neutral endopeptidase; fMLF, N-formylmethionyl-leucyl-phenylalanine; FPR, N-formylpeptide receptor; FPRL1R, FPR-like whereas the other, known as FPR2, is a functional receptor for 1 receptor; HEK, human embryonic kidney. fMLF (low affinity compared with mouse FPR) and serum amyloid

A (38, 39). Here we use gene transfected cell lines and FPR knockout mice to prove that spinorphin blocks fMLF-induced chemotaxis of neutrophils by specifically antagonizing FPR. Materials and Methods Peptides fMLF was purchased from Sigma (St. Louis, MO). Spinorphin was syn- thesized by the Peptide Synthesis and Analysis Unit, Research Technolo- gies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD). HPLC analysis indicated that the peptide was 93% pure. Both fMLF and spinorphin were dissolved in DMSO, and 10 mM stocks were kept at Ϫ20°C until use. Mouse leukocyte purification FIGURE 1. Spinorphin is a functional ligand for the fMLF receptor subtype FPR, but not for the related receptor FPR2. Calcium mobilization Development of an FPR knockout mouse has been reported previously was measured as relative cell fluorescence in HEK 293 cells transfected ϩ/Ϫ ϫ ϩ/Ϫ (40). Mice used in this study were littermates from FPR FPR with mouse FPR and mouse FPR2. The arrows indicate test molecules, matings of an F backcross of FPRϩ/Ϫ 129/Sv with wild-type C57BL/6 1 concentrations, and time of addition for each tracing. Each tracing corre- mice. Leukocytes were harvested from the peritoneal cavity after thioglycolate irritation, as described previously (40). Cells obtained after 3 h were sponds to the receptor indicated to the left of the row in which it is found. Ͼ90% neutrophils, as determined by the morphologic appearance of Diff- Data are from a single representative experiment repeated at least three Quick-stained preparations. times. Downloaded from Cell lines Human embryonic kidney (HEK) 293 cell lines expressing mouse FPR and quential stimulation experiments, again monitoring calcium flux. mouse FPR2 have been previously described (37). Cells were cultured in A saturating concentration of spinorphin added first (500 ␮M) DMEM plus 10% FBS and 1 mg/ml G418 at 37°C and were collected for caused a marked reduction in the response of the cells to 5 ␮M analysis in PBS at room temperature. fMLF given second, and 5 ␮M fMLF added first almost com- http://www.jimmunol.org/ Intracellular Ca2ϩ measurements pletely desensitized the cells to 300 ␮M spinorphin given second (Fig. 3). This interference pattern suggests usage of the same sig- Cells were incubated in PBS (107 cells/ml) containing 2.5 ␮M fura-2/AM (Molecular Probes, Eugene, OR) for 30 min at 37°C in the dark. Cells were naling pathway by these two agonists washed twice and resuspended in HBSS. Cells (3 ϫ 106), continuously stirred in a 2-ml cuvette at 37°C, were stimulated by fMLF or spinorphin FPR is a functional neutrophil receptor for spinorphin in a fluorometer (Photon Technology, South Brunswick, NJ). The data We previously reported that FPR and FPR2 are both expressed in were recorded every 200 ms as the relative ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm. primary mouse neutrophils (38). To test whether our results from

Chemotaxis by guest on September 27, 2021 Leukocytes were resuspended at 1.5 ϫ 106 cells/ml in RPMI 1640 sup- plemented with 1% BSA and 20 mM HEPES. Spinorphin and fMLF were loaded at varying concentrations in the lower compartment of a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD). The chamber was soaked in 1% SDS overnight and washed before each experiment. A 3-␮m pore size polyvinylpyrrolidone-free polycarbonate filter was used. Fifty microliters of 1.5 ϫ 106 cells/ml was loaded in the upper compartment, and the chemotaxis chamber was incubated at 37°C, 100% humidity, and 5% CO2 for 1 h. The filter was then removed, washed, fixed, and stained. Cells that migrated through the filter were counted microscopically under high power. All conditions were tested in triplicate. Results Spinorphin is a partial agonist at mouse FPR To identify specific spinorphin receptors, we used a calcium flux assay to test as candidates the two known mouse N-formylpeptide receptor subtypes (FPR and FPR2), expressed in HEK 293 cells by stable transfection with appropriate plasmids. Untransfected HEK 293 cells do not normally express these receptors or respond to fMLF and did not respond to spinorphin (not shown). As expected, each cell line responded specifically to fMLF with a typical rise in the intracellular Ca2ϩ concentration, followed by a rapid return to resting levels (Fig. 1). As previously reported (38), each receptor could be distinguished pharmacologically by the EC50 of fMLF for activation and by reactivity to selective agonists (data not shown). Neither cell line responded to stimulation with irrelevant chemoattractant agonists, such as the chemokine macrophage inflammatory ␤ peptide-1 . FIGURE 2. Potency of spinorphin for activation of mouse FPR. HEK Spinorphin also induced calcium flux in FPR-transfected cells, 293 cells expressing mouse FPR were stimulated with spinorphin at the Ϯ but not in cells expressing FPR2 (Fig. 1); the EC50 was 128 24 concentration indicated to the right of each tracing, and calcium flux was ␮M(n ϭ 3; Fig. 2). To confirm that spinorphin and fMLF share measured as the relative change in cell fluorescence. Data are from a single the same signaling pathway via FPR, we performed real-time se- experiment repeated six times. The Journal of Immunology 6611

added ﬁrst dramatically reduced the response of FPRϩ/ϩ neutrophils to 1 ␮M fMLF given second. This concentration of fMLF induces a maximal response in HEK 293 cells transfected with mouse FPR. Conversely, 200 nM fMLF abolished the response of the cells to 100 ␮M spinorphin given second (Fig. 4C). These results are consistent with the idea that FPR functions as a shared calcium-mobilizing mouse neutrophil receptor for spinorphin and fMLF. Moreover, the failure of 100 ␮M spinorphin to induce calcium ﬂux in FPRϪ/Ϫ neutrophils or to block the re- Ϫ/Ϫ ␮ sponse of FPR neutrophils to 10 M fMLF, which is the EC50 for activation of FPR2, strongly suggests that spinorphin is neither an agonist nor an antagonist of endogenous FPR2 (Fig. 4C). Thus, there is agreement between the gain- and loss-of-function genetic tests of these receptors in transfected HEK 293 cells and primary mouse neutrophils.

Spinorphin acts as an antagonist at FPR to block fMLF induction of neutrophil chemotaxis Downloaded from We next tested whether spinorphin can affect neutrophil chemotaxis. In control experiments, fMLF was approximately 100-fold more potent in inducing chemotaxis of thioglycolate-elicited peritoneal neutrophils from FPRϩ/ϩ mice compared with cells from FPRϪ/Ϫ mice (Fig. 5), which confirms our previous report (38). fMLF appears to induce chemotaxis of FPRϪ/Ϫ neutrophils via http://www.jimmunol.org/ FPR2, since both these cells and FPR2-transfected HEK 293 cells respond chemotactically to fMLF over a similar dose range. To understand the spinorphin results that follow, it is important to realize that the dose-response curve for fMLF induction of chemotaxis in wild-type neutrophils has two optima at 1 and 100 ␮M FIGURE 3. Desensitization of FPR by spinorphin. HEK 293 cells ex- (Fig. 5A), reflecting the well-known bell shape of chemotaxis dose- pressing mouse FPR were stimulated sequentially with spinorphin and response curves and the responsiveness of FPR and FPR2 in these fMLF at the times and concentrations indicated, and calcium flux was cells at low and high concentrations of fMLF, respectively (38). measured as the relative change in cell fluorescence. Data are from a single experiment repeated at least six times. Although spinorphin was an effective calcium-mobilizing ago- by guest on September 27, 2021 nist for neutrophils from FPRϩ/ϩ mice, it was an extremely poor direct chemoattractant of these cells (Fig. 5), which agrees with studying the cloned receptors in HEK 293 cell transfectants accu- previously reported results using human neutrophils (26) and pro- rately reflect the properties of the receptors when expressed endo- vides an additional example of the nonequivalence of calcium flux genously in primary cells, we compared functional responses from and chemotaxis pathways in neutrophils. The maximal chemotac- thioglycolate-elicited peritoneal neutrophils obtained from wild tic index was consistently Ͻ2 at optimal spinorphin concentrations type (ϩ/ϩ) and FPR knockout (Ϫ/Ϫ) mice (Fig. 4). In neutrophils (10–50 ␮M; Fig. 5A). Spinorphin-induced chemotaxis was not ob- from FPRϩ/ϩ mice, spinorphin induced dose-dependent, saturable served in neutrophils from FPRϪ/Ϫ mice, indicating that the ac- calcium flux responses (Fig. 4A), whereas neutrophils from tivity, albeit weak, was specifically mediated by FPR (Fig. 5B). In FPRϪ/Ϫ mice did not respond even at 100 ␮M spinorphin (Fig. contrast to this weak intrinsic agonist activity, spinorphin specif- 4B). In sequential stimulation experiments, 100 ␮M spinorphin ically and effectively antagonized fMLF-induced chemotaxis of

FIGURE 4. Spinorphin is an agonist at endogenous FPR expressed in mouse neutrophils. Calcium mobilization in thioglycolate-elicited peritoneal neutrophils obtained from wild-type (A and C) and FPR-knockout (B) mice. A, Dose response of spinorphin in FPRϩ/ϩ neutrophils. B, Speciﬁcity of spinorphin for endogenous FPR. C, Cross-desensitization of calcium ﬂux responses by fMLF and spinorphin in FPRϩ/ϩ neutrophils. Data are from a single representative experiment representative of three independent experiments. 6612 SPINORPHIN ANTAGONISM OF N-FORMYLPEPTIDE RECEPTORS

neutrophils to an unrelated agonist, the chemokine SDF-1 (Fig. 6B). When an fMLF concentration that is both selective for FPR over FPR2 and optimal for FPR-mediated neutrophil chemotaxis was used to stimulate neutrophils from FPRϩ/ϩ mice, spinorphin in-

hibited the activity in a dose-dependent manner. The IC50 was approximately 30 ␮M (Fig. 7A). At the highest concentration of spinorphin tested (100 ␮M) the inhibitory efﬁcacy was about 70%. In contrast, when an fMLF concentration that is optimal for FPR2- mediated neutrophil chemotaxis was used to stimulate neutrophils from FPRϪ/Ϫ mice, the activity was unaffected by spinorphin throughout the concentration range tested (Fig. 7B). This is consistent with the calcium ﬂux experiments in mouse receptor-transfected HEK 293 cells and FPRϪ/Ϫ mouse neutrophils and supports the conclusion that spinorphin is highly selective for mouse FPR compared with FPR2. Spinorphin has also been reported to block human neutrophil oxidant production triggered by fMLF (26). However, fMLF induction of oxidant production in mouse neutrophils was weak, and Downloaded from spinorphin quenched the Diogenes reagent that is most sensitive for detecting superoxide protection; therefore, analysis of spinorphin modulation of this pathway could not be accomplished.

Discussion In this study we have used genetic gain- and loss-of-function cri- http://www.jimmunol.org/ teria to prove that the hemorphin family member spinorphin blocks fMLF induction of mouse neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR. Spinorphin also exhibited partial agonist activity at FPR, since it was able to FIGURE 5. Spinorphin is a weak chemoattractant for mouse neutro- directly induce calcium flux and to weakly induce chemotaxis in phils. Chemotaxis was measured as the number of cells migrated per high Ϫ Ϫ wild-type mouse neutrophils, but not in FPR / neutrophils. Our power field (hpf) for wild-type (ϩ/ϩ) and FPR knockout (Ϫ/Ϫ) neutrophils in response to the indicated concentrations of fMLF and spinorphin. results are consistent with and provide a specific molecular mech- Ϯ anism for the findings of Yamamoto et al. (26), who reported that Data are the mean SE of triplicate determinations and are representative by guest on September 27, 2021 of two independent experiments. spinorphin could inhibit fMLF induction of human neutrophil chemotaxis, superoxide generation, and exocytosis and could inhibit fMLF binding to human neutrophils. That result did not specify a neutrophils from FPRϩ/ϩ mice (Fig. 6). This agrees with the re- molecular mechanism of spinorphin action because of the exis- ported ability of spinorphin to block fMLF-induced chemotaxis of tence of at least one other fMLF receptor subtype, FPR2, which we human neutrophils (26). Selectivity for FPR was suggested by the now show does not appear to interact with spinorphin, and because ability of 100 ␮M spinorphin to inhibit chemotaxis of wild-type of the possibility that spinorphin could cross-desensitize fMLF re- neutrophils over the full chemotactic concentration range of fMLF ceptors through an independent pathway. (10–1000 nM) for FPR (Fig. 6A) but not at 10 ␮M fMLF, which FPR is the first membrane receptor identified for spinorphin. induces chemotaxis via FPR2, not FPR (38), as well as by the Other members of the hemorphin family have been shown to bind inability of spinorphin to affect chemotaxis of wild-type mouse angiotensin receptors (14, 15), which, like FPR, are members of

FIGURE 6. Spinorphin is a speciﬁc antagonist of fMLF-induced neutrophil migration. Migration was measured in vitro for thioglycolate-elicited peritoneal neutrophils obtained from wild-type mice. A, Spinorphin antagonism of chemotaxis induced by in- creasing concentrations of fMLF. B, Spinor- phin speciﬁcity for FPR. The name and concentrations of stimuli in the lower chemotaxis well are indicated below each bar. Data are the mean Ϯ SE of triplicate determinations from a single experiment and are representative of two experiments. The Journal of Immunology 6613

and FPR2. The approximately 50 known members of the chemokine family typically have strong agonist activity, although two endogenous chemokine antagonists active at CXCR3 have recently been reported (49). Although the role of endogenous antagonists in the regulation of the inflammatory response remains undefined in vivo, the identification of specific spinorphin antagonism at FPR increases the biological plausibility of such a mechanism. Based on the specificity of FPR for phagocytes and the fact that spinorphin is cleaved from ␤-hemoglobin, such a mechanism is most likely to operate in the setting of acute inflammation and may be of general impor- tance rather than restricted to a specific tissue type.

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3. Brantl, V., C. Gramsch, F. Lottspeich, A. Henschen, K. H. Jaeger, and A. Herz. Downloaded from 1985. Novel opioid peptides derived from mitochondrial cytochrome b: cyto- chrophins. Eur. J. Pharmacol. 111:293. 4. Duethman, D., N. Dewan, and J. M. Conlon. 2000. Isolation of the opioid peptide leu-val-val-hemorphin-7 from bronchoalveolar lavage fluid of a patient with non- small cell lung cancer. Peptides 21:137. 5. Karelin, A. A., M. M. Philippova, E. V. Karelina, and V. T. Ivanov. 1994. Iso- lation of endogenous hemorphin-related hemoglobin fragments from bovine brain. Biochem. Biophys. Res. Commun. 202:410. FIGURE 7. Potency of spinorphin antagonism of fMLF-induced neu- 6. Glamsta, E. L., L. Morkrid, I. Lantz, and F. Nyberg. 1993. Concomitant increase http://www.jimmunol.org/ trophil chemotaxis. Migration was measured in vitro for thioglycolate-elic- in blood plasma levels of immunoreactive hemorphin-7 and ␤-endorphin follow- ited peritoneal neutrophils obtained from wild-type (A) and FPR knockout ing long distance running. Regul. Pept. 49:9. (B) mice. A, Spinorphin dose dependence for antagonism of chemotaxis 7. Glamsta, E. L., A. Marklund, U. Hellman, C. Wernstedt, L. Terenius, and induced by a concentration of fMLF selective for FPR. The indicated con- F. Nyberg. 1991. Protein isolation and characterization of a hemoglobin-derived opioid peptide from the human pituitary gland. Regul. Pept. 34:169. Ϫ6 centrations of spinorphin were incubated with 10 M fMLF in the lower 8. Cerpa-Poljak, A., J. Lahnstein, K. E. Mason, G. A. Smythe, and M. W. Duncan. well of the chemotaxis chamber. B, Lack of spinorphin antagonism of 1997. Mass spectrometric identification and quantification of hemorphins ex- chemotaxis induced by a concentration of fMLF selective for FPR2. The tracted from human adrenal and pheochromocytoma tissue. J. Neurochem. 68: indicated concentrations of spinorphin were incubated with 10Ϫ4 M fMLF 1712. 9. Glamsta, E. L., B. Meyerson, J. Silberring, L. Terenius, and F. Nyberg. 1992. in the lower well of the chemotaxis chamber. Data are the mean Ϯ SE of

Isolation of a hemoglobin-derived opioid peptide from cerebrospinal fluid of by guest on September 27, 2021 triplicate determinations from a single experiment and are representative of patients with cerebrovascular bleedings. Biochem. Biophys. Res. Commun. 184: two independent experiments. 1060. 10. Sanderson, K., M. Thornwall, G. Nyberg, E. L. Glamsta, and F. Nyberg. 1994. Reversed-phase high-performance liquid chromatography for the determination of haemorphin-like immunoreactivity in human cerebrospinal fluid. J. Chro- the peptidergic group of G protein-coupled receptors (41). How- matogr. 676:155. 11. Dagouassat, N., I. Garreau, F. Sannier, Q. Zhao, and J. M. Piot. 1996. Generation ever, interaction of spinorphin with angiotensin receptors and in- of VV-hemorphin-7 from globin by peritoneal macrophages. FEBS Lett. 382:37. teraction of other hemorphins with FPR have not been reported. 12. Barkhudaryan, N., J. Kellermann, A. Galoyan, and F. Lottspeich. 1993. High molecular weight aspartic endopeptidase generates a coronaro-constrictory pep- FPR ligands were originally thought to be restricted to formylated tide from the ␤-chain of hemoglobin. FEBS Lett. 329:215. peptides potentially derived from bacterial and mitochondrial pro- 13. Fruitier, I., I. Garreau, and J. M. Piot. 1998. Cathepsin D is a good candidate for teins (42), but are now known to also include several nonformy- the specific release of a stable hemorphin from hemoglobin in vivo: VV-hemorphin-7. Biochem. Biophys. Res. Commun. 246:719. lated peptides, all of which, with the exception of spinorphin, are 14. Moeller, I., R. A. Lew, F. A. Mendelsohn, A. I. Smith, M. E. Brennan, T. J. Tetaz, synthetic peptides derived from random peptide libraries or HIV and S. Y. Chai. 1997. The globin fragment LVV-hemorphin-7 is an endogenous proteins (43, 44). These peptides imply that there may also exist ligand for the AT4 receptor in the brain. J. Neurochem. 68:2530. 15. Moeller, I., S. Y. Chai, I. Smith, R. Lew, and F. A. Mendelsohn. 1998. Haemor- nonformylated endogenous peptide agonists for FPR, but to date phin peptides may be endogenous ligands for brain angiotensin AT4 receptors. none has been found. Clin. Exp. Pharmacol. Physiol. 25(Suppl.):S68. Limiting phagocyte accumulation and activation in the tissue is 16. Zhao, Q., and J. M. Piot. 1997. Investigation of inhibition angiotensin-converting enzyme (ACE) activity and opioid activity of two hemorphins, LVV-hemor- crucial in preventing inappropriate inflammatory responses and phin-5 and VV-hemorphin-5, isolated from a defined peptic hydrolysate of bo- disease; however, to date the mechanisms that negatively regulate vine hemoglobin. Neuropeptides 31:147. 17. Lantz, I., E. L. Glamsta, L. Talback, and F. Nyberg, F. 1991. Hemorphins derived phagocyte chemotaxis are not well defined. In addition to inter- from hemoglobin have an inhibitory action on angiotensin converting enzyme rupting ligand-receptor binding, endogenous chemoattractant activity. FEBS Lett. 287:39. blocking agents may work by scavenging (45, 46), degrading, or 18. Nishimura, K., and T. Hazato. 1993. Protein isolation and identification of an endogenous inhibitor of enkephalin-degrading enzymes from bovine spinal cord. processing the chemoattractant (25, 47) or by desensitizing the Biochem. Biophys. Res. Commun. 194:713. receptor through phosphorylation and/or internalization mecha- 19. Ueda, H., S. Matsunaga, M. Inoue, Y. Yamamoto, and T. Hazato. 2000. Complete nisms (48). Spinorphin has been shown to block fMLF binding to inhibition of purinoceptor agonist-induced nociception by spinorphin, but not by morphine. Peptides 21:1215. neutrophils (26), but since it is a partial agonist at FPR, it could 20. Look, A. T., R. A. Ashmun, L. H. Shapiro, and S. C. Peiper. 1989. Human also work by desensitizing and down-regulating the receptor. We myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J. Clin. Invest. 83:1299. refer to it as an FPR antagonist to emphasize its very weak che- 21. Ziaber, J., Z. Baj, J. Pasnik, H. Chmielewski, and H. Tchorzewski. 2000. In- motactic agonist activity at this receptor and strong inhibition of creased expression of neutral endopeptidase (NEP) and aminopeptidase N (APN) fMLF-induced chemotaxis. To date very few examples of endog- on peripheral blood mononuclear cells in patients with multiple sclerosis. Immu- nol. Lett. 71:127. enous antagonists of chemoattractant receptors have been identi- 22. Juillerat-Jeanneret, L., J. D. Aubert, and P. Leuenberger. 1997. Peptidases in fied. Apart from spinorphin no others have been reported for FPR human bronchoalveolar lining fluid, macrophages, and epithelial cells: dipeptidyl 6614 SPINORPHIN ANTAGONISM OF N-FORMYLPEPTIDE RECEPTORS

(amino)peptidase IV, aminopeptidase N, and dipeptidyl (carboxy)peptidase (an- 36. Gao, J.-L., H. Chen, J. D. Filie, C. A. Kozak, and P. M. Murphy. 1998. Differ- giotensin-converting enzyme). J. Lab. Clin. Med. 130:603. ential expansion of the N-formylpeptide receptor gene cluster in human and 23. Chen, H., C. A. Kinzer, and W. E. Paul. 1996. p161, a murine membrane protein mouse. Genomics 51:270. expressed on mast cells and some macrophages, is mouse CD13/aminopeptidase 37. Takano, T., S. Fiore, J. F. Maddox, H. R. Brady, N. A. Petasis, and C. N. Serhan. N. J. Immunol. 157:2593. 1997. Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues 24. Iwamoto, I., A. Kimura, K. Ochiai, H. Tomioka, and S. Yoshida. 1991. Distri- are potent inhibitors of acute inflammation: evidence for anti-inflammatory re- bution of neutral endopeptidase activity in human blood leukocytes. J. Leukocyte ceptors. J. Exp. Med. 185:1693. Biol. 49:116. 38. Hartt, J. K., G. Barish, P. M. Murphy, and J.-L. Gao. 1999. N-formylpeptides 25. Painter, R. G., R. Dukes, J. Sullivan, R. Carter, E. G. Erdos, and A. R. Johnson. induce two distinct concentration optima for mouse neutrophil chemotaxis by 1988. Function of neutral endopeptidase on the cell membrane of human neu- differential interaction with two N-formylpeptide receptor (FPR) subtypes: mo- trophils. J. Biol. Chem. 263:9456. lecular characterization of FPR2, a second mouse neutrophil FPR. J. Exp. Med. 26. Yamamoto, Y., T. Kanazawa, M. Shimamura, M. Ueki, and T. Hazato. 1997. 190:741. Inhibitory effects of spinorphin, a novel endogenous regulator, on chemotaxis, 39. Liang, T. S., J. M. Wang, P. M. Murphy, and J.-L. Gao. 2000. Serum Amyloid Ϫ O2 generation, and exocytosis by N-formylmethionyl-leucyl-phenylalanine A is a chemotactic agonist at FPR2, a low-affinity N-formylpeptide receptor on (FMLP)-stimulated neutrophils. Biochem. Pharmacol. 54:695. mouse neutrophils. Biochem. Biophys. Res. Commun. 270:331. 27. Yamamoto, Y., H. Kanazawa, M. Shimamura, M. Ueki, and T. Hazato. 1998. 40. Gao, J.-L., E. J. Lee, and P. M. Murphy. 1999. Impaired anti-bacterial host de- Inhibitory action of spinorphin, an endogenous regulator of enkephalin-degrading fense in mice lacking the N-formylpeptide receptor. J. Exp. Med. 189:657. enzymes, on carrageenan-induced polymorphonuclear neutrophil accumulation in 41. Murphy, P. M. 1994. The molecular biology of leukocyte chemoattractant recep- mouse air-pouches. Life Sci. 62:1767. tors. Annu. Rev. Immunol. 12:593. 28. Boulay, F., M. Tardif, L. Brouchon, and P. Vignais. 1990. The human 42. Murphy, P. M. 1996. The N-formylpeptide chemotactic receptor. In Chemoat- N-formylpeptide receptor: characterization of two cDNA isolates and evidence tractant Ligands and Their Receptors. R. Horuk, ed. CRC Press, Boca Raton, for a new subfamily of G-protein-coupled receptors. Biochemistry. 29:11123. p. 269. 29. Ye, R. D., S. L. Cavanagh, O. Quehenberger, E. R. Prossnitz, and C. G. Cochrane. 43. Chen, J., H. S. Bernstein, M. Chen, L. Wang, M. Ishii, C. W. Turck, and 1992. Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide S. R. Coughlin. 1995. Tethered ligand library for discovery of peptide agonists. receptor. Biochem. Biophys. Res. Commun. 184:582. J. Biol. Chem. 270:23398. 30. Murphy, P. M., T. Ozcelik, R. T. Kenney, H. L. Tiffany, D. McDermott, and 44. Su, S. B., W. Gong, J.-L. Gao, W.-P. Shen, M. C. Grimm, X. Deng, Downloaded from U. Francke. 1992. A structural homologue of the N-formyl peptide receptor: P. M. Murphy, J. J. Oppenheim, and J. M. Wang. 1999. T20/DP178, an ectodo- characterization and chromosomal mapping of a peptide chemoattractant receptor main peptide of human immunodeficiency virus type 1 gp41, is an activator of gene family. J. Biol. Chem. 267:7637. human phagocyte N-formyl peptide receptor. Blood. 93:3885. 31. Bao, L., N. P. Gerard, R. L. Eddy, Jr., T. B. Shows, and C. Gerard. 1992. Map- 45. Darbonne, W. C., G. C. Rice, M. A. Mohler, T. Apple, C. A. Hebert, ping of genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), A. J. Valente, and J. B. Baker. 1991. Red blood cells are a sink for interleukin 8, and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chro- a leukocyte chemotaxin. J. Clin. Invest. 88:1362. mosome 19. Genomics 13:437. 46. Sylvester, I., T. Yoshimura, M. Sticherling, J. M. Schroder, M. Ceska, P. Peichl,

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