of the Formyl Peptide Receptor Family Members Leukocytes by ...

The Journal of Immunology

An Annexin 1 N-Terminal Peptide Activates Leukocytes by Triggering Different Members of the Formyl Peptide Receptor Family1 Stefanie Ernst, Carsten Lange, Andreas Wilbers, Verena Goebeler, Volker Gerke,2 and Ursula Rescher2 The human N-formyl peptide receptor (FPR) is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. FPR is the founding member of a subfamily of G protein-coupled receptors thought to function in inflammatory processes. The other two members, FPR-like (FPRL)1 and FPRL2, have a greatly reduced affinity for bacterial peptides or do not bind them at all, with FPRL2 being considered an orphan receptor so far. In this study we show that a peptide derived from the N-terminal domain of the anti-inflammatory protein annexin 1 (lipocortin 1) can activate all three FPR family members at similar concentrations. The annexin 1 peptide initiates chemotactic responses in human monocytes that express all three FPR family members and also desensitizes the cells toward subsequent stimulation with bacterial peptide agonists. Experiments using HEK 293 cells stably expressing a single FPR family member reveal that all three receptors can be activated and desensitized by the N-terminal annexin 1 peptide. These observations identify the annexin 1 peptide as the first endogenous ligand of FPRL2 and indicate that annexin 1 participates in regulating leukocyte emigration into inflamed tissue by activating and desensitizing different receptors of the FPR family. The Journal of Immunology, 2004, 172: 7669 –7676.

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he recruitment of leukocytes into inflamed tissue is a precisely regulated process requiring the involvement of several soluble or surface-bound mediators of the directed leukocyte migration. Following inflammatory activation, these mediators control leukocyte rolling on and adhesion to the endothelium as well as control the subsequent extravasation from the blood vessel into the tissue (1, 2). One protein that has received considerable attention as an endogenous anti-inflammatory agent is annexin 1, formerly termed lipocortin 1 (3). It inhibits granulocyte extravasation in several models of inflammation (4 –11) and annexin 1 knockout mice show an altered regulation of inflammation with a significantly enhanced neutrophil extravasation (12). Annexin 1 is a member of the annexin family of Ca2⫹-lipid binding proteins, which are structurally defined by a highly conserved protein core domain harboring Ca2⫹ and phospholipid binding sites and an N-terminal region that is unique for a given annexin (13). The anti-inflammatory activities elicited by exogenously applied annexin 1 are mediated through its unique N-terminal domain. Peptides derived from this domain, which are most likely generated at sites of inflammation and in coculture of neutrophils with activated endothelial cells, are not only true mimetics of the annexin 1 action in all inflammation models tested, but

Institute for Medical Biochemistry, Center for Molecular Biology of Inflammation, Mu¨nster, Germany Received for publication August 11, 2003. Accepted for publication April 6, 2004. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Interdisciplinary Center for Clinical Research of the University of Mu¨nster Interdisziplina¨res Zentrum fu¨r Klinische Forschung, C22 (to U.R. and Vo.G.), and the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 293, Project A3. 2 Address correspondence and reprint requests to Drs. Volker Gerke and Ursula Rescher, Institute for Medical Biochemistry, Center for Molecular Biology of Inflammation, von Esmarch-Strasse 56, D-48149 Mu¨nster, Germany. E-mail addresses: [email protected] and [email protected]

Copyright © 2004 by The American Association of Immunologists, Inc.

most likely are also the active entities with respect to the antiinflammatory actions described for annexin 1 (5, 7–11). Recently, it was shown that the inhibitory effect of the N-terminal annexin 1 peptides as well as full-length annexin 1 on the transendothelial migration of granulocytes is at least in part due to a peptidetriggered desensitization of the N-formyl peptide receptor (FPR)3 on human granulocytes. This identified annexin 1 as an endogenous ligand of the receptor with the interaction mediated through annexin 1 N-terminal peptides (14). FPR is a seven transmembrane, G protein-coupled receptor activated by chemoattractant bacterial peptides of the prototype fMLP. Following fMLP binding downstream signaling mediated through associated G proteins results in cytoskeletal rearrangements required for directed granulocyte migration in the fMLP gradient (15–17). FPR is the founding member of the human FPR subfamily that so far consists of the three proteins FPR, FPR-like (FPRL)1, and FPRL2. FPRL1, also known as the lipoxin A4 receptor, is 69% homologous at the amino acid level to FPR and both receptors are expressed in monocytes and granulocytes. FPRL2 shares 83% amino acid identity with FPRL1 and is expressed in monocytes but not granulocytes (18, 19, 16). Whereas the prototype receptor for fMLP, FPR, binds fMLP with high affinity and is activated by nanomolar concentrations of fMLP, FPRL1 is a low affinity fMLP receptor and responds only to high fMLP concentrations in the micromolar range (20). FPRL1, however, is also activated by a variety of other ligands including the serum amyloid A (SAA) protein (21) and the lipid lipoxin A4 (22). Moreover, FPRL1 has recently been proposed to be a key component of lipidand peptide-mediated anti-inflammatory circuits because it responds to aspirin-triggered lipoxins as well as glucocorticoid-induced annexin 1 with a resulting inhibition of neutrophil extravasation (23). In contrast to FPR and FPRL1, FPRL2 is an orphan

3 Abbreviations used in this paper: FPR, N-formyl peptide receptor; FPRL, FPR-like; SAA, serum amyloid A; F-actin, filamentous actin.

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7670 receptor and does not bind fMLP. The only FPRL2 ligand identified so far is the unnatural synthetic peptide WKYMVM (24). Because annexin 1 might also signal through a low affinity fMLP receptor on granulocytes or other leukocytes, we investigated whether it can act through FPRL1 or FPRL2. We show that the mimetic N-terminal annexin 1 peptide Ac1-25 is able to activate and desensitize not only FPR but also FPRL1 and FPRL2. Human embryonic kidney (HEK) 293 cells solely expressing FPRL1 or FPRL2 respond to the annexin 1 peptide with Ca2⫹ fluxes and chemotaxis revealing that the peptide is an agonist for all members of the FPR subfamily and the first endogenous ligand for FPRL2.

Materials and Methods Peptides and Abs The synthetic chemotactic peptide fMLP and its analog NfNleLFNleYK (which has a slightly higher affinity for FPR) were obtained from SigmaAldrich (St. Louis, MO). The annexin 1 peptide (Ac1-25, N-acetyl-AMVSEFLKQAWFIENEEQEYVQTVK), which was acetylated during synthesis to mimic the physiological situation (25) and the unnatural synthetic peptide WKYMVM or the D-methionine containing WKYMVm peptides were purchased from Advanced Biotechnology Centre (London, U.K.). All experiments were reproduced with Ac1-25 from different syntheses and preparations to exclude possible effects of potential impurities.

Isolation of human PBLs Peripheral blood granulocytes and monocytes were isolated from buffy coats using Ficoll-Paque gradient centrifugation (26). Erythrocytes were removed from sedimented granulocytes through hypotonic lysis.

Stable expression of FPR, FPRL1, and FPRL2 in HEK 293 cells Cloning and stable expression of the FPR has been previously described (14). The cDNAs encoding the human receptors FPRL1 and FPRL2 were obtained by PCR using a human leukocyte cDNA library as template (Clontech Laboratories, Palo Alto, CA). To create FPRL fusion proteins C-terminal-tagged with a 10-aa myc epitope (FPRL1myc, FPRL2myc), a PCR was performed using oligonucleotide primers introducing a XhoI restriction site immediately 5⬘ of the initial ATG and a myc epitope sequence followed by a translational Stop signal and an EcoRI site 3⬘ to the last codon of the receptor DNA. For FPRL1, oligonucleotides used were 5⬘-gatcctcgagga caagatggaaaccaacttctccact-3⬘ and 5⬘-gatcgaattcttacaagtcctcttcagatatcagct tttgctcggccattgcctgtaa-3⬘; FPRL2 was amplified with oligonucleotides 5⬘gatcctcgaggacaagatggaaaccaacttctccatt-3⬘ and 5⬘-gatcgaattcttacaagtcctctt cagatatcagcttttgctcggccattgcttgtaa-3⬘. The PCR products were inserted into the pcDNA3.1 vector (Invitrogen, Karlsruhe, Germany). All constructs were confirmed by sequencing (SEQLAB, Go¨ ttingen, Germany). Plasmids encoding FPRL1 or FPRL2 were transfected into HEK 293 cells by electroporation as previously described (14) and cultured in supplemented RPMI 1640 in the presence of 800 ␮g/ml geneticin (G418; PAA Laboratories, Co¨ lbe, Germany) to maintain selection. Resistant clones were analyzed for receptor expression by immunofluorescence using the monoclonal 9E10 Ab (Alexis, Lausen, Switzerland) specific for the myc epitope.

Fluorescence microscopy of stained actin Granulocytes were allowed to adhere to coverslips by a 20 min incubation at 37°C in unsupplemented RPMI 1640. Nonadherent cells were removed by washing. The remaining cells were then incubated in the presence or absence of fMLP or the annexin 1 peptide. After washing, cells were fixed for 4 min in ⫺20°C methanol and stained for filamentous actin (F-actin) using rhodamine-phalloidin. Subsequently, the cells were washed extensively in PBS and mounted using Mowiol containing 4% n-propylgallat. Images were acquired using a cooled CCD camera (Micromax; Princeton Instruments, Trenton, NJ) installed on a DM RXA fluorescence microscope (Leica, Wetzlar, Germany).

Calcium mobilization assay Monocytes, granulocytes, stably transfected HEK 293 cells expressing FPR (FPR-293), FPRL1 (FPRL1-293), FPRL2 (FPRL2-293), or parental HEK 293 cells, respectively, were loaded with fura 2-AM (Molecular Probes, Leiden, The Netherlands) by incubating 107 cells/ml with 1 ␮M (granulocytes), 3 ␮M (HEK 293), or 5 ␮M (monocytes) fura 2-AM in HBSS supplemented with 2 mM CaCl2 at 37°C for 30 min (HEK 293,

ANNEXIN 1 ACTIVATES FPRLs monocytes) or 45 min (granulocytes) in the dark. Subsequently, the cells were washed twice and resuspended (2–5 ⫻ 106 cells/ml) in HBSS containing 2 mM Ca2⫹. Stimulants were added at the time points and concentrations indicated. Mobilization of intracellular Ca2⫹ was measured by recording the ratio of fluorescence emission at 510 nm after sequential excitation at 340 and 380 nm.

Chemotaxis assay Migration of human monocytes, HEK 293 cells stably expressing FPR, FPRL1, or FPRL2 or parental HEK 293 cells, respectively, was assessed using a microchemotaxis chamber (NeuroProbe, Gaithersburg, MD) according to the manufacturer’s protocol. Briefly, chemotactic factors diluted in assay medium (RPMI 1640, 1% BSA, 20 mM HEPES, pH 7.4) were added in the wells of the lower compartment and the cell suspension was placed in the wells of the upper compartment of the chamber. The two compartments were separated by a filter (10 ␮m pores; NeuroProbe). For migration of the transfected cells, filters were coated with fibronectin. Cells were allowed to migrate for 90 min (monocytes) or 120 min (293 cells) at 37°C in humidified air with 5% CO2. Subsequently, the filter was removed, stained with DiffQuick and the migrated cells were counted. To assess migration of granulocytes, chemoattractants diluted in 600 ␮l assay medium were placed into 24-well cluster plates. Granulocytes (5 ⫻ 105 cells/ 100 ␮l assay medium) were added to inserted Transwell chambers containing polycarbonate membranes (5 ␮m pore size; Corning, SchipholRijk, The Netherlands), allowed to migrate for 30 min, and counted using a Coulter counter (Fullerton, CA) with gates set for granulocytes. Results were expressed as the mean chemotactic index (fold increase of cells migrating in response to stimuli compared with assay medium alone). All experimental setups were performed at least three times, with at least triplicate parallel samples. Statistical significance of the results was evaluated by unpaired Student’s t tests.

Results Annexin 1 peptide-induced activation of human leukocytes Previous studies revealing the anti-inflammatory effect of annexin 1 in different models of inflammation have demonstrated that the activities displayed by full-length annexin 1 are faithfully and completely retained within the N-terminal peptide Ac1-25 (for example, see Refs. 27, 9, 23). Moreover, proteolytic cleavage at position 26 of the N-terminal domain was previously shown to occur in different cells and N-terminal proteolysis was markedly induced upon neutrophil extravasation with only 25% of neutrophil annexin 1 remaining intact (28, 29). Because this strongly suggested that the N-terminal peptide Ac1-25 is the physiological and pathophysiological active compound, Ac1-25 was used throughout the study. With this N-terminal peptide (as well as full-length annexin 1) we could show previously that its inhibitory effect on the transendothelial migration of granulocytes is due to an interaction with and desensitization of FPR (14). To further characterize the ability of annexin 1 to activate leukocytes, we analyzed whether the Nterminal peptide Ac1-25 can also act as a chemoattractant. As shown in Fig. 1A, the annexin 1 peptide induced the directional migration of human peripheral blood granulocytes and monocytes in a dose-dependent manner. When equal concentrations of Ac1-25 were present in both the lower and the upper wells of the migration chamber, no increase in cell migration was observed (data not shown). Thus, the observed migration induced by Ac1-25 was based on chemotaxis rather than chemokinesis. Granulocytes stimulated by chemoattractants respond by rapidly adopting a polarized morphology, with distinctive leading and trailing edges, and localized actin polymerization. Comparison of the granulocyte morphology and F-actin content upon stimulation with the prototypical bacterial chemoattractant fMLP or with the annexin 1 peptide Ac1-25, respectively, revealed that both FPR agonists caused rearrangements of F-actin and a substantial alteration in cell shape, leading to cell polarization (Fig. 1B). Thus, in addition to inhibiting granulocyte migration by desensitizing FPR (14), annexin 1 can also elicit chemotaxis most likely by activating FPR or a related family member.


FIGURE 1. Directional migration of human peripheral blood granulocytes and monocytes. A, Chemotactic activity of the annexin 1 peptide Ac1-25 for human granulocytes and monocytes. Migration of granulocytes (䡺) or monocytes (f) in response to different peptide concentrations is presented as the average chemotaxis index ⫾ SEM of triplicate wells. ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01. B, The annexin 1 peptide Ac1-25 alters neutrophil morphology and F-actin distribution. Rhodamine-phalloidin staining of F-actin in unstimulated granulocytes (a) or cells treated with 50 ␮M Ac1-25 for 2 min (b) or 5 min (c). Cells were fixed following treatment and analyzed by fluorescence microscopy. Note that Ac1-25 causes remodeling of the actin cytoskeleton, cell polarization, and spreading. Bar represents 10 ␮m.

To determine the specificity in the activation mediated by the annexin 1 peptide and to elucidate the range of leukocyte responses elicited, we performed desensitization experiments using human monocytes which are known to express all three members of the FPR subfamily (24). Desensitization induced by one agonist

FIGURE 2. Dose-response curves of intracellular Ca2⫹ mobilization in monocytes. Monocytes loaded with fura 2-AM were stimulated with increasing concentrations of the fMLP analog NfNleLFNleYK (A), fMLP (B), WKYMVM (C) or Ac1-25 (D). Fluorescence ratios of representative recordings are presented. Arrows indicate agonist addition.

7671 toward another generally indicates activation of the same receptor. To measure such homologous desensitization in the case of FPR/ FPRLs on human monocytes, we recorded intracellular Ca2⫹ mobilization in response to different peptide ligands, the fMLP analog NfNleLFNleYK and fMLP used at low or high concentrations to trigger FPR or FPRL1, respectively, the artificial WKYMVM peptide known to activate FPRL2 and FPRL1 (but not FPR) (24) and the annexin 1 peptide Ac1-25. As shown in Fig. 2, stimulation with all peptides elicited a dose-dependent sustained increase in intracellular Ca2⫹. When monocytes were first stimulated with the known FPR family agonists, the fMLP analog NfNleLFNleYK, fMLP or WKYMVM given at saturating concentrations (Fig. 2), no significant second Ca2⫹ mobilization was observed in response to a second stimulation with the same agonist (data not shown). In contrast, in each case a response was obtained with the annexin 1 peptide as second challenge (Fig. 3, A–C). These results suggest that the annexin 1 peptide might either use a completely different receptor or act agonistically on all three members of the FPR family, thereby activating the remaining two susceptible receptors when one family member is desensitized upon binding of the specific peptide ligand. The fact that when cells were restimulated with NfNleLFNleYK, fMLP, or WKYMVM after a first challenge with the annexin 1 peptide at saturating concentrations (100 ␮M), no significant second rise in intracellular Ca2⫹ could be observed (Fig. 3, D–F) indicates that Ac1-25 was able to activate all FPR family members. Likewise, cells stimulated with annexin 1 peptide were unable to generate a second rise in intracellular Ca2⫹ upon a second challenge with this peptide (data not shown). To further test this hypothesis, we aimed at activating and desensitizing all FPR/FPRL receptors expressed in leukocytes. We therefore used the synthetic D-methionine containing peptide WKYMVm, which had been shown to activate all receptors of the FPR family (24, 30, 31). When monocytes or granulocytes were exposed to WKYMVm, the cells were unable to respond to a second stimulation with either the same agonist (Fig. 4, A and E), NfNleLFNleYK (Fig. 4, B and F) or fMLP (Fig. 4, C and G),

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ANNEXIN 1 ACTIVATES FPRLs

FIGURE 3. Densensitization of Ca2⫹ mobilization in monocytes triggered with FPR/FPRL agonists. Cells were loaded with fura 2-AM and the fluorescence ratios were recorded to analyze the agonist-induced rise in intracellular Ca2⫹. Stimulation with the fMLP analog NfNleLFNleYK (A), fMLP (B), or WKYMVM (C) at the concentrations indicated did not desensitize the cells toward a second challenge with Ac1-25. No significant second peak indicative of a second stimulation was observed in cells first activated with Ac1-25 and then restimulated with the fMLP analog NfNleLFNleYK (D), fMLP (E), or WKYMVM (F) at the concentrations indicated (homologous desensitization). Curves show representative experiments. Arrows indicate the time point of agonist addition.

respectively. This indicates that after the first stimulation with WKYMVm, the cells were subsequently unable to mobilize intracellular Ca2⫹ through the FPR family receptors due to homolo-

gous desensitization. Activation by WKYMVm also blocked annexin 1 peptide-induced Ca2⫹ mobilization (Fig. 4, D and H), indicating that both peptides act on the same receptors. Thus, the

FIGURE 4. Simultaneous desensitization of all FPR family members inhibits Ac1-25 induced Ca2⫹ mobilization. Fura 2-AM loaded monocytes (A–D) or granulocytes (E–H) were first stimulated with WKYMVm, an agonist for all known FPRs. WKYMVm was used at 4 ␮M, a concentration fully desensitizing the cells toward a second WKYMVm challenge (data not shown). Cells were then challenged with an agonist specific for a given receptor. Arrows indicate agonist addition. Tracings of representative experiments are shown.


FIGURE 5. The annexin 1 peptide Ac1-25 activates all members of the FPR family. HEK 293 cells stably expressing FPR, FPRL1, or FPRL2, respectively, were loaded with fura 2-AM and stimulated with various concentrations of Ac1-25. For comparison, FPR-293 cells (A) were additionally triggered with the fMLP analog NfNleLFNleYK, FPRL1–293 cells (B) with fMLP, and FPRL2–293 cells (C) with WKYMVM used at the concentrations indicated. In the case of WKYMVM, micromolar concentrations were used to ensure full receptor activation in this cell system. Fluorescence ratios of representative recordings are presented. Arrows indicate agonist addition. To simplify the illustration, only one tracing for a given concentration of the known agonist (NfNleLFNleYK, fMLP, WKYMVM) is included.

annexin 1 peptide Ac1-25 appears to stimulate monocytes and granulocytes through all FPR family receptors. The annexin 1 peptide is an agonistic ligand of all members of the human FPR family To elucidate whether the annexin 1 peptide can indeed activate all three FPR family members, we next measured intracellular Ca2⫹ mobilization upon Ac1-25 stimulation of HEK 293 cells stably expressing only FPR, FPRL1, or FPRL2. We did not observe Ca2⫹ mobilization in the parental HEK 293 cells with any of the peptides tested, i.e., Ac1-25 or the known agonists NfNleLFNleYK, fMLP, and WKYMVM, respectively (data not shown). As ex-

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FIGURE 6. Cross-desensitization of HEK 293 cells stably expressing FPR, FPRL1, or FPRL2, respectively. HEK 293 cells stably expressing the receptors indicated; FPR (A and B), FPRL1 (C and D), FPRL2 (E and F) were loaded with fura 2-AM and subjected to Ca2⫹ mobilization recordings following different peptide challenges. To identify cross-desensitization, the cells were first triggered with the annexin 1 peptide Ac1-25 followed by the receptor-specific agonistic peptide (A, C, and E) or subjected to peptide stimulation in the opposite order, i.e., first the specific agonist and then Ac1-25 (B, D, and F). Fluorescence ratios of representative recordings are given. Note the desensitization of each FPR by Ac1-25. Some residual Ca2⫹ mobilization is seen when high fMLP concentrations are used as a second challenge (C), possibly due to a minimal receptor reactivation during the time between the first and second challenge.

pected, low nanomolar concentrations of the fMLP analog NfNleLFNleYK or micromolar concentrations of the annexin 1 peptide induced Ca2⫹ transients in FPR expressing HEK 293 cells (Fig. 5A). This reflects the ⬃10,000-fold higher affinity of FPR for fMLP as compared with the annexin peptide (14). In contrast, cells stably expressing the low affinity fMLP receptor FPRL1, responded only to higher fMLP concentrations in the micromolar range (Fig. 5B), whereas the FPRL2 transfected cells failed to mobilize intracellular Ca2⫹ upon fMLP stimulation even when micromolar concentration of the formylated peptide were used (data not shown). The synthetic WKYMVM peptide, which had

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ANNEXIN 1 ACTIVATES FPRLs FPRL1- and FPRL2-mediated chemotaxis is elicited by the annexin 1 peptide Because the annexin 1 peptide initiated a migratory response in monocytes, we next examined the ability of the Ac1-25 peptide to elicit chemotaxis in the HEK transfectants. Control cells, i.e., nontransfected HEK 293 cells, did not migrate in response to any of the peptides tested (data not shown). The fMLP analog NfNleLFNleYK induced migration of FPR-293 cells at nanomolar concentrations (Fig. 7A), whereas FPRL1-293 cells only responded to high (micromolar) levels of fMLP in a chemotactic manner (Fig. 7B). FPRL2-expressing 293 cells failed to migrate in response to fMLP (data not shown) but migrated upon exposure to WKYMVM. When challenged with the annexin 1 peptide, all three ectopically expressed receptors mediated a potent migratory response, indicating that Ac1-25 can induce chemotaxis through FPR, FPRL1, and FPRL2, respectively (Fig. 7).

Discussion

FIGURE 7. Annexin 1 can use all members of the FPR family to induce chemotaxis. Various concentrations of chemoattractant were added in the lower wells of a chemotaxis chamber. HEK 293 cells stably expressing FPR, FPRL1, or FPRL2, respectively (106 cells/ml), were placed in the upper wells. Following incubation the number of cells that had migrated toward the chemoattractant source was determined as described in Materials and Methods. Cell migration is expressed as the average chemotaxis index (mean ⫾ SEM) of six wells. A, Migration of FPR-293 cells in response to Ac1-25 or the fMLP analog NfNleLFNleYK is shown. B, The migration of FPRL1-293 cells in response to Ac1-25 or fMLP and (C) migration of FPRL2-293 cells in response to Ac1-25 or the synthetic peptide WKYMVM. ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01

been reported to activate both FPRL1 and FPRL2 (24), induced mobilization of Ca2⫹ in the FPRL2 expressing cells (Fig. 5C). Interestingly, the annexin 1 peptide not only triggered Ca2⫹ release in the FPR-293 cells but also induced Ca2⫹ fluxes in both, FPRL1–293 and FPRL2-293 cells, in a dose-dependent manner (Fig. 5, B and C). This shows that Ac1-25 is an agonist of both FPRL receptors and also identifies the peptide as the first endogenous ligand of FPRL2, so far considered an orphan receptor. In each case the annexin 1 peptide concentrations required to obtain a marked Ca2⫹ flux were in the low micromolar range, corresponding to the levels shown to elicit chemotaxis in granulocytes and monocytes (as previously mentioned). Desensitization experiments revealed that annexin 1 peptide-mediated activation of each of the receptors desensitized the respective cells toward a subsequent stimulation by the known specific agonist and vice versa (Fig. 6), suggesting desensitization due to two agonists sharing the same receptor.

In this study, we have demonstrated that a peptide derived from the unique N-terminal domain of annexin 1 elicits chemotaxis in human peripheral blood granulocytes and monocytes. Moreover, we could show that this peptide is capable of activating all three members of the human FPR family, FPR, FPRL1, and FPRL2. In each case analyzed, this activation induced a chemotactic response indicating that the annexin 1 derived peptide can function as an endogenous and activating ligand of all three FPRs known in humans. Although fMLP is a high affinity agonist for FPR, it binds to FPRL1 with low affinity and transduces signals via this receptor only when applied at high concentrations (18, 19). FPRL1 is a rather promiscuous receptor that is activated by a broad spectrum of unrelated agonists, including the SAA protein (21) and even a lipid metabolite, lipoxin A4 (22). It was shown recently to participate in mediating the anti-inflammatory activities of aspirin and glucocorticoids by responding to aspirin-triggered lipoxin A4 and dexamethasone-induced annexin 1 (Ac1-25) with a resulting down-regulation of neutrophil diapedesis (23). The second FPR homologue, termed FPRL2, is expressed in monocytes, but not in granulocytes. Although it shares 83% amino acid identity with FPRL1, FPRL2 is not activated by fMLP (24, 19) and the only reported agonist has been the synthetic hexapeptide WKYMVM. Our observations identify the annexin 1-derived peptide Ac1-25 as the first endogenous ligand of FPRL2. In most cases, chemotactic responses are elicited through high affinity ligand-receptor interactions whereas binding of the annexin 1 peptide most likely represents a low affinity ligand-receptor interaction. Previously identified ligand-G protein-coupled receptor interactions of relatively low affinity include those of different chemokines and their receptors, e.g., macrophage inflammatory protein-1␤ or RANTES and their interactions with CCR3 or CCR4, respectively (32, 33). Low affinity FPR (or FPRL) ligands might be essential for leukocyte recruitment toward the focus of inflammation where high concentrations of the chemoattractant are met and in which high affinity ligand-receptor interactions would result in receptor saturation, desensitization and/or sequestration. This scenario would require high (micromolar) extracellular concentrations of the annexin 1 peptide in close proximity to the focus of inflammation. Such concentrations are higher than annexin 1 levels reported in normal serum. However, annexin 1 expression is induced by glucocorticoids and IL-6 (34 –36) and systemic annexin 1 concentrations have been shown to increase at least 10-fold in a number of inflammatory and infectious diseases (37–39). Furthermore, leukocytes have been reported to release annexin 1 under certain conditions (40) and thus may produce relatively high concentrations of the protein in their microenvironment. Finally, annexin 1

The Journal of Immunology could be released by tissue or cell damage locally generating high extracellular concentrations. In such cases the released annexin 1 is most likely proteolytically cleaved and the active N-terminal peptides are generated. In fact, annexin 1 added to activated human granulocytes is quickly cleaved within its N-terminal domain, most likely by a liberated granulocyte protease (U. Rescher and A. Wilbers, unpublished observation). A substantial degree of N-terminal annexin 1 proteolysis also occurs within cells, e.g., in neutrophils upon extravasation into the subendothelium (29). Collectively, these data indicate that the N-terminal annexin 1 peptide is the physiologically active entity released from cells or generated extracellularly at sites of inflammation. The importance of the free N-terminal peptide is also underscored by recent crystal structure data of full-length annexin 1. In this structure a substantial part of the N-terminal domain is buried in the protein core being inaccessible for protein (e.g., FPR/L) interaction and it requires high Ca2⫹ concentrations to release this part (41, 42). Thus, the proteolytically generated N-terminal peptide is most likely the annexin 1 derivative regulating leukocyte activities through FPR family member interactions and its concentration at local inflammatory sites is likely to be within the range necessary to attract and activate leukocytes. Interestingly, other known ligands of FPRL1, namely SAA and lipoxin A4, also show markedly increased levels during inflammation (43, 44). Apart from inducing chemotaxis, the annexin 1 N-terminal peptide can also desensitize FPR family members thereby rendering leukocytes unresponsive to additional stimuli and thus limiting the degree of inflammation. Such anti-inflammatory and antimigratory activity is observed when the annexin 1 peptide is applied exogenously in mouse models of inflammation (9, 23) or when it is present at the luminal side of the endothelium in in vitro models of human granulocyte diapedesis (14). The balance between the anti-migratory and chemotactic activities of annexin 1 could depend on the receptors being used (FPR, FPRL1, or FPRL2) and the type of inflammation occurring. Although annexin 1 released during granulocyte extravasation (40) could trigger through its N-terminal peptide receptor desensitization and thereby reduce the extent of inflammation occurring in response to bacterial infections, annexin 1 peptides liberated during noninfectious tissue damage, i.e., in the absence of bacterial peptides, could chemoattract leukocytes.

Acknowledgment We thank Kris Palczewski (University of Washington, Seattle, WA) for critical reading of the manuscript.

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