Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

Yoshitaka Sogawa; Takao Ohyama; Hiroaki Maeda; Kazuki Hirahara

doi:10.1111/j.1365-2567.2010.03367.x

. 2011 Mar;132(3):441–450. doi: 10.1111/j.1365-2567.2010.03367.x

Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

Yoshitaka Sogawa ¹, Takao Ohyama ², Hiroaki Maeda ³, Kazuki Hirahara ³

PMCID: PMC3044910 PMID: 21039475

Abstract

It has been reported that the stimulation of neutrophils with N-formyl-Met-Leu-Phe (fMLF), an agonist for formyl peptide receptor (Fpr) 1, renders cells unresponsive to other chemoattractants in vitro. This is known as cross-desensitization, but its functional relevance in neutrophil migration in vivo has not been investigated. Here, we show that precedent stimulation of mouse neutrophils with compound 43, a non-peptidyl agonist for mouse Fpr1 and Fpr2, rendered the cells unresponsive to a second stimulation with C5a, leukotriene B₄, or keratinocyte-derived cytokine (KC) in calcium mobilization and chemotaxis assays in vitro. The expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on the surface of neutrophils was concomitantly diminished by stimulating the cells with the compound. Moreover, oral administration of the compound to mice before they were exposed to lipopolysaccharide (LPS) aerosol resulted in a dose-dependent reduction in the neutrophil count in bronchoalveolar lavage fluid. The expression of CXCR2 on blood neutrophils was also reduced in the compound-administered mice. The recipient mice that underwent adoptive transfer of fluorescence-labelled neutrophils that had been incubated with the compound showed a substantial decrease in neutrophil counts in bronchoalveolar lavage fluid after they were exposed to LPS, when compared with the control mice to which vehicle-treated neutrophils had been transferred. These results are consistent with the idea that the agonist for Fpr1 and Fpr2 induced cross-desensitization in neutrophils and attenuated neutrophil migration into the airways. Our results also revealed the unpredicted effect of an Fpr1 and Fpr2 dual agonist, which may act as a functional antagonist for multiple chemoattractant receptors in vivo.

Keywords: chemokine receptors, inflammation, lung/respiratory, neutrophils

Introduction

Polymorphonuclear neutrophil migration is a critical cellular response in innate immunity. It is widely accepted that neutrophil migration is regulated by chemotactic responses which are induced by the interactions of chemoattractants and their specific receptors expressed on the cell surface membrane. The formyl peptide receptor (FPR) is one of the most extensively studied G protein-coupled receptors involved in neutrophil chemotaxis (reviewed by Ye et al.¹). Human neutrophils express FPR1 and FPR2/ALX (receptor for lipoxin A4 and aspirin-triggered lipoxins, previously known as FPRL1), but not FPR3.² FPR1 was identified as a high-affinity receptor for N-formyl-Met-Leu-Phe (fMLF), a prototypical N-formyl peptide from Escherichia coli. It is well known that fMLF induces calcium flux and chemotaxis of neutrophils. FPR2/ALX was identified as a low-affinity receptor for fMLF, but accumulating evidence has revealed that the receptor interacts with a wide variety of structurally unrelated ligands.³ The biological effects of these ligands on neutrophil chemotaxis vary depending on the ligand. Interestingly, lipoxin A₄, which is thought to be an endogenous lipid ligand for FPR2/ALX, exerts anti-inflammatory and pro-resolving effects including the inhibition of neutrophil migration.¹^,⁴^–⁶ In mice, eight formyl peptide receptor genes have been identified,³^,⁷ and mouse Fpr1 and Fpr2 are considered to be the receptors homologous to human FPR1 and FPR2/ALX, respectively.³^,⁸ Mouse neutrophils also express both Fpr1 and Fpr2.⁸ The biological functions of these receptors in neutrophil migration in vivo, however, are not fully understood. Previous studies using Fpr1-deficient mice⁹^,¹⁰ demonstrated the critical role of Fpr1 in host defence, but no difference was observed in neutrophil migration towards infected tissues between the gene-deficient and wild-type mice, calling into question the significance of Fpr1 in neutrophil migration. In Fpr2-deficient mice, a critical role for mFpr2 in the progression of allergic airway inflammation with eosinophil infiltration in the lungs has been demonstrated.¹¹ However, in terms of neutrophil migration in interleukin (IL)-1β-induced air pouch or zymosan peritonitis assays, no difference was observed between Fpr2-deficient mice and wild-type mice.¹² Thus, the biological roles of Fpr1 and Fpr2 in neutrophil migration in vivo remain elusive.

To study the function of Fprs in neutrophil migration, we focused on the unique feature of fMLF that can induce cross-desensitization in neutrophils in vitro. Cross-desensitization is the heterologous desensitization of chemoattractant receptors; that is, stimulation of neutrophils with one chemoattractant renders the cells unresponsiveness to subsequent stimulation with unrelated chemoattractants. Indeed, the stimulation of FPR1 with fMLF desensitized not only FPR1 but also C5aR and chemokine (C-X-C motif) receptor 2 (CXCR2), and inhibited neutrophil responses, such as calcium mobilization and chemotaxis, which are induced by C5a or IL-8.¹³^–¹⁷ It has also been reported that fMLF induces the internalization of CXCR2 from the cell surface as a result of cross-desensitization.¹⁷ C5a and IL-8 can also cross-desensitize FPR1, but there is a hierarchy in the ability to induce cross-desensitization among chemoattractants, with a rank order of fMLF > C5a > IL-8.¹⁴^,¹⁶ Therefore, stimulation of FPR1 appears to have a dominant effect on the induction of cross-desensitization in neutrophils. The phenomenon of cross-desensitization has only been documented in studies using human cells in vitro, and it has not been demonstrated whether or not an Fpr1 agonist can induce cross-desensitization and inhibit neutrophil migration in vivo or even in vitro in mice. This is partly because the agonistic activity of fMLF to mouse Fpr1 is weaker than that to human FPR1.¹⁸ It is also because peptides are generally unstable and rapidly degrade in vivo, which has made it difficult to address this issue in mice until a potent and stable non-peptidyl agonist for mouse Fprs is found.

In a previous study, we reported that the non-peptidyl pyrazolone compound 43 (Cpd43) is a dual agonist for human FPR1 and FPR2/ALX and is also a dual agonist for mouse Fpr1 and Fpr2.¹⁹ It was reported that this compound did not show significant interactions with a series of unrelated protein targets, including CXCR2 and mitogen-activated P38α kinase.²⁰ In addition, the compound was orally active in mice.²⁰ Cpd43 is thus the first potent, orally active compound to show agonistic activity for mouse Fpr1, as well as Fpr2. This prompted us to test the hypothesis that stimulation of Fpr1 and Fpr2 induces cross-desensitization in vitro and inhibits neutrophil migration in vivo in mice exposed to lipopolysaccharide (LPS) aerosol, which is a well-described mouse model for studying neutrophil migration into the airway.²¹

In this study, we found that neutrophils stimulated with Cpd43 lost their chemotactic responses to chemoattractants in vitro, and that oral administration of Cpd43 to mice inhibited LPS-induced neutrophil migration into the airways. These results are consistent with the idea that the agonist for mouse Fpr1 and Fpr2 induced cross-desensitization in neutrophils and attenuated neutrophil migration into the airways. Our results also revealed the unpredicted effect of an Fpr1 and Fpr2 agonist, which may act as a functional antagonist for multiple chemoattractant receptors.

Materials and methods

Reagents

The N-formyl peptide fMLF, leukotriene B₄ (LTB₄), Histopaque-1077, Histopaque-1119, and LPS (from Salmonella enterica serotype Minnesota) were purchased from Sigma-Aldrich (St Louis, MO). A peptide WKYMVM (Trp-Lys-Tyr-Met-Val-Met-NH2) was purchased from Tocris (Ellisville, MO). Recombinant mouse keratinocyte-derived cytokine (KC) and mouse C5a (mC5a) were purchased from R&D Systems (Minneapolis, MN). All cell culture media used in this study were purchased from Life Technologies (Carlsbad, CA). Fluo 4-AM and Calcein-AM were purchased from Dojindo Laboratories (Kumamoto, Japan). Phycoerythrin (PE)-conjugated anti-Gr-1 monoclonal antibody (mAb) was purchased from BD Biosciences (Palo Alto, CA). PE-conjugated anti-mouse CXCR2 mAb was purchased from R&D Systems. N-(4-chlorophenyl)- N‘-(5-isopropyl-1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)urea (Cpd43) was synthesized at Medicinal Chemistry Research Laboratories at Daiichi Sankyo (Tokyo, Japan).

Mice

Male BALB/c mice 5–7 weeks of age were purchased from Charles River Laboratories Japan (Kanagawa, Japan). All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Daiichi Sankyo.

Cell preparation

Mouse bone marrow cells were harvested by flushing marrow from femurs and tibias with RPMI-1640 medium. Mouse neutrophils were isolated from bone marrow cells by density centrifugation with Histopaque-1077 and Histopaque-1119. The purity of isolated neutrophils was routinely > 95% as assessed by light microscopic analysis of the cells stained with Diff-Quick (Wako Pure Chemical Industries, Osaka, Japan), and > 98% viable as assessed by a trypan blue exclusion test.

Measurement of calcium flux

Mouse bone marrow cells were labelled with PE-conjugated anti-Gr-1 mAb, and then incubated with 4 μm Fluo4-AM and 0·16% pluronic acid (Life Technologies) at 37° in 5% CO₂ for 30 min. The cells were washed and re-suspended in Iscove's modified Dulbecco's medium (IMDM) with 0·5% bovine serum albumin (BSA). The cells were pre-stimulated with Cpd43 at room temperature for 30 min and subsequently stimulated with 100 ng/ml KC, 1 ng/ml mC5a, or 10 nm LTB₄ without a washout step. Calcium flux in Gr-1⁺ neutrophil population was detected as a change in fluorescence intensity in neutrophils with a flow cytometer (Cytomics FC500; Beckman Coulter, Fullerton, CA). The mean fluorescence intensity (arbitrary units) in the 5-second period immediately before and after stimulation was calculated. Assays were performed in duplicate. Results are expressed as mean fluorescence intensity.

Chemotaxis assays

Chemotaxis assays were performed using HTS Transwell-96 plates with 3-μm pores (Corning, Lowell, MA). Mouse neutrophils isolated from bone marrow were re-suspended in RPMI-1640 medium supplemented with 20 mm HEPES. The cells were incubated with Calcein-AM at 37° for 30 min, then washed, and re-suspended in RPMI-1640 medium supplemented with 2% fetal bovine serum (FBS) and 55 μm 2-mercaptoethanol (2-ME). The cells were incubated with various concentrations of Cpd43 at 37° for 30 min, and then washed twice to remove the compound. The cells were added into the upper wells (2 × 10⁵ cells/well) and 1 μg/ml KC, 10 ng/ml mC5a or 10 nm LTB₄ was added to the lower wells. The plates were incubated at 37° for 30 min. After removing the upper wells, migrated cells were lysed with sodium dodecyl sulphate (SDS), and the resultant fluorescence intensity was measured with a plate reader (ARVO SX1420; PerkinElmer, Waltham, MA). Assays were performed in triplicate. Results are expressed as the percentage of net migrated cells.

Flow cytometric analysis on cell surface receptors

Mouse neutrophils isolated from bone marrows or mouse whole blood were incubated with or without various concentrations of Cpd43 at room temperature for 30 min, and subsequently stained with PE-conjugated anti-CXCR2 at room temperature for another 30 min in the presence of Cpd43. The erythrocytes in the whole blood were lysed using a mouse erythrocyte lysing kit (R&D Systems). The stained cells were washed with wash buffer included in the erythrocyte lysing kit, and then fixed with 2% paraformaldehyde. The expression of CXCR2 on neutrophils was analysed with FACSCanto (BD Biosciences). The gated cells were all Gr-1 positive (> 98%). Data were analysed using Flowjo software (Tree Star, Ashland, OR).

LPS-induced neutrophil migration into the airway

Mice were exposed to aerosols of 0·1% [weight/volume (w/v)] LPS in saline generated with an ultrasonic nebulizer (NE-U12; Omron, Kyoto, Japan) for 30 min in a chamber. The mice in the control group were not exposed to LPS. At 4 hr after LPS exposure, the mice were anaesthetized using pentobarbital. The trachea was cannulated, the lungs were lavaged three times with 1 ml of saline, and 3 ml of bronchoalveolar lavage fluid (BALF) in total was collected. The BALF was centrifuged and re-suspended in 0·3 ml of saline. Cell counts were obtained using a haematology analyser, XT-2000i (Sysmex, Hyogo, Japan). Cpd43 suspended in 0·5% methyl cellulose was orally administered 15 min before LPS exposure. Only 0·5% methyl cellulose was administered to mice in the control group and the vehicle group. In some experiments, the mice were killed at 1–4 hr after LPS exposure, and BALF and blood were collected. The numbers of neutrophils in BALF and blood were analysed with XT-2000i. For the flow cytometric analysis, mouse blood was collected at 1 hr after LPS exposure. Whole blood cells were washed with phosphate-buffered saline (PBS) containing 0·5% BSA and stained with PE-conjugated anti-mouse CXCR2 mAb in Hanks’ balanced salt solution (HBSS) supplemented with 10 mm HEPES, 2 mm ethylenediaminetetraacetic acid (EDTA), 2% FBS, penicillin, and streptomycin on ice for 30 min. Fixation of the cells and the analysis of receptor expression on neutrophils were performed as described above.

Cell transfer experiment

Mouse bone marrow cells were harvested from normal mice and incubated with 5 μm Calcein-AM and either 10 μm Cpd43 or vehicle (0.1% dimethyl sulfoxide) in RPMI-1640 medium at 37° for 30 min. The cells were washed and re-suspended in PBS. Recipient mice were intravenously injected with the labelled cells from the tail vein (1 × 10⁷ cells in 0·2 ml PBS/animal) and exposed to an aerosol of 0·1% LPS in saline immediately after cell transfer. The mice in the control group were not exposed to LPS. Four hours after LPS exposure, BALF was collected. The cells in BALF were counted using a haematology analyser, XT-2000i, and the percentage of labelled neutrophils was determined with FACSCanto. The concentration of labelled cells in BALF was calculated by multiplying the concentration of total neutrophils in BALF and the percentage of labelled cells in the neutrophil population in BALF.

Statistics

Statistical analysis was applied to data obtained from in vivo animal experiments. Student's t-test was used to compare the control and vehicle-administered groups, and Dunnett's test was used to compare the vehicle-treated and compound-administered groups in experiments depicted in Fig. 4. Student's t-test was used to compare the vehicle-treated group and the compound-treated group in experiments depicted in Fig. 7. P values < 0·05 were considered significant.

Cpd43 inhibits neutrophil migration into the airways in lipopolysaccharide (LPS)-exposed mice. Mice were exposed to LPS aerosol for 30 min. Cpd43 or vehicle was administered 15 min before LPS exposure. The mice in the control group were not exposed to LPS. At 4 hr after LPS exposure, bronchoalveolar lavage fluid (BALF) was collected. The concentrations of neutrophils in BALF are shown (mean ± standard error). **P<0·01 (versus the control group); †P<0·05, and ‡P<0·01 (versus the vehicle-treated group); n=6 (control group, n=4) per group. The experiment was repeated twice with similar results.

Neutrophils treated with Cpd43 *in vitro* do not migrate into the airways in lipopolysaccharide (LPS)-exposed recipient mice. Mouse bone marrow cells from normal mice were incubated with 5 μm Calcein-AM and either 10 μm Cpd43 or vehicle at 37° for 30 min. Recipient mice were intravenously injected with the labelled cells from the tail vein and exposed to a 0·1% LPS aerosol immediately after cell transfer. The mice in the control group were not exposed to LPS. Four hours after LPS exposure, bronchoalveolar lavage fluid (BALF) was collected. The cells in the BALF were counted and the percentage of labelled neutrophils was determined using a flow cytometer. Data (mean ± standard error) are expressed as the concentration of labelled cells in the neutrophil population in BALF. ‡P<0·01 (Student's t-test, versus the vehicle group); n=5 per group. The experiment was repeated twice with similar results. N.D., not detected.

Results

Cpd43 induces cross-desensitization of intracellular calcium mobilization in neutrophils

We previously reported that Cpd43 has agonistic activities for mouse Fpr1 and Fpr2, with 50% effective concentration (EC₅₀) values of 0·35 and 0·89 μm, respectively, in aequorin assays measuring calcium mobilization (Table 1 and Sogawa et al.¹⁹). In this study, we first investigated whether Cpd43 would induce cross-desensitization in mouse neutrophils. Mouse bone marrow-derived neutrophils were pretreated with or without Cpd43 for 30 min and subsequently stimulated with other chemoattractants, and calcium mobilization at the second stimulation was measured. The pretreatment with Cpd43 dose-dependently inhibited calcium mobilization in neutrophils stimulated with mC5a, LTB₄ or KC (Fig. 1). All the cells showed calcium mobilization in response to 100 μm ATP stimulation even after pretreatment with 10 μm Cpd43 (data not shown). Thus, Cpd43 induced cross-desensitization of chemoattractant-stimulated calcium mobilization in mouse neutrophils.

Table 1.

Pharmacological parameters of Cpd43 for mouse formyl peptide receptor 1 (Fpr1) and Fpr2

	Ca²⁺ mobilization EC₅₀ (nm)¹

Compound	mFpr1	mFpr2
Cpd43	350	890
fMLF	170	31 000
WKYMVM	510	25

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Fifty percent effective concentration (EC₅₀) values were calculated from the results previously reported by Sogawa et al.¹⁹ in which calcium mobilization in Chinese hamster ovary cells expressing either mouse Fpr1 or Fpr2 was measured. Two peptides were used as control agonists.

fMLF, N-formyl-Met-Leu-Phe.

Cpd43 induces cross-desensitization of the Ca²⁺ response in neutrophils. Bone marrow cells were labelled with phycoerythrin (PE)-conjugated anti-Gr-1 antibody (Ab) and then loaded with Fluo-4AM. The cells were prestimulated with various concentrations of Cpd43 for 30 min and subsequently stimulated with 100 ng/ml keratinocyte-derived cytokine (KC), 1 ng/ml mC5a, or 10 nm leukotriene B₄ (LTB₄). The calcium flux after the second stimulation in the Gr-1⁺ neutrophil population was analysed using a flow cytometer. Data (mean ± standard error) are expressed as the mean fluorescence intensity in the 5-second period immediately before (white bar) and after (black bar) stimulation. The experiment was performed in duplicate and repeated twice with similar results. AU, arbitrary units.

Cpd43 attenuates the chemotactic activity of neutrophils

We next investigated the effect of cross-desensitization induced by Cpd43 on neutrophil chemotaxis. As Cpd43 itself induces mouse neutrophil chemotaxis with a maximal response at a concentration of 0·1 μm,¹⁹ we pretreated neutrophils with various concentrations of Cpd43 and washed it out before using the cells in chemotaxis assays to circumvent the effect of its chemotactic activity. Pretreatment with Cpd43 dose-dependently attenuated the chemotactic responses of mouse neutrophils to mC5a, LTB₄ or KC (Fig. 2). The inhibitory effect of Cpd43 was strong at concentrations of ≥ 0·1 μm. As it has been reported that fMLF induced the internalization of CXCR2 as a result of cross-desensitization in human neutrophils,¹⁷ we also investigated the expression of CXCR2, the receptor for KC, on mouse neutrophils treated with Cpd43. The expression of CXCR2 on the surface of bone marrow-derived neutrophils was diminished by stimulating the cells with Cpd43 in vitro in a concentration-dependent manner (Fig. 3a). Before we investigated the effect of Cpd43 in vivo, we tested whether it would induce the same effect on blood neutrophils. The expression of CXCR2 on blood neutrophils was also diminished by incubation with Cpd43 (Fig. 3b). The expression of receptors for mC5a and LTB₄ was not tested because there were no available mAbs that could specifically detect the molecules. The expression of CD11b, one of the surface activation markers on neutrophils, on bone marrow-derived and blood neutrophils was increased by the incubation of cells with Cpd43 (data not shown). Therefore, prior stimulation with Cpd43 inhibited neutrophil chemotaxis to several well-known chemoattractants, and Cpd43-treated neutrophils also showed a concomitantly decreased expression of CXCR2. These results indicate that Cpd43 induced cross-desensitization in mouse neutrophils in vitro.

Pretreatment of Cpd43 inhibits neutrophil chemotaxis induced by several chemoattractants. Mouse neutrophils isolated from bone marrow were preincubated with various concentrations of Cpd43 at 37° for 30 min and washed twice to remove the compound. Chemotaxis of these cells was induced with 1 μg/ml keratinocyte-derived cytokine (KC), 10 ng/ml mC5a or 10 nm leukotriene B₄ (LTB₄). Data (mean ± standard error) are expressed as the percentage of net migrated cells. The experiment was performed in triplicate and repeated twice with similar results.

Cpd43 diminishes the expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on neutrophils. (a, b) Mouse neutrophils isolated from (a) bone marrow or (b) whole blood were incubated with or without various concentrations of Cpd43 at room temperature for 30 min and subsequently stained with anti-CXCR2 monoclonal antibody (mAb). The erythrocytes in the whole blood were lysed. The expression of the receptor on neutrophils was analysed using a flow cytometer. Representative data from two different experiments with similar results are shown. Each histogram represents cells treated with Cpd43 at 0 μm (solid line), 0·1 μm (light grey), 1 μm (grey) or 10 μm (black). The dotted line represents the isotype control.

Cpd43 inhibits neutrophil migration in vivo

Next, we investigated whether Cpd43 would affect neutrophil migration in vivo in mice exposed to LPS aerosol. Exposure to LPS aerosol induced neutrophil migration into the airways, which was detected as an increased number of neutrophils in BALF. When mice were orally administered Cpd43 at doses of 3, 10, 30 or 100 mg/kg prior to LPS exposure, the number of neutrophils in BALF dose-dependently and significantly decreased (Fig. 4). To study the effect of Cpd43 on neutrophil recruitment into the airways more precisely, we performed a histological examination of lung tissues from LPS-exposed mice which were administered vehicle or Cpd43 at 4 hr after LPS exposure. We could not detect a significant number of neutrophils in any section of the lung specimens stained with haematoxylin and eosin in either group (data not shown). Next, we conducted a time–course analysis of the numbers of neutrophils in BALF and blood. In vehicle-treated mice, the numbers of neutrophils in BALF steadily increased and those in blood increased more rapidly following LPS exposure (Fig. 5). In contrast, the numbers of neutrophils in both BALF and blood did not increase in mice administered Cpd43. Thus, neutrophil mobility induced by LPS exposure in mice was inhibited in Cpd43-administered mice.

Time–course analysis of neutrophil counts in (a) bronchoalveolar lavage fluid (BALF) and (b) blood in lipopolysaccharide (LPS)-exposed mice. Mice were exposed to LPS aerosol for 30 min. Cpd43 or vehicle was administered 15 min before LPS exposure. The mice in the control group were not exposed to LPS. BALF and blood were collected at the indicated time-points. Data are the mean ± standard error; n=3–5 per group. The experiment was repeated twice with similar results.

As Cpd43-treated neutrophils showed a diminished expression of CXCR2 as a result of cross-desensitization in vitro, we investigated the expression of CXCR2 on neutrophils in blood collected from LPS-exposed mice. CXCR2 expression on mouse blood neutrophils diminished in Cpd43-administered mice compared with that in vehicle-administered mice (Fig. 6), suggesting that Cpd43 induced cross-desensitization of chemoattractant receptors in neutrophils in vivo as well as in vitro.

Administration of Cpd43 to mice decreases chemokine (C-X-C motif) receptor 2 (CXCR2) expression on neutrophils. Mice were exposed to LPS aerosol for 30 min. Cpd43 (30 mg/kg) or vehicle was administered 15 min before LPS exposure. An hour after LPS exposure, blood was collected. The expression of CXCR2 on neutrophils was analysed using a flow cytometer. Representative data from two different experiments (n=2–3) with similar results are shown. The histogram represents cells from the vehicle-treated mouse (solid line) and the Cpd43-treated mouse (black). The dotted line represents the isotype control.

Cpd43 exerts an anti-migratory effect in vivo by directly affecting neutrophils

To determine whether Cpd43 directly affected neutrophils and inhibited their migration in vivo, we conducted a cell transfer experiment. Fluorescence-labelled neutrophils treated with or without 10 μm Cpd43 were transferred into recipient mice. The concentration of 10 μm was set to induce sufficient cross-desensitization in neutrophils. The recipient mice were exposed to LPS aerosol immediately after cell transfer and BALF was collected 4 hr after LPS exposure. The fluorescence-labelled neutrophils were not detected in BALF from the control mice which were not exposed to LPS, but a significant number of labelled cells were detected in BALF from LPS-exposed mice. Compared with the control group, the fluorescence-labelled neutrophils in BALF significantly decreased in mice into which Cpd43-treated cells had been transferred (Fig. 7). There was no significant difference in the rates of fluorescence-labelled neutrophils in blood between the two groups of recipient mice into which either vehicle-treated cells or Cpd43-treated cells had been transferred (data not shown). These results suggested that neutrophils treated with Cpd43 lost the ability to migrate into the airways in mice. The inhibitory effect of Cpd43 on neutrophil migration in vivo was clearly shown in this cell transfer experiment.

Discussion

In this study, we showed that Cpd43, an Fpr1 and Fpr2 dual agonist, rendered mouse neutrophils unresponsive to other chemoattractants in vitro. Moreover, Cpd43 dose-dependently inhibited neutrophil migration into the airways in LPS-exposed mice, possibly through the induction of cross-desensitization. Our results showed for the first time that stimulation of Fpr1 and Fpr2 induced cross-desensitization in mouse neutrophils and attenuated neutrophil migration.

The indication of cross-desensitization which mediated marked inhibition of neutrophil migration in vivo was surprising because the phenomenon of cross-desensitization has previously been demonstrated only under in vitro conditions. Although fMLF-induced cross-desensitization in vitro has been well documented,¹³^–¹⁷ in most cases it has been reported that the administration of fMLF in vivo induces typical effects of chemoattractants on neutrophils, such as neutrophil infiltration into the airway induced by the inhalation of fMLF²²^–²⁴ and transient neutropenia induced by the intravenous administration of fMLF.²⁵^,²⁶ Among these studies, only Ley et al.²⁵ reported the possibility of an inhibitory effect of fMLF on neutrophil migration towards chemoattractants. They stated that the intravenous administration of fMLF to rabbits inhibited IL-8-induced extravasation of leukocytes, but the mechanism by which fMLF inhibited leukocyte extravasation was not mentioned in the report.

We first demonstrated that Cpd43 inhibited neutrophil responses to chemoattractants in calcium mobilization and chemotaxis assays in vitro. The Cpd43-treated neutrophils showed diminished expression of CXCR2. These results are consistent with observations in studies describing cross-desensitization in human cells induced by fMLF.¹³^–¹⁷ In calcium mobilization assays, we showed that the inhibitory effect of Cpd43 on KC- and LTB₄-induced calcium mobilization was stronger than that on mC5a-induced calcium mobilization. These results are consistent with the hierarchy of the ability to induce cross-desensitization (fMLF > C5a > IL-8),¹⁴ which means that the C5a-induced signal is more resistant to the induction of cross-desensitization. In addition to the flow cytometric analysis of CXCR2, we also investigated the mRNA expression of CXCR2 in Cpd43-treated neutrophils isolated from bone marrow. We found that mRNA expression was slightly decreased at 0·5 hr and was decreased by about half at 1 and 2 hr in response to incubation of bone marrow neutrophils with Cpd43 (Fig. S1). The moderate inhibition of CXCR2 mRNA expression by Cpd43 cannot explain the greatly diminished CXCR2 expression on the cell surface of bone marrow neutrophils within 1 hr (Fig. 3a), which supports the idea that the loss CXCR2 expression resulted from the receptor internalization associated with the cross-desensitization. Finally, we confirmed the high specificity of Cpd43 for FPRs in ligand-binding assays using a wide range of human receptors (Y. Sogawa, T. Ohyama, H. Maeda, K. Hirahara, manuscript in preparation). These data indicate that Cpd43 inhibited neutrophil chemotaxis towards several chemoattractants in vitro by inducing cross-desensitization of chemoattractant receptors on neutrophils.

Importantly, Cpd43 dramatically inhibited neutrophil migration into the airway in the LPS-exposed mice. As the neutrophil count in blood did not change at the time-points we tested, it is unlikely that the inhibition of neutrophil infiltration into the airway was a reflection of the reduced number of neutrophils in blood, which might have been caused by any detrimental effect of Cpd43 on neutrophils in vivo. In fact, oral administration of Cpd43 to mice did not show any distinct toxicity up to 300 mg/kg (data not shown). We also measured the concentration of KC in BALF, but it did not differ between the Cpd43- and vehicle-administered mice (data not shown). In addition, Cpd43 did not inhibit KC or tumour necrosis factor-α production by LPS-stimulated splenocytes in vitro (data not shown). These results suggest that the inhibition of neutrophil migration into BALF by Cpd43 was not attributable to the suppression of chemoattractant production. The phenomenon of reduced expression of CXCR2 on blood neutrophils in Cpd43-administered mice was consistent with our in vitro results, demonstrating that treatment with Cpd43 decreased CXCR2 expression on neutrophils in the wake of cross-desensitization induction. The concentration of Cpd43 unbound to plasma protein in blood increased and reached as high as 0·7 μm 0·5 hr after oral administration of Cpd43 at a dose of 30 mg/kg (data not shown), which was sufficient to exert agonistic activities for both Fpr1 and Fpr2. Thus, it was sufficient for induction of cross-desensitization in neutrophils in mice. These results are consistent with the idea that the oral administration of Cpd43 inhibited neutrophil migration through the induction of cross-desensitization of chemoattractant receptors in neutrophils in vivo.

We further conducted a cell transfer experiment and more directly demonstrated that neutrophils exposed to Cpd43 rapidly lost the ability to migrate in LPS-exposed mice, even in the absence of Cpd43 in the circulation. These results support the notion that Cpd43 directly inhibited the migratory activity of neutrophils. It is also conceivable that Cpd43 has some effect on neutrophil mobilization from bone marrow to blood by inducing cross-desensitization to CXCR2 and CXCR4, as it has been reported that these receptors are involved in the retention of granulocytes in bone marrow,²⁷^–³⁰ and that the stimulation of FPR2/ALX cross-desensitizes CXCR4.³¹^,³² Further studies will be required to elucidate the full effect of Cpd43 on neutrophil mobilization in vivo.

As Cpd43 has agonistic activity for both Fpr1 and Fpr2, it remains unclear how each receptor is involved in the induction of cross-desensitization in neutrophils in vitro and the inhibition of neutrophil migration in vivo. It is straightforward to consider that both Fpr1 and Fpr2 are involved in the mechanism of inducing cross-desensitization of chemoattracting receptors on neutrophils. Previous studies using cells transfected with a combination of FPR1 and other chemoattractant receptors indicated that cross-desensitization induced by fMLF in vitro was mediated by FPR1.¹³^,³³^,³⁴ It is reasonable to speculate that FPR2/ALX is also capable of inducing cross-desensitization in neutrophils, because it has been reported that some FPR2/ALX selective agonists induced cross-desensitization of CCR5 and CXCR4 in human monocytes and cells transfected with FPR2/ALX in combination with CCR5 or CXCR4.³¹^,³²^,³⁵ In addition, the Fpr2-dependent inhibitory effect of Cpd43 on neutrophil migration in the IL-1β-induced air pouch assay described in a study using Fpr2-deficient mice¹² implies that Fpr2-mediated cross-desensitization is operative in vivo as a mechanism of inhibition by Cpd43, although induction of cross-desensitization by Cpd43 was not investigated in that study. Obtaining highly selective ligands/antibody for each receptor and/or receptor-deficient mice will help to answer the question regarding the roles of Fpr1 and Fpr2 in the induction of cross-desensitization.

With regard to developing therapeutic agents, we also revealed an unpredicted effect of the Fpr1 and Fpr2 agonist, in that it may act as a functional antagonist for multiple chemoattractant receptors in vivo. It has been reported that agonists for receptors that are associated with leucocyte trafficking act as functional antagonists and effectively inhibit leucocyte migration. For example, a sphingosine 1-phosphate receptor agonist, such as fingolimod (FTY-720), exerts immunosuppressive activity by modulating lymphocyte trafficking.³⁶^–³⁹ As stimulation of FPR can desensitize multiple chemoattractant receptors as if each receptor is functionally antagonized by its specific ligand, FPR agonists should work as broad-spectrum functional antagonists for multiple chemoattracting receptors. Cpd43 inhibited neutrophil migration into the airways in LPS-exposed mice in which CXCR2 on neutrophils was involved in the migration,⁴⁰ and the CXCR2 antagonist also significantly inhibited neutrophil migration in LPS-exposed mice.⁴¹ Furthermore, we also found that Cpd43 markedly inhibited zymosan-induced neutrophil accumulation in the peritoneal cavity in mice (Fig. S2) in which C5aR on neutrophils was involved in the accumulation.⁴² These results indicate that Cpd43 inhibited neutrophil migration in circumstances in which CXCR2 and C5aR on neutrophils usually mediate neutrophil migration in vivo. This supports our idea that FPR agonists act as functional antagonists for multiple chemoattracting receptors through the induction of cross-desensitization of the receptors. This may suggest a new strategy for developing novel medicines that could effectively inhibit neutrophil migration, although it would be necessary to investigate the therapeutic effects of FPR agonists in various disease models, especially in more chronic or severe models. Recently, a number of non-peptidyl compounds acting as FPR agonists have been reported,²⁰^,⁴³^–⁴⁶ and more potent therapeutic agents than selective antagonists for chemoattractant receptors may develop from these.

In conclusion, we have found that Cpd43, an Fpr1 and Fpr2 dual agonist, induced cross-desensitization of chemoattractant receptors in mouse neutrophils. Moreover, Cpd43 inhibited neutrophil migration in mice, possibly through the induction of cross-desensitization in neutrophils. In addition, our findings suggest that the FPR1 and FPR2/ALX dual agonist has therapeutic potential for treatment of diseases involving neutrophilic inflammation.

Acknowledgments

The authors thank Dr Hitoshi Kurata for providing Cpd43; Dr Akiko Shimizugawa and Ms Miyuki Nagasaki for their assistance in performing the assays; and Dr Takeshi Shiiki for his assistance in performing the pharmacokinetic assays. We are also grateful to Dr Shinichi Kurakata for his critical review of the manuscript and for providing helpful advice.

Glossary

Abbreviations:

ALX: receptor for lipoxin A4 and aspirin-triggered lipoxins
BALF: bronchoalveolar lavage fluid
fMLF: N-formyl-Met-Leu-Phe
FPR (Fpr): formyl peptide receptor
KC: keratinocyte-derived cytokine
LPS: lipopolysaccharide
LTB₄: leukotriene B₄
mC5a: mouse C5a

Disclosures

The authors have no financial conflict of interest.

Supporting information

Additional supporting information may be found in the online version of this article:

Figure S1. The effect of Cpd43 on mRNA expression of chemokine (C-X-C motif) receptor 2 (CXCR2) in neutrophils. Neutrophils purified from bone marrow cells were incubated with various concentration of Cpd43 in duplicate. mRNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized with the High Capacity RNA-to-cDNA Kit (Life Technologies). Real-time polymerase chain reaction (PCR) for quantification of mRNA was performed using TaqMan Gene Expression Assays including primer and probe sets for detection of each target and the 7900HT Fast Real-Time PCR System (Life Technologies). Results were normalized to β-glucuronidase (GUS) expression. Relative expression data are the mean ± standard error for duplicate samples. The experiment was repeated twice with similar results.

imm0132-0441-SD1.tif^{(60.6KB, tif)}

Figure S2. Cpd43 inhibits neutrophil migration into the peritoneal cavity in zymosan-administered mice. Peritonitis was induced by intraperitoneal administration of 1 mg of zymosan in 0·5 ml of saline to mice. The mice in the control group were administered 0·5 ml of saline without zymosan. Compounds were orally administered 30 min before zymosan administration. At 4 hr after zymosan administration, the cells that were exuded into the peritoneal cavity were harvested. The counts of neutrophils in the peritoneal exuded cells are shown (mean ± standard error). **P < 0·01 (versus the control group) by Student's t-test; †P < 0·05, and ‡P < 0·01 (versus the vehicle-treated group) by Dunnett's test (n = 5 per group). The experiment was repeated twice with similar results.

imm0132-0441-SD2.tif^{(64.6KB, tif)}

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

imm0132-0441-SD1.tif^{(60.6KB, tif)}

imm0132-0441-SD2.tif^{(64.6KB, tif)}

[b1] 1.Ye RD, Boulay F, Wang JM, Dahlgren C, Gerard C, Parmentier M, Serhan CN, Murphy PM. International union of basic and clinical pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family. Pharmacol Rev. 2009;61:119–61. doi: 10.1124/pr.109.001578. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b2] 2.Murphy PM, Ozcelik T, Kenney RT, Tiffany HL, McDermott D, Francke U. A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family. J Biol Chem. 1992;267:7637–43. [PubMed] [Google Scholar]

[b3] 3.Migeotte I, Communi D, Parmentier M. Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses. Cytokine Growth Factor Rev. 2006;17:501–19. doi: 10.1016/j.cytogfr.2006.09.009. [DOI] [PubMed] [Google Scholar]

[b4] 4.Devchand PR, Arita M, Hong S, Bannenberg G, Moussignac RL, Gronert K, Serhan CN. Human ALX receptor regulates neutrophil recruitment in transgenic mice: roles in inflammation and host defense. FASEB J. 2003;17:652–9. doi: 10.1096/fj.02-0770com. [DOI] [PubMed] [Google Scholar]

[b5] 5.Bonnans C, Fukunaga K, Levy MA, Levy BD. Lipoxin A(4) regulates bronchial epithelial cell responses to acid injury. Am J Pathol. 2006;168:1064–72. doi: 10.2353/ajpath.2006.051056. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b6] 6.Maderna P, Godson C. Lipoxins: resolutionary road. Br J Pharmacol. 2009;158:947–59. doi: 10.1111/j.1476-5381.2009.00386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b7] 7.Gao JL, Chen H, Filie JD, Kozak CA, Murphy PM. Differential expansion of the N-formylpeptide receptor gene cluster in human and mouse. Genomics. 1998;51:270–6. doi: 10.1006/geno.1998.5376. [DOI] [PubMed] [Google Scholar]

[b8] 8.Hartt JK, Barish G, Murphy PM, Gao JL. N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR. J Exp Med. 1999;190:741–7. doi: 10.1084/jem.190.5.741. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b9] 9.Gao JL, Lee EJ, Murphy PM. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor. J Exp Med. 1999;189:657–62. doi: 10.1084/jem.189.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b10] 10.Gauthier JF, Fortin A, Bergeron Y, Dumas MC, Champagne ME, Bergeron MG. Differential contribution of bacterial N-formyl-methionyl-leucyl- phenylalanine and host-derived CXC chemokines to neutrophil infiltration into pulmonary alveoli during murine pneumococcal pneumonia. Infect Immun. 2007;75:5361–7. doi: 10.1128/IAI.02008-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b11] 11.Chen K, Le Y, Liu Y, et al. A critical role for the g protein-coupled receptor mFPR2 in airway inflammation and immune responses. J Immunol. 2010;184:3331–5. doi: 10.4049/jimmunol.0903022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b12] 12.Dufton N, Hannon R, Brancaleone V, et al. Anti-inflammatory role of the murine formyl-peptide receptor 2: ligand-specific effects on leukocyte responses and experimental inflammation. J Immunol. 2010;184:2611–9. doi: 10.4049/jimmunol.0903526. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b13] 13.Didsbury JR, Uhing RJ, Tomhave E, Gerard C, Gerard N, Snyderman R. Receptor class desensitization of leukocyte chemoattractant receptors. Proc Natl Acad Sci U S A. 1991;88:11564–8. doi: 10.1073/pnas.88.24.11564. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b14] 14.Tomhave ED, Richardson RM, Didsbury JR, Menard L, Snyderman R, Ali H. Cross-desensitization of receptors for peptide chemoattractants. Characterization of a new form of leukocyte regulation. J Immunol. 1994;153:3267–75. [PubMed] [Google Scholar]

[b15] 15.Blackwood RA, Hartiala KT, Kwoh EE, Transue AT, Brower RC. Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor. J Leukoc Biol. 1996;60:88–93. doi: 10.1002/jlb.60.1.88. [DOI] [PubMed] [Google Scholar]

[b16] 16.Kitayama J, Carr MW, Roth SJ, Buccola J, Springer TA. Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils. J Immunol. 1997;158:2340–9. [PubMed] [Google Scholar]

[b17] 17.Sabroe I, Williams TJ, Hebert CA, Collins PD. Chemoattractant cross-desensitization of the human neutrophil IL-8 receptor involves receptor internalization and differential receptor subtype regulation. J Immunol. 1997;158:1361–9. [PubMed] [Google Scholar]

[b18] 18.Gao JL, Murphy PM. Species and subtype variants of the N-formyl peptide chemotactic receptor reveal multiple important functional domains. J Biol Chem. 1993;268:25395–401. [PubMed] [Google Scholar]

[b19] 19.Sogawa Y, Shimizugawa A, Ohyama T, Maeda H, Hirahara K. The pyrazolone originally reported to be a formyl peptide receptor (FPR) 2/ALX-selective agonist is instead an FPR1 and FPR2/ALX dual agonist. J Pharmacol Sci. 2009;111:317–21. doi: 10.1254/jphs.09196sc. [DOI] [PubMed] [Google Scholar]

[b20] 20.Burli RW, Xu H, Zou X, et al. Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents. Bioorg Med Chem Lett. 2006;16:3713–8. doi: 10.1016/j.bmcl.2006.04.068. [DOI] [PubMed] [Google Scholar]

[b21] 21.Reutershan J, Basit A, Galkina EV, Ley K. Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2005;289:L807–15. doi: 10.1152/ajplung.00477.2004. [DOI] [PubMed] [Google Scholar]

[b22] 22.Cavarra E, Martorana PA, de Santi M, Bartalesi B, Cortese S, Gambelli F, Lungarella G. Neutrophil influx into the lungs of beige mice is followed by elastolytic damage and emphysema. Am J Respir Cell Mol Biol. 1999;20:264–9. doi: 10.1165/ajrcmb.20.2.3235. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

Yoshitaka Sogawa

Takao Ohyama

Hiroaki Maeda

Kazuki Hirahara

Abstract

Introduction

Materials and methods

Reagents

Mice

Cell preparation

Measurement of calcium flux

Chemotaxis assays

Flow cytometric analysis on cell surface receptors

LPS-induced neutrophil migration into the airway

Cell transfer experiment

Statistics

Figure 4.

Figure 7.

Results

Cpd43 induces cross-desensitization of intracellular calcium mobilization in neutrophils

Table 1.

Figure 1.

Cpd43 attenuates the chemotactic activity of neutrophils

Figure 2.

Figure 3.

Cpd43 inhibits neutrophil migration in vivo

Figure 5.

Figure 6.

Cpd43 exerts an anti-migratory effect in vivo by directly affecting neutrophils

Discussion

Acknowledgments

Glossary

Abbreviations:

Disclosures

Supporting information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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