Identification of peptides that antagonize formyl peptide receptor-like 1-mediated signaling

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1's agonists (Trp-Lys-Tyr-Met-Val-D-Met-CONH(2) (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH(2) (WRWWWW (WRW(4))) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW(4) inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW(4) to the FPR family, we observed that WRW(4) specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid beta42 (Abeta42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW(4) on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Abeta42 peptide in human neutrophils. Abeta42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW(4), which also completely inhibited the internalization of Abeta42 peptide in human macrophages. WRW(4) is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.     

Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are approximately 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.     

The N-formylpeptide receptor (FPR) and a second G(i)-coupled receptor mediate fMet-Leu-Phe-stimulated activation of NADPH oxidase in murine neutrophils

N-Formylypeptides such as fMet-Leu-Phe (fMLF) potently induce superoxide production through NADPH oxidase activation. The receptors that mediate this response have not been defined. Here, we provide definitive proof using a mouse model that formyl peptide receptor (FPR) is a receptor, but not the only receptor, that mediates fMLF-induced oxidase activation. In wild-type (FPR(+/+)) mouse neutrophils, superoxide production is dependent on the concentration of fMLF with an EC(50) of approximately 5 microM and a peak at approximately 50 microM. In contrast, FPR-deficient (FPR(-/-)) mouse neutrophils produced markedly less superoxide with an EC(50) of approximately 50 microM and a peak at approximately 200 microM. Yet, FPR(+/+) and FPR(-/-) neutrophils showed similar oxidase activation kinetics and G(i) protein-dependent pharmacological sensitivities. These results suggested that a second receptor, likely FPR2, mediates superoxide production at high concentrations of fMLF. This less sensitive second pathway may permit continued oxidant generation in response to formyl peptides when FPR is desensitized in high concentrations of the chemotactic gradient.     

The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2, an orphan receptor sharing 83% amino acid identity with FPRL1. FPRL1 is a promiscuous receptor activated by serum amyloid A and by different synthetic peptides, including the hexapeptide Trp-Lys-Tyr-Met-Val-d-Met-NH(2) (WKYMVm). By measuring calcium flux in HL-60 cells transfected with FPR, FPRL1, or FPRL2, we show that WKYMVm activated all three receptors, whereas the l-conformer WKYMVM activated exclusively FPRL1 and FPRL2. The functionality of FPRL2 was further assessed by the ability of HL-60-FPRL2 cells to migrate toward nanomolar concentrations of hexapeptides. The half-maximal effective concentrations of WKYMVM for calcium mobilization in HL-60-FPRL1 and HL-60-FPRL2 cells were 2 and 80 nm, respectively. Those of WKYMVm were 75 pm and 3 nm. The tritiated peptide WK[3,5-(3)H(2)]YMVM bound to FPRL1 (K(D) approximately 160 nm), but not to FPR. The two conformers similarly inhibited binding of (125)I-labeled WKYMVm to FPRL2-expressing cells (IC(50) approximately 2.5-3 micrometer). Metabolic labeling with orthophosphoric acid revealed that FPRL1 was differentially phosphorylated upon addition of the l- or d-conformer, indicating that it induced different conformational changes. In contrast to FPRL1, FPRL2 was already phosphorylated in the absence of agonist and not evenly distributed in the plasma membrane of unstimulated cells. However, both receptors were internalized upon addition of either of the two conformers. Taken together, the results indicate that neutrophils are activated by WKYMVM through FPRL1 and that FPRL2 is a chemotactic receptor transducing signals in myeloid cells.     

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O₂^?) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-α (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O₂^? relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O₂^? by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O₂^? by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-δ isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.     

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-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-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 concentrations 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.     

F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.     

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.

A cDNA of 1650 base pairs was isolated by screening an HL-60 granulocyte library with an N-formyl peptide receptor (NFPR) cDNA probe under low stringency conditions. The cDNA encodes a protein of 351 amino acids tentatively named FPR2, with a calculated molecular weight of 39 kDa. Sequence analysis revealed that FPR2 is 69% identical in sequence to the human NFPR and shares extensive homology to several other chemoattractant receptors. FPR2 expressed in transfected cells mediated formyl peptide-stimulated calcium mobilization at micromolar concentrations of ligand. FPR2 messenger is detected in granulocytic HL-60 cells, but not in undifferentiated HL-60 cells. These findings suggest that FPR2 is a novel receptor for formyl peptide ligand and a new member of the chemoattractant receptor gene family.     

Small G-proteins Ras,Rac and Rho in the regulation of the neutrophil respiratory burst induced by formyl peptide

Formylated peptides specifically activate many of the neutrophil functions; their action is mediated via formyl peptide receptors (FPRs). FPRs belong to the family of receptors having seven transmembrane-spanning domains and coupled with G-proteins (GPCR). About a dozen of highly homologous genes of FPRs were found to be localized in mouse chromosome 17. By binding with labeled N-formyl-Met-Leu-Phe (fMLF), FPRs are classified as receptors with high (FPR1) and low (FPR2 and FPR3/FPRL1) affinity to formyl peptide. Binding of formyl peptide with FPRs triggers the complex signaling events, the most studied are: activation of phospholipase C (PLC) with subsequent calcium signaling; launching of mitogen activated protein kinases (MAPKs) cascade pathway, and activation of phosphoinositol-3-kinase (PI3K) cascades. As we have shown previously, the priming of the respiratory burst of mice neutrophils occurs under the cell activation by fMLF in high doses only, i.e., it is necessary to activate low affinity FPRs. Besides, the usage of the specific MEK and p38MAPK inhibitors induced significant suppression of the response to 1 μM fMLM, while the response to 50 μM fMLF increased in the presence of the inhibitors. We suggest that there is a signal divergence upon activation of high and low affinity fMLF receptors, and small G protein dependent signaling pathways could be alternative to activate NADPH oxidase. Here we demonstrate that Ras-proteins participate in the respiratory burst activation, especially in activation via the high affinity fMLF receptors. Activation of the Rho- and Rac-proteins induced the down-regulation of the respiratory burst under the stimulation of high affinity FPRs. The inhibition of the Rho-proteins almost completely suppressed the respiratory burst activated via the high and low affinity receptors, probably due to inability to assemble of the cytoskeleton proteins and NADPH oxidase components.     

A monocyte-specific peptide from herpes simplex virus type 2 glycoprotein G activates the NADPH-oxidase but not chemotaxis through a G-protein-coupled receptor distinct from the members of the formyl peptide receptor family

We have recently identified a peptide derived from the secreted portion of the HSV-2 glycoprotein G, gG-2p20, to be proinflammatory. Based on its ability to activate neutrophils and monocytes via the formyl peptide receptor (FPR) to produce reactive oxygen species (ROS) that down-regulate NK cell function, we suggested it to be of importance in HSV-2 pathogenesis. We now describe the effects of an overlapping peptide, gG-2p19, derived from the same HSV-2 protein. Also, this peptide activated the ROS-generating NADPH-oxidase, however, only in monocytes and not in neutrophils. Surprisingly, gG-2p19 did not induce a chemotactic response in the affected monocytes despite using a pertussis toxin-sensitive, supposedly G-protein-coupled receptor. The specificity for monocytes suggested that FPR and its homologue FPR like-1 (FPRL1) did not function as receptors for gG-2p19, and this was also experimentally confirmed. Surprisingly, the monocyte-specific FPR homologue FPRL2 was not involved either, and the responsible receptor thus remains unknown so far. However, the receptor shares some basic signaling properties with FPRL1 in that the gG-2p19-induced response was inhibited by PBP10, a peptide that has earlier been shown to selectively inhibit FPRL1-triggered responses. We conclude that secretion and subsequent degradation of the HSV-2 glycoprotein G can generate several peptides that activate phagocytes through different receptors, and with different cellular specificities, to generate ROS with immunomodulatory properties.     

The synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met is a potent chemotactic agonist for mouse formyl peptide receptor

Formyl peptides are potent neutrophil chemoattractants. In humans and rabbits, the formyl peptide receptor (FPR) binds N-formyl-Met-Leu-Phe (fMLF) with high affinity (K(d) approximately 1 nM). The mouse FPR (mFPR) is a low-affinity receptor for fMLF (K(d) approximately 100 nM); therefore, other agonists for this receptor may exist. Using mFPR-transfected rat basophilic leukemia cells, we found that a recently identified synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a potent agonist for mFPR. WKYMVm induced calcium mobilization with an EC(50) of 1.2-1.5 nM. Optimal chemotaxis was achieved with 1 nM of WKYMVm, but it required 100 nM of fMLF. WKYMVm stimulated rapid and potent phosphorylation of the mitogen-activated protein kinases extracellular signal-related kinases 1 and 2 when used at 50 nM. Pertussis toxin only partially blocked calcium mobilization and production of inositol 1,4,5-trisphosphate in the stimulated mFPR cells, suggesting the possibility that this receptor couples to Galpha proteins other than Gi and Go. Competitive binding and desensitization data suggest that both peptides interact with the same receptor but may use nonoverlapping binding sites because WKYMVm was unable to effectively displace [(3)H]fMLF bound to mFPR. These results provide evidence for the presence of an alternative potent agonist for mFPR, and suggest a potential usage of WKYMVm for probing the ligand-receptor interactions with the murine formyl peptide receptor homologs.     

The HIV-1 cell entry inhibitor T-20 potently chemoattracts neutrophils by specifically activating the N-formylpeptide receptor

T-20, a synthetic peptide corresponding to the heptad repeat sequence of HIV-1 gp41, blocks HIV-1 entry by targeting gp41, and is currently in clinical trials as an anti-retroviral agent. We recently reported that in vitro T-20 also functions as a phagocyte chemoattractant and a chemotactic agonist at the phagocyte N-formylpeptide receptor (FPR). Here we show that T-20 is also a potent chemotactic agonist in vitro at a related human phagocyte receptor FPRL1R. To test the relative importance of FPR and FPRL1R in primary cells, we identified the corresponding mouse T-20 receptors, mFPR and FPR2, which are both expressed in neutrophils, and compared T-20 action on neutrophils from wild type and mFPR knockout mice. Surprisingly, although T-20 activates mFPR and FPR2 in transfected cells with equal potency and efficacy in both calcium flux and chemotaxis assays, neutrophils from mFPR knockout mice did not respond to T-20. These results provide genetic evidence that FPR is the major phagocyte T-20 receptor in vivo and point to the potential feasibility of studying T-20 effects on immunity in a mouse model. This may help define the cause of local inflammation after T-20 injection that has recently been reported in Phase I clinical trials.     

An antimicrobial cathelicidin peptide, human CAP18/LL-37, suppresses neutrophil apoptosis via the activation of formyl-peptide receptor-like 1 and P2X7

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. An antibacterial cathelicidin, human cationic antibacterial protein of 18 kDa/LL-37, not only exhibits potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also functions as a chemoattractant for immune cells, including neutrophils. During bacterial infections, the life span of neutrophils is regulated by various pathogen- and host-derived substances. In this study, to further evaluate the role of LL-37 in innate immunity, we investigated the action of LL-37 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, LL-37 dose dependently (0.01-5 microg/ml) suppressed neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/2, expression of Bcl-x(L) (an antiapoptotic protein), and inhibition of caspase 3 activity. Interestingly, LL-37-induced suppression of neutrophil apoptosis was attenuated by the antagonists for formyl-peptide receptor-like 1 (FPRL1) and P2X7 nucleotide receptor. Of importance, the agonists for FPRL1 and P2X7 apparently suppressed neutrophil apoptosis. Collectively, these observations indicate that LL-37 cannot only kill bacteria, but also modulate (suppress) neutrophil apoptosis via the activation of FPRL1 and P2X7 in bacterial infections. Suppression of neutrophil apoptosis results in the prolongation of their life span, and may be advantageous for host defense against bacterial invasion.     

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.     

Involvement of Phospholipase D 1 and 2 in the subcellular localization and activity of formyl-peptide-receptors in the human colonic cell line HT29

Epithelial cells of the alimentary tract play a central role in the mucosal host defence against pathogens and in the recognition of agonists that interact with mucosal surfaces. In particular, the formyl peptide receptor (FPR) family and their three human subtypes: FPR, formyl-peptide-receptor-like-1 (FPRL1) and FPRL2, are involved in the host defence against pathogens that mediate epithelial responses thus upregulating inflammation. To elucidate the mechanisms by which FPR function, we examined the influence of phospholipase D (PLD) 1 and 2 on the activity and signal transduction of human enterocytes cell line HT29. PLD is a key enzyme involved in secretion, endocytosis and receptor signalling. We inhibited PLD1 and 2 by small interference RNA (siRNA) and determined the activity of formyl peptide receptors using Western blotting and cAMP level measurements. We then analyzed the distribution of formyl peptide receptors FPR, FPRL1 and FPRL2 compared to a control. In this study, we demonstrated that the depletion of PLD1 and 2 resulted in a marked reduction of formyl peptide receptor activity due to inhibited extracellular-signal regulated kinases 1/2 (ERK1/2), phosphorylation and cAMP level reduction. In addition, we observed an intracellular accumulation of FPR, FPRL1 and FPRL2 as a result of receptor recycling inhibition using fluorescence microscopy. The constitutive internalization rate was unaffected. Our results support the importance of PLD1 and 2 in formyl peptide receptor function and the role of endocytosis, receptor recycling and reactivation for receptor activity.     

Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions

Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.     

Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor

Clostridium difficile may induce antibiotic‐associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB‐induced rise in intracellular‐free Ca²⁺ and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR‐1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR‐1. Domain analyses revealed that the N‐terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro‐inflammatory ligand effect can be triggered even by cleaved and, thus, non‐cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved ‘non‐cytotoxic’ fragments and (iii) an interaction of the N‐terminal glucosyltransferase domain instead of the C‐terminal receptor binding domain of TcdB with target cells.