By
From the * Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, and the Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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N-formylpeptides derive from bacterial and mitochondrial proteins, and bind to specific receptors on mammalian phagocytes. Since binding induces chemotaxis and activation of phagocytes in vitro, it has been postulated that N-formylpeptide receptor signaling in vivo may be important in antimicrobial host defense, although direct proof has been lacking. Here we test this hypothesis in mice lacking the high affinity N-formylpeptide receptor (FPR), created by targeted
gene disruption. FPR/
mice developed normally, but had increased susceptibility to challenge with Listeria monocytogenes, as measured by increased mortality compared with wild-type
littermates. FPR
/
mice also had increased bacterial load in spleen and liver 2 d after infection,
which is before development of a specific cellular immune response, suggesting a defect in innate immunity. Consistent with this, neutrophil chemotaxis in vitro and neutrophil mobilization into peripheral blood in vivo in response to the prototype N-formylpeptide fMLF
(formyl-methionyl-leucyl-phenylalanine) were both absent in FPR
/
mice. These results indicate that FPR functions in antibacterial host defense in vivo.
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Introduction |
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Perhaps the most widely used probes for studying mechanisms of leukocyte chemotaxis are the N-formylpeptides, particularly the prototype formyl-methionyl-leucyl-phenylalanine (fMLF).1 However, after over 20 years of research, their biological significance is still unknown (for review see reference 1). At one extreme, they could simply mimic in vitro an endogenous leukocyte chemoattractant not yet described, and have no biological role themselves. At the other, they may be very important in antibacterial host defense. The latter view has substantial indirect support because N-formylpeptides are products of bacterial proteins (2), and because phagocytes are critical effectors of antibacterial host defense.
The N-formylpeptide signaling pathway has been studied in detail in vitro. Specific phagocyte receptors have been characterized biochemically, and two human genes, designated FPR1 and FPRL1, that encode specific N-formylpeptide receptor subtypes, have been cloned (5). Both are members of the 7-transmembrane domain receptor superfamily, and both couple to pertussis toxin-sensitive G proteins (10). They each have 69% deduced amino acid sequence identity, and are commonly referred to as FPR (formyl peptide receptor) and the FPRL1 (FPR-like 1) receptor, respectively. Both are expressed in human neutrophils and monocytes (11), both bind fMLF (5, 8), and, when activated, both induce calcium mobilization in transfected cells (6, 8, 11). However, there are several major differences: FPR binds fMLF with an ~1,000-fold higher affinity than FPRL1 (8); FPR, but not FPRL1, has been shown to be a chemotactic receptor (12); and FPRL1, but not FPR, has been shown to be a functional lipoxin A4 receptor (13). One other related human gene, named FPRL2, has been identified by cross-hybridization, but the function of the putative receptor is undefined (9, 11). All three genes are clustered on chromosome 19q13.3 (14).
In mice, an orthologue of FPR1 and five structurally related genes have been identified by cross-hybridization with human FPR and FPRL1 probes, indicating that the FPR gene cluster has undergone differential lineage-specific expansion in mammals (15). This has created problems in defining orthologous genes in the two species. The mouse orthologue of human FPR1 is clearly encoded by the mouse gene Fpr1. The encoded receptor is 77% identical in amino acid sequence to human FPR and responds to fMLF in calcium flux assays, although the EC50 is ~100-fold higher than for human FPR under comparable conditions (15). Like FPRL1, one of the related mouse genes, designated Fpr-rs1, encodes a lipoxin A4 receptor. However, neither this nor the other four related genes has been shown to encode a receptor specific for N-formylpeptides or other chemoattractant ligands (17).
Although several antagonists of fMLF have been reported (18), they have not been developed extensively as pharmacologic tools for defining N-formylpeptide biology. We have taken an alternative genetic approach, through analysis of mice lacking FPR, by targeted disruption of Fpr1.
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Materials and Methods |
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Generation of FPR/
Mice.
Isolation of Mouse Leukocytes.
Total leukocytes (80-85% mononuclear cells and 15-20% neutrophils) were isolated from citrated peripheral blood obtained from mouse tails; erythrocytes were lysed in ACK lysis buffer. To obtain enriched populations of neutrophils and macrophages, the peritoneal cavity was washed with PBS 3 and 72 h, respectively, after peritoneal injection with 2 ml (for neutrophils) or 1 ml (for macrophages) of thioglycollate. The purity of both populations was >90% as assessed by light microscopy of Diff-quick-stained () cytospin preparations.Analysis of mRNA Expression.
Total RNA was prepared from peritoneal exudate neutrophils with RNA STAT-60 kit (Tel-Test, Inc.) according to the manufacturer's instructions. The cDNA was synthesized with cDNA Cycle kit (Invitrogen). Wild-type and mutated FPR mRNA was screened by PCR using primers FPR310 (5'-ATGTTCTAGGAGTCTACAAGATGG, a sense oligonucleotide located 20 bp before the start codon), AFPR660 (5'-ATATATGAATTTGCACATGAACCA, an antisense oligonucleotide located in the deleted part of the ORF in the targeting construct), and SNEO2 (5'-CGCTCCCGATTCGCAGCGCATCGC, a sense oligonucleotide of the neo gene). Primers FPR310 and AFPR660 amplify 350 bp of wild-type cDNA, and primers FPR310 and SNEO2 amplify 430 bp of mutated cDNA. The PCR conditions were 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 70°C for 30 s, with a final extension at 72°C for 10 min.[Ca2+]i Measurements.
Cells (107/ml) were loaded with FURA-2 (Molecular Probes) as previously described (21). 2 × 106 cells in 2 ml HBSS were placed in a continuously stirred cuvette at 37°C in a fluorimeter (Photon Technology Inc.). The data are presented as the relative ratio of fluorescence excited alternately at 340 and 380 nm every 0.5 s, and monitored at 510 nm, in response to fMLF () or recombinant mouse macrophage inflammatory protein (MIP)-1Chemotaxis.
Chemotaxis was analyzed using polyvinylpyrrolidone-free polycarbonate membranes with 3-µm pores in 48-well chambers (NeuroProbe). fMLF in HBSS was placed in the lower chamber, and 50 µl of thioglycollate-elicited peritoneal neutrophils suspended in HBSS (1.5 × 106/ml) were placed in the upper chamber. After incubation for 45 min at 37°C, the membrane was removed, rinsed with PBS, fixed, and stained with Diff-Quick. Cells were counted in five randomly selected fields at 1,000-fold magnification.Neutrophil Mobilization to Peripheral Blood.
Mice were injected subcutaneously with 200 µl of 2 µM fMLF. Complete peripheral blood leukocyte counts and differentials were determined for tail vein collections taken before injection and 1.5 h after injection.Challenge with Listeria monocytogenes.
The experimental procedure has been described previously (22). In brief, log-phase culture of L. monocytogenes strain EGD was grown in brain-heart infusion broth (Difco Labs.), aliquoted in 1-ml volumes, and stored at ![]() |
Results and Discussion |
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To inactivate FPR,
we replaced a 150-bp ORF fragment of Fpr1 with a neomycin resistance cassette (neor) by homologous recombination in 129/Sv embryonic stem cells (Fig. 1 a). The deleted
region is from the first extracellular loop to the fourth
transmembrane segment predicted from the FPR sequence (codons 101-150). The mutated and wild-type alleles were
distinguished by an XbaI restriction fragment length polymorphism, 8.5 versus 7.8 kb respectively, of genomic
DNA by Southern hybridization (Fig. 1 b). Consistent
with this, mutant FPR mRNA was detected in neutrophils
from +/ and
/
, but not +/+, mice, whereas normal FPR mRNA was detected in neutrophils from +/
and
+/+, but not
/
, mice (Fig. 1 c).
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The genotypic frequencies for 264 total progeny of eight
+/ mating pairs were: 24.4% +/+, 49.6% +/
, and
26.0%
/
, which is not significantly different from Mendelian expectation for an autosomal gene. FPR
/
mice
were viable and fertile, and exhibited normal growth, development, anatomy, and behavior compared with +/+
littermates. In particular, no defects in the complete blood
count or differential white blood cell count were detected.
These mice have been observed for >17 mo, and have not
exhibited defects in hemostasis or healing of tail wounds, or
increased susceptibility to spontaneous infection when derived in a specific pathogen-free environment. Thus either FPR is not involved in these processes or it functions redundantly with other factors, or else, under unstressed conditions, its function is compensated by other factors in mutant mice.
We first used the
knockout mice to test whether FPR mediates fMLF signaling in primary leukocytes. First, we observed that the normal fMLF-induced calcium flux in thioglycollate-elicited peritoneal (TP) neutrophils (Fig. 2 a) and PBMCs (data not
shown) from +/+ mice was absent in cells from /
mice. As a positive control, a robust response to the CC
chemokine MIP-1
was detected in the same cell types
from both +/+ and
/
mice, consistent with previous
observations (23). TP neutrophils from FPR
/
mice also
failed to chemotax in response to fMLF (100 nM-1 µM) in
vitro, whereas neutrophils from +/+ mice exhibited a typical bell-shaped dose-response curve (Fig. 2 b). Similar results were obtained from TP macrophages, but the response of +/+ macrophages to fMLF was much lower than
the response of +/+ neutrophils (data not shown). These
in vitro results indicate that FPR mediates mouse neutrophil chemotactic and calcium flux responses to fMLF, which has not been shown previously, and verify gene inactivation at the functional level.
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We next tested the effects of fMLF in vivo in +/+ and
/
mice. We found that subcutaneous injection of +/+
mice with fMLF specifically induced a 70% increase in peripheral blood neutrophil concentrations when measured
90 min after injection (Fig. 2 c). The response was absent
in
/
mice, indicating that FPR is involved. The CC
chemokine MIP-1
is also able to induce this response, through the receptor CCR1 (23). We also injected 1, 10, and 100 nmoles of fMLF intraperitoneally, but did not observe local accumulation of leukocytes in either +/+ or
/
mice when the peritoneal cavity was washed 4 h after injection. Presumably, the peptide is scavenged or degraded
by factors present at this site. In contrast, intraperitoneal
injection with thioglycollate, a nonspecific inflammatory
stimulus, induced peritoneal exudation, with neutrophil
and macrophage predominance at 4 and 72 h, respectively, consistent with previous reports (23). However, no significant difference was observed for this response between
FPR
/
and FPR+/+ mice. Reduced leukocyte mobilization in response to thioglycollate has been reported in several chemokine receptor knockout mice (24) and in
C5a receptor knockout mice (29), all of which bind endogenous chemoattractants. Thus, thioglycollate challenge in
FPR knockout mice does not reveal evidence for the widely
held speculation that an endogenous ligand for FPR exists.
To test the role of FPR in host defense, we
compared the susceptibility of FPR/
and FPR+/+ mice
to infection with L. monocytogenes. This organism was chosen because it produces an acute infection in mice with
well-characterized immunopathogenesis (30), involving both
innate and acquired arms of the immune system, and because, like other bacteria, it produces N-formylpeptides (31).
It is not known whether Listeria-derived N-formylpeptides
are released in vivo in amounts required to activate FPR;
however, this is also unknown for other bacteria. FPR
/
mice exhibited a markedly higher rate and extent of mortality compared with FPR+/+ littermates (Fig. 3 a). 73% of
FPR
/
mice injected with 2 × 104 CFU died by day 4 in
contrast to only 6% in FPR+/+ mice. Only 6% of FPR
/
mice survived beyond 6 d, whereas 50% of FPR+/+ mice
remained healthy. The survival curve of +/
mice fell
between those of
/
and +/+ mice, suggesting a gene
dosage effect for this phenotype. Consistent with this, we
observed intermediate responsiveness of neutrophils from
+/
mice versus
/
and +/+ mice to fMLF in the
calcium flux assay (data not shown).
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Early mortality suggests a defect in innate immunity. To
test this further, we infected mice with 2 × 104 CFU and
measured bacterial burden 2 d later, a time when nonspecific immune responses control infection (30). Consistent
with a defect in innate immunity, FPR/
mice showed
32- and 45-fold more bacteria in spleen and liver, respectively, at this time, relative to +/+ control mice (Fig. 3 b).
Although our results show that FPR deficiency causes
increased susceptibility to Listeria infection, they do not
clearly identify its mechanism of action. The histopathologic appearance of the liver and spleen was indistinguishable in infected FPR+/+ and FPR/
mice killed on days 1, 2, and 3 after infection, and in infected animals with discordant genotypes that were allowed to die naturally. In
both cases, neutrophilic abscesses were observed, suggesting that FPR deficiency does not affect normal neutrophil
trafficking to these organs in this model. Additional work
will be needed to identify the precise mechanism of action
of FPR in host defense in this model.
It has been demonstrated previously that, as in FPR-deficient mice, neutrophil-depleted mice rapidly succumb to
overwhelming listeriosis after experimental challenge, with
marked increases in bacterial burden in both liver and
spleen (32, 33). Disruption of other genes that regulate phagocyte function, including the genes encoding gp91phox (a
component of the phagocyte NADPH oxidase; reference
34), the IFN- receptor (35), and the chemokine receptors
CCR2 (24) and CCR5 (25), has also been linked to increased susceptibility to Listeria. Since not all FPR
/
mice
died from Listeria challenge in our experiments, these and other genes may compensate, at least partially, when FPR
is absent.
In summary, we have found that FPR/
mice have no
obvious developmental defects and do not develop spontaneous infection when derived in specific pathogen-free
conditions. This suggest that, under these conditions, FPR
is dispensable. However, when challenged with L. monocytogenes, FPR-deficient mice have accelerated mortality and
increased bacterial burden in liver and spleen early after infection, which suggests a role for FPR in host defense, specifically through regulation of innate immunity. This is
consistent with expression of FPR on phagocytes. Additional work will be needed to establish whether FPR is a
host defense factor versus other types of microorganisms,
and a factor in inflammation induced by noninfectious
agents, as well as to establish whether its mechanism of action in listeriosis involves binding of N-formylpeptides and
trafficking of phagocytes.
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Footnotes |
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Address correspondence to Ji-Liang Gao, Laboratory of Host Defenses, NIAID, Bldg. 10, Rm. 11N113, National Institutes of Health, Bethesda, MD 20892. Phone: 301-496-2877; Fax: 301-402-4369; E-mail: jgao{at}nih.gov
Received for publication 31 August 1998 and in revised form 25 November 1998.
We thank Karen Elkins for Listeria strains, helpful advice, and comments on the manuscript, and Heiner Westphal for helpful advice.
Abbreviations used in this paper fMLF, formyl-methionyl-leucyl-phenylalanine; FPR, high affinity N-formylpeptide receptor; FPRL1, low affinity N-formylpeptide receptor or lipoxin A4 receptor; MIP, macrophage inflammatory protein; ORF, open reading frame; TP neutrophils, thioglycollate-elicited peritoneal neutrophils.
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