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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.

Hartt JK, Barish G, Murphy PM, Gao JL - J. Exp. Med. (1999)

Bottom Line: The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides.The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2.Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

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Distribution of FPR2 mRNA in mouse leukocytes. A Northern blot containing total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG (3h TGPC and 72h TGPC) was serially hybridized to a 32P-labeled Fpr-rs2 ORF probe encoding FPR2 and a 32P-labeled actin probe, both under high stringency conditions. 3- and 72-h TG-elicited peritoneal cells were enriched in neutrophils and macrophages, respectively (>90%). After hybridization with Fpr-rs2 and actin, the blot was exposed for 3 and 2 d, respectively, to x-ray film. The positions of 18S and 28S ribosomal bands are indicated at the right. The 72h TGPC lane was deliberately overloaded with RNA, as indicated by the actin hybridization, to test for low abundance mRNA.
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Figure 1: Distribution of FPR2 mRNA in mouse leukocytes. A Northern blot containing total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG (3h TGPC and 72h TGPC) was serially hybridized to a 32P-labeled Fpr-rs2 ORF probe encoding FPR2 and a 32P-labeled actin probe, both under high stringency conditions. 3- and 72-h TG-elicited peritoneal cells were enriched in neutrophils and macrophages, respectively (>90%). After hybridization with Fpr-rs2 and actin, the blot was exposed for 3 and 2 d, respectively, to x-ray film. The positions of 18S and 28S ribosomal bands are indicated at the right. The 72h TGPC lane was deliberately overloaded with RNA, as indicated by the actin hybridization, to test for low abundance mRNA.

Mentions: Previously, we reported that Fpr-rs2 is expressed in unfractionated peripheral blood leukocytes 22. To determine the expression pattern in finer detail, we used Northern blot hybridization to probe total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG. The elicited peritoneal cell populations were markedly enriched in neutrophils and macrophages at 3 and 72 h, respectively (>90% pure). The residual 10% of cells were mainly mononuclear cells and neutrophils in the 3- and 72-h cell populations, respectively. Specific mRNA bands were detected in both the 3- and 72-h TG-elicited peritoneal cells. In both cases, two classes of transcripts (∼1.5 and 1.9 kb) were observed, and the signal strength was similar for each class within each sample. However, signals from the 3-h cells were much stronger than from the 72-h cells (Fig. 1). Note that 72-h RNA was deliberately overloaded relative to 3-h RNA, as revealed by hybridization with an actin probe. A reasonable interpretation of these results, consistent with the functional data that follows, is that Fpr-rs2 is primarily expressed in neutrophils, and the weak signal in the 72-h cells is due to neutrophils, which make up a small minority of the total cell population. An alternative explanation is that monocytes express Fpr-rs2 in the circulation, but not after extravasation.


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.

Hartt JK, Barish G, Murphy PM, Gao JL - J. Exp. Med. (1999)

Distribution of FPR2 mRNA in mouse leukocytes. A Northern blot containing total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG (3h TGPC and 72h TGPC) was serially hybridized to a 32P-labeled Fpr-rs2 ORF probe encoding FPR2 and a 32P-labeled actin probe, both under high stringency conditions. 3- and 72-h TG-elicited peritoneal cells were enriched in neutrophils and macrophages, respectively (>90%). After hybridization with Fpr-rs2 and actin, the blot was exposed for 3 and 2 d, respectively, to x-ray film. The positions of 18S and 28S ribosomal bands are indicated at the right. The 72h TGPC lane was deliberately overloaded with RNA, as indicated by the actin hybridization, to test for low abundance mRNA.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195614&req=5

Figure 1: Distribution of FPR2 mRNA in mouse leukocytes. A Northern blot containing total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG (3h TGPC and 72h TGPC) was serially hybridized to a 32P-labeled Fpr-rs2 ORF probe encoding FPR2 and a 32P-labeled actin probe, both under high stringency conditions. 3- and 72-h TG-elicited peritoneal cells were enriched in neutrophils and macrophages, respectively (>90%). After hybridization with Fpr-rs2 and actin, the blot was exposed for 3 and 2 d, respectively, to x-ray film. The positions of 18S and 28S ribosomal bands are indicated at the right. The 72h TGPC lane was deliberately overloaded with RNA, as indicated by the actin hybridization, to test for low abundance mRNA.
Mentions: Previously, we reported that Fpr-rs2 is expressed in unfractionated peripheral blood leukocytes 22. To determine the expression pattern in finer detail, we used Northern blot hybridization to probe total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG. The elicited peritoneal cell populations were markedly enriched in neutrophils and macrophages at 3 and 72 h, respectively (>90% pure). The residual 10% of cells were mainly mononuclear cells and neutrophils in the 3- and 72-h cell populations, respectively. Specific mRNA bands were detected in both the 3- and 72-h TG-elicited peritoneal cells. In both cases, two classes of transcripts (∼1.5 and 1.9 kb) were observed, and the signal strength was similar for each class within each sample. However, signals from the 3-h cells were much stronger than from the 72-h cells (Fig. 1). Note that 72-h RNA was deliberately overloaded relative to 3-h RNA, as revealed by hybridization with an actin probe. A reasonable interpretation of these results, consistent with the functional data that follows, is that Fpr-rs2 is primarily expressed in neutrophils, and the weak signal in the 72-h cells is due to neutrophils, which make up a small minority of the total cell population. An alternative explanation is that monocytes express Fpr-rs2 in the circulation, but not after extravasation.

Bottom Line: The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides.The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2.Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

Show MeSH
Related in: MedlinePlus