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Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

Tiffany HL, Lautens LL, Gao JL, Pease J, Locati M, Combadiere C, Modi W, Bonner TI, Murphy PM - J. Exp. Med. (1997)

Bottom Line: Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein.These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model.The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors.

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 human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

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An HL-60 clone 15  cell line model of endogenous  CCR8 expression and function.  HL-60 clone 15 cells were cultured for 6 d in the presence of  butyric acid 0.5 μM and for the  final 4 d in the presence of IL-5  10 ng/ml, which induces differentiation to an eosinophilic phenotype. (A) CCR8 mRNA expression. A Northern blot  containing 10 μg total RNA  from undifferentiated (U) and  differentiated (D) cells was hybridized with a CCR8 ORF  probe (top) and washed under  high stringency conditions. The  blot was then exposed to x-ray  film using an intensifying screen  for 20 h. The corresponding region of the ethidium bromide– stained gel is shown in the lower  panel. (B) Calcium flux response  to I-309. Fura-2–loaded undifferentiated (top tracing, U) and  differentiated (lower tracing, D)  cells were stimulated with I-309  50 nM. (C) Potency of I-309 for  calcium flux. Data are from a  single experiment representative  of three separate experiments.  (D) Distinct receptor usage by  I-309 and other CC chemokines.  Differentiated cells were stimulated with the indicated chemokines 50 nM and Fura-2 fluoresence was monitored.
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Figure 4: An HL-60 clone 15 cell line model of endogenous CCR8 expression and function. HL-60 clone 15 cells were cultured for 6 d in the presence of butyric acid 0.5 μM and for the final 4 d in the presence of IL-5 10 ng/ml, which induces differentiation to an eosinophilic phenotype. (A) CCR8 mRNA expression. A Northern blot containing 10 μg total RNA from undifferentiated (U) and differentiated (D) cells was hybridized with a CCR8 ORF probe (top) and washed under high stringency conditions. The blot was then exposed to x-ray film using an intensifying screen for 20 h. The corresponding region of the ethidium bromide– stained gel is shown in the lower panel. (B) Calcium flux response to I-309. Fura-2–loaded undifferentiated (top tracing, U) and differentiated (lower tracing, D) cells were stimulated with I-309 50 nM. (C) Potency of I-309 for calcium flux. Data are from a single experiment representative of three separate experiments. (D) Distinct receptor usage by I-309 and other CC chemokines. Differentiated cells were stimulated with the indicated chemokines 50 nM and Fura-2 fluoresence was monitored.

Mentions: The clone 15 variant of HL-60 cells can be induced by butyric acid and IL-5 treatment to differentiate within 2 d to cells having many of the characteristics of peripheral blood eosinophils, including expression of eosinophil-specific granule proteins (20, 21). Using Northern blot analysis, we were unable to detect mRNA for CCR8 in the uninduced cells, and the cells did not respond to I-309 in either the calcium flux or chemotaxis assays (Fig. 4, A–C). However, when the cells were cultured in the presence of butyric acid and IL-5, a 4.6-kb band was detected by Northern blot using a CCR8 ORF probe (Fig. 4 A).


Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

Tiffany HL, Lautens LL, Gao JL, Pease J, Locati M, Combadiere C, Modi W, Bonner TI, Murphy PM - J. Exp. Med. (1997)

An HL-60 clone 15  cell line model of endogenous  CCR8 expression and function.  HL-60 clone 15 cells were cultured for 6 d in the presence of  butyric acid 0.5 μM and for the  final 4 d in the presence of IL-5  10 ng/ml, which induces differentiation to an eosinophilic phenotype. (A) CCR8 mRNA expression. A Northern blot  containing 10 μg total RNA  from undifferentiated (U) and  differentiated (D) cells was hybridized with a CCR8 ORF  probe (top) and washed under  high stringency conditions. The  blot was then exposed to x-ray  film using an intensifying screen  for 20 h. The corresponding region of the ethidium bromide– stained gel is shown in the lower  panel. (B) Calcium flux response  to I-309. Fura-2–loaded undifferentiated (top tracing, U) and  differentiated (lower tracing, D)  cells were stimulated with I-309  50 nM. (C) Potency of I-309 for  calcium flux. Data are from a  single experiment representative  of three separate experiments.  (D) Distinct receptor usage by  I-309 and other CC chemokines.  Differentiated cells were stimulated with the indicated chemokines 50 nM and Fura-2 fluoresence was monitored.
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Figure 4: An HL-60 clone 15 cell line model of endogenous CCR8 expression and function. HL-60 clone 15 cells were cultured for 6 d in the presence of butyric acid 0.5 μM and for the final 4 d in the presence of IL-5 10 ng/ml, which induces differentiation to an eosinophilic phenotype. (A) CCR8 mRNA expression. A Northern blot containing 10 μg total RNA from undifferentiated (U) and differentiated (D) cells was hybridized with a CCR8 ORF probe (top) and washed under high stringency conditions. The blot was then exposed to x-ray film using an intensifying screen for 20 h. The corresponding region of the ethidium bromide– stained gel is shown in the lower panel. (B) Calcium flux response to I-309. Fura-2–loaded undifferentiated (top tracing, U) and differentiated (lower tracing, D) cells were stimulated with I-309 50 nM. (C) Potency of I-309 for calcium flux. Data are from a single experiment representative of three separate experiments. (D) Distinct receptor usage by I-309 and other CC chemokines. Differentiated cells were stimulated with the indicated chemokines 50 nM and Fura-2 fluoresence was monitored.
Mentions: The clone 15 variant of HL-60 cells can be induced by butyric acid and IL-5 treatment to differentiate within 2 d to cells having many of the characteristics of peripheral blood eosinophils, including expression of eosinophil-specific granule proteins (20, 21). Using Northern blot analysis, we were unable to detect mRNA for CCR8 in the uninduced cells, and the cells did not respond to I-309 in either the calcium flux or chemotaxis assays (Fig. 4, A–C). However, when the cells were cultured in the presence of butyric acid and IL-5, a 4.6-kb band was detected by Northern blot using a CCR8 ORF probe (Fig. 4 A).

Bottom Line: Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein.These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model.The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors.

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 human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

Show MeSH
Related in: MedlinePlus