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Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemokine that is a structural and functional homologue of human MCP-1.

Sarafi MN, Garcia-Zepeda EA, MacLean JA, Charo IF, Luster AD - J. Exp. Med. (1997)

Bottom Line: Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes.MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5.These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Unit, Massachusetts General Hospital, Boston, USA.

ABSTRACT
The chemokines are a large family of cytokines that control the recruitment of leukocytes in immune and inflammatory responses. We describe the isolation of a novel murine CC chemokine that, based on its biological and structural features, we have named monocyte chemoattractant protein (MCP)-5. MCP-5 mapped to the CC chemokine cluster on mouse chromosome 11 and was most closely related to human MCP-1 in structure (66% amino acid identity). Purified recombinant MCP-5 protein was a potent chemoattractant for peripheral blood monocytes, was only weakly active on eosinophils at high doses, and was inactive on neutrophils. MCP-5 induced a calcium flux in peripheral blood mononuclear cells, but not in purified murine eosinophils or neutrophils. Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes. MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5. Constitutive expression of MCP-5 mRNA was detected predominantly in lymph nodes, and its expression was markedly induced in macrophages activated in vitro and in vivo. Moreover, MCP-5 expression was up-regulated in the lungs of mice following aerosolized antigen challenge of sensitized mice, and during the host response to infection with Nippostrongylus brasiliensis. These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.

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Identification of CCR2 as a functional MCP-5 receptor.  HEK-293 cells stably expressing human CCR2b (A–C) or murine  CCR2 (D and E) were loaded with indo-1 AM, and intracellular calcium concentrations were monitored by ratio fluorescence in response  to the indicated concentrations of MCP-5 (A and D), JE (B and E), and  MCP-1 (C). Shown is one of three similar experiments.
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Figure 4: Identification of CCR2 as a functional MCP-5 receptor. HEK-293 cells stably expressing human CCR2b (A–C) or murine CCR2 (D and E) were loaded with indo-1 AM, and intracellular calcium concentrations were monitored by ratio fluorescence in response to the indicated concentrations of MCP-5 (A and D), JE (B and E), and MCP-1 (C). Shown is one of three similar experiments.

Mentions: The signaling and desensitization studies performed on mononuclear cells suggested that MCP-5 activated the same receptor as MCP-1 and JE. Furthermore, the inability of MCP-5 to induce a calcium flux in murine eosinophils suggested that MCP-5 does not signal through the two receptors known to be expressed on eosinophils, CCR3, and CCR1 (21). To directly test these hypotheses, we examined the ability of MCP-5 to activate a number of cloned human CC chemokine receptors stably expressed in HEK-293 cells. MCP-5 (100 nM) reproducibly induced a robust intracellular calcium flux in cells expressing CCR2 (Fig. 4), but not CCR1, CCR3, or CCR5 (data not shown). In control experiments, we confirmed functional expression of the cloned receptors in these MCP-5 unresponsive targets by demonstrating that the CCR1 and CCR5 cell lines responded to MIP-1α, and the CCR3 line responded to eotaxin (data not shown). Doseresponse experiments demonstrated that MCP-5 was an excellent ligand for human CCR2b (EC50 ⩽5 nM), as was MCP-1, and both were more potent ligands than JE (Fig. 4 A–C). Since the MCP-5 we have described is a murine protein, it was of interest to directly compare it to JE for activation of the murine MCP-1 receptor (CCR2). As shown in Fig. 4 B, JE induced a more robust intracellular calcium flux than MCP-5 in HEK-293 cells stably expressing murine CCR2. No response was seen to MCP-5 in cells transfected with murine CCR5, although these cells did respond to the positive control MIP-1α (data not shown). These data raise the possibility that the natural murine receptor for MCP-5 is yet to be cloned. Desensitization studies using cells transfected with human CCR2b revealed that MCP-5 blocked subsequent responses to MCP-5, MCP-1, and JE (Fig. 5). Similar results were obtained when the initial agonist was MCP-1 or JE (Fig. 5). However, MCP-1 did not completely desensitize the CCR2b transfectants to a subsequent MCP-5 stimulation (Fig. 5). These data are consistent with the desensitization responses observed on human PBMC (Fig. 3), and with the hypothesis that MCP-5 is a full agonist for the human MCP-1 receptor (CCR2b).


Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemokine that is a structural and functional homologue of human MCP-1.

Sarafi MN, Garcia-Zepeda EA, MacLean JA, Charo IF, Luster AD - J. Exp. Med. (1997)

Identification of CCR2 as a functional MCP-5 receptor.  HEK-293 cells stably expressing human CCR2b (A–C) or murine  CCR2 (D and E) were loaded with indo-1 AM, and intracellular calcium concentrations were monitored by ratio fluorescence in response  to the indicated concentrations of MCP-5 (A and D), JE (B and E), and  MCP-1 (C). Shown is one of three similar experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196097&req=5

Figure 4: Identification of CCR2 as a functional MCP-5 receptor. HEK-293 cells stably expressing human CCR2b (A–C) or murine CCR2 (D and E) were loaded with indo-1 AM, and intracellular calcium concentrations were monitored by ratio fluorescence in response to the indicated concentrations of MCP-5 (A and D), JE (B and E), and MCP-1 (C). Shown is one of three similar experiments.
Mentions: The signaling and desensitization studies performed on mononuclear cells suggested that MCP-5 activated the same receptor as MCP-1 and JE. Furthermore, the inability of MCP-5 to induce a calcium flux in murine eosinophils suggested that MCP-5 does not signal through the two receptors known to be expressed on eosinophils, CCR3, and CCR1 (21). To directly test these hypotheses, we examined the ability of MCP-5 to activate a number of cloned human CC chemokine receptors stably expressed in HEK-293 cells. MCP-5 (100 nM) reproducibly induced a robust intracellular calcium flux in cells expressing CCR2 (Fig. 4), but not CCR1, CCR3, or CCR5 (data not shown). In control experiments, we confirmed functional expression of the cloned receptors in these MCP-5 unresponsive targets by demonstrating that the CCR1 and CCR5 cell lines responded to MIP-1α, and the CCR3 line responded to eotaxin (data not shown). Doseresponse experiments demonstrated that MCP-5 was an excellent ligand for human CCR2b (EC50 ⩽5 nM), as was MCP-1, and both were more potent ligands than JE (Fig. 4 A–C). Since the MCP-5 we have described is a murine protein, it was of interest to directly compare it to JE for activation of the murine MCP-1 receptor (CCR2). As shown in Fig. 4 B, JE induced a more robust intracellular calcium flux than MCP-5 in HEK-293 cells stably expressing murine CCR2. No response was seen to MCP-5 in cells transfected with murine CCR5, although these cells did respond to the positive control MIP-1α (data not shown). These data raise the possibility that the natural murine receptor for MCP-5 is yet to be cloned. Desensitization studies using cells transfected with human CCR2b revealed that MCP-5 blocked subsequent responses to MCP-5, MCP-1, and JE (Fig. 5). Similar results were obtained when the initial agonist was MCP-1 or JE (Fig. 5). However, MCP-1 did not completely desensitize the CCR2b transfectants to a subsequent MCP-5 stimulation (Fig. 5). These data are consistent with the desensitization responses observed on human PBMC (Fig. 3), and with the hypothesis that MCP-5 is a full agonist for the human MCP-1 receptor (CCR2b).

Bottom Line: Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes.MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5.These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Unit, Massachusetts General Hospital, Boston, USA.

ABSTRACT
The chemokines are a large family of cytokines that control the recruitment of leukocytes in immune and inflammatory responses. We describe the isolation of a novel murine CC chemokine that, based on its biological and structural features, we have named monocyte chemoattractant protein (MCP)-5. MCP-5 mapped to the CC chemokine cluster on mouse chromosome 11 and was most closely related to human MCP-1 in structure (66% amino acid identity). Purified recombinant MCP-5 protein was a potent chemoattractant for peripheral blood monocytes, was only weakly active on eosinophils at high doses, and was inactive on neutrophils. MCP-5 induced a calcium flux in peripheral blood mononuclear cells, but not in purified murine eosinophils or neutrophils. Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes. MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5. Constitutive expression of MCP-5 mRNA was detected predominantly in lymph nodes, and its expression was markedly induced in macrophages activated in vitro and in vivo. Moreover, MCP-5 expression was up-regulated in the lungs of mice following aerosolized antigen challenge of sensitized mice, and during the host response to infection with Nippostrongylus brasiliensis. These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.

Show MeSH
Related in: MedlinePlus