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Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW - J. Exp. Med. (1997)

Bottom Line: High expression was also detected in normal thymus and less expression in lung and spleen.Unlike most other CC chemokines, MDC is encoded on human chromosome 16.MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

View Article: PubMed Central - PubMed

Affiliation: Icos Corporation, Bothell, Washington 98021, USA.

ABSTRACT
A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

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Northern analysis of MDC mRNA expression in human  cells. Migration of MDC, 28S RNA, and 18S RNA is indicated. (A) Peripheral blood monocytes were allowed to differentiate into macrophages  by incubation on plastic tissue culture dishes for 6 d (41). Total RNA was  isolated from individual dishes on the indicated days, fractionated through  a formaldehyde agarose gel, blotted, and probed with the radiolabeled  MDC cDNA. The blot was subsequently probed for GAPDH message.  (B) Expression of MDC in differentiated HL-60 cells. Cells were treated  with PMA to induce differentiation to monocytic cells or with DMSO to  induce granulocytic cells. MDC expression was analyzed by Northern  blotting as in A. (C) Northern blot of MDC expression in human peripheral blood monocytes (Mono.), macrophages derived from these monocytes (Mac.), dendritic cells derived from PBMC of two different donors  (DC-1 and DC-2), and natural killer cells derived from PBMC (NK). (D)  Expression of MDC in cell cultures. Lung epithelial cell line A549, lung  fibroblast line IMR90, and I-HUVEC without and with TNF-α stimulation; PBMC without and with stimulation by PHA and PMA; and  monocyte derived macrophages after 6 d in culture. After probing with  MDC, the filter was stripped and probed sequentially with MCP-1 and  GAPDH.
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Figure 4: Northern analysis of MDC mRNA expression in human cells. Migration of MDC, 28S RNA, and 18S RNA is indicated. (A) Peripheral blood monocytes were allowed to differentiate into macrophages by incubation on plastic tissue culture dishes for 6 d (41). Total RNA was isolated from individual dishes on the indicated days, fractionated through a formaldehyde agarose gel, blotted, and probed with the radiolabeled MDC cDNA. The blot was subsequently probed for GAPDH message. (B) Expression of MDC in differentiated HL-60 cells. Cells were treated with PMA to induce differentiation to monocytic cells or with DMSO to induce granulocytic cells. MDC expression was analyzed by Northern blotting as in A. (C) Northern blot of MDC expression in human peripheral blood monocytes (Mono.), macrophages derived from these monocytes (Mac.), dendritic cells derived from PBMC of two different donors (DC-1 and DC-2), and natural killer cells derived from PBMC (NK). (D) Expression of MDC in cell cultures. Lung epithelial cell line A549, lung fibroblast line IMR90, and I-HUVEC without and with TNF-α stimulation; PBMC without and with stimulation by PHA and PMA; and monocyte derived macrophages after 6 d in culture. After probing with MDC, the filter was stripped and probed sequentially with MCP-1 and GAPDH.

Mentions: Because the MDC clone was isolated from a human macrophage cDNA library, its expression during differentiation of monocytes into macrophages was examined. Human monocytes from a single donor were cultured on a series of tissue culture plates, and cells from individual plates were harvested after 0, 2, 4, or 6 d. Under these conditions, monocytes differentiate into macrophages by day 6 (36, 41). A Northern blot of RNA from the cells harvested at each time point was probed with the MDC cDNA. No signal was detectable in RNA from freshly isolated monocytes, whereas a very strong signal was generated from cells that had differentiated into macrophages after 6 d of culture (Fig. 4 A).


Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW - J. Exp. Med. (1997)

Northern analysis of MDC mRNA expression in human  cells. Migration of MDC, 28S RNA, and 18S RNA is indicated. (A) Peripheral blood monocytes were allowed to differentiate into macrophages  by incubation on plastic tissue culture dishes for 6 d (41). Total RNA was  isolated from individual dishes on the indicated days, fractionated through  a formaldehyde agarose gel, blotted, and probed with the radiolabeled  MDC cDNA. The blot was subsequently probed for GAPDH message.  (B) Expression of MDC in differentiated HL-60 cells. Cells were treated  with PMA to induce differentiation to monocytic cells or with DMSO to  induce granulocytic cells. MDC expression was analyzed by Northern  blotting as in A. (C) Northern blot of MDC expression in human peripheral blood monocytes (Mono.), macrophages derived from these monocytes (Mac.), dendritic cells derived from PBMC of two different donors  (DC-1 and DC-2), and natural killer cells derived from PBMC (NK). (D)  Expression of MDC in cell cultures. Lung epithelial cell line A549, lung  fibroblast line IMR90, and I-HUVEC without and with TNF-α stimulation; PBMC without and with stimulation by PHA and PMA; and  monocyte derived macrophages after 6 d in culture. After probing with  MDC, the filter was stripped and probed sequentially with MCP-1 and  GAPDH.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196293&req=5

Figure 4: Northern analysis of MDC mRNA expression in human cells. Migration of MDC, 28S RNA, and 18S RNA is indicated. (A) Peripheral blood monocytes were allowed to differentiate into macrophages by incubation on plastic tissue culture dishes for 6 d (41). Total RNA was isolated from individual dishes on the indicated days, fractionated through a formaldehyde agarose gel, blotted, and probed with the radiolabeled MDC cDNA. The blot was subsequently probed for GAPDH message. (B) Expression of MDC in differentiated HL-60 cells. Cells were treated with PMA to induce differentiation to monocytic cells or with DMSO to induce granulocytic cells. MDC expression was analyzed by Northern blotting as in A. (C) Northern blot of MDC expression in human peripheral blood monocytes (Mono.), macrophages derived from these monocytes (Mac.), dendritic cells derived from PBMC of two different donors (DC-1 and DC-2), and natural killer cells derived from PBMC (NK). (D) Expression of MDC in cell cultures. Lung epithelial cell line A549, lung fibroblast line IMR90, and I-HUVEC without and with TNF-α stimulation; PBMC without and with stimulation by PHA and PMA; and monocyte derived macrophages after 6 d in culture. After probing with MDC, the filter was stripped and probed sequentially with MCP-1 and GAPDH.
Mentions: Because the MDC clone was isolated from a human macrophage cDNA library, its expression during differentiation of monocytes into macrophages was examined. Human monocytes from a single donor were cultured on a series of tissue culture plates, and cells from individual plates were harvested after 0, 2, 4, or 6 d. Under these conditions, monocytes differentiate into macrophages by day 6 (36, 41). A Northern blot of RNA from the cells harvested at each time point was probed with the MDC cDNA. No signal was detectable in RNA from freshly isolated monocytes, whereas a very strong signal was generated from cells that had differentiated into macrophages after 6 d of culture (Fig. 4 A).

Bottom Line: High expression was also detected in normal thymus and less expression in lung and spleen.Unlike most other CC chemokines, MDC is encoded on human chromosome 16.MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

View Article: PubMed Central - PubMed

Affiliation: Icos Corporation, Bothell, Washington 98021, USA.

ABSTRACT
A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

Show MeSH
Related in: MedlinePlus