Limits...
Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

Mueller CG, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Briere F, Lebecque S, Liu YJ - J. Exp. Med. (1997)

Bottom Line: It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction.In situ hybridization showed distinct expression of this gene in the germinal center.This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough Laboratory for Immunological Research, 69571 Dardilly, France.

ABSTRACT
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

Show MeSH
Restricted expression  profile of decysin by RT-PCR.  (A) Decysin is not detected in  PMA/ionomycin-activated human cell lines TF1 (myeloid precursor cell), JURKAT (T cell),  CHA, MRC5 (kidney epithelial  and lung fibroblastic cells), and  JY (B cell), but is expressed in  PMA/ionomycin-activated stem  cell–derived DCs. PCR with  specific primers to decysin (Fig.  3 A) and β actin was performed  on reverse transcribed RNA  from the cell lines, stem cell– derived DCs harvested at days 6  and 12 of cell culture (6) as well  as GCDC library subtracted (Fig. 1, lane 7) and nonsubtracted (Fig. 1, lane 5). In the subtracted library, β actin could not be amplified. (B) A low level of  cDNA is seen in PBMCs, monocytes and B cells. GCDCs were activated for 24 h by αCD40 antibody G28-5 in complete medium. Peripheral blood  mononuclear cells, monocytes, and T lymphocytes were obtained from human peripheral blood, and B lymphocytes from human tonsils.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199019&req=5

Figure 2: Restricted expression profile of decysin by RT-PCR. (A) Decysin is not detected in PMA/ionomycin-activated human cell lines TF1 (myeloid precursor cell), JURKAT (T cell), CHA, MRC5 (kidney epithelial and lung fibroblastic cells), and JY (B cell), but is expressed in PMA/ionomycin-activated stem cell–derived DCs. PCR with specific primers to decysin (Fig. 3 A) and β actin was performed on reverse transcribed RNA from the cell lines, stem cell– derived DCs harvested at days 6 and 12 of cell culture (6) as well as GCDC library subtracted (Fig. 1, lane 7) and nonsubtracted (Fig. 1, lane 5). In the subtracted library, β actin could not be amplified. (B) A low level of cDNA is seen in PBMCs, monocytes and B cells. GCDCs were activated for 24 h by αCD40 antibody G28-5 in complete medium. Peripheral blood mononuclear cells, monocytes, and T lymphocytes were obtained from human peripheral blood, and B lymphocytes from human tonsils.

Mentions: In the case of the human cell lines and stem cell-derived DCs (Fig. 2 A) 50 μg of total RNA was treated with 20 units DNAse (Boehringer Mannheim GmbH, Mannheim, Germany) in the presence of RNAse inhibitor (RNAsin; Promega, Madison, WI) in standard reaction conditions. After phenol/chloroform extraction, the RNA was reverse transcribed using Superscript™II (GIBCO BRL, Gaithersburg, MD) and a dT(12-18) oligonucleotide, according to instructions (GIBCO BRL). The reaction was stopped, and nucleic acids were ethanol precipitated and resuspended in water. About 20 ng cDNA was used in one PCR reaction. For all other cells, total RNA was prepared from ∼100,000 cells, reverse transcribed using SuperscriptTMII (GIBCO BRL) and a random hexaoligonucleotide (pd[N]6; Pharmacia) in a 20 μl reaction. 1 μl was used in a PCR reaction. The 50 μl PCR reaction contained 100 ng primers, 200 μM dNTP (Pharmacia, Uppsala, Sweden), and 0.5 units AmpliTaq™ polymerase (Roche Molecular Systems Inc., Branchburg, NJ) in standard PCR buffer (Roche Molecular Systems) and was subjected to 28 or 35 cycles of denaturing (30 s, 94°C), annealing (30 s, 60°C), and extension (2 min, 72°C) on the Perkin-Elmer thermal cycler 480. Decysin primers U137 and L677 are shown in Fig. 3 A, β actin primers were purchased (Stratagene, La Jolla, CA). The Marathon™ kit (Clontech) was used for RACE PCR and performed as recommended by the supplier. The outward primers were 5′-CCCATCAGACCAGATTTCCATACCTACC (upstream) and 5′-CCCATCTTCGGTTGCTGTTATTGAGGCT (downstream), and were used with the Marathon™ recommended cycling program 1. The two distinct PCR products were cloned into the T/A-vector and sequenced.


Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

Mueller CG, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Briere F, Lebecque S, Liu YJ - J. Exp. Med. (1997)

Restricted expression  profile of decysin by RT-PCR.  (A) Decysin is not detected in  PMA/ionomycin-activated human cell lines TF1 (myeloid precursor cell), JURKAT (T cell),  CHA, MRC5 (kidney epithelial  and lung fibroblastic cells), and  JY (B cell), but is expressed in  PMA/ionomycin-activated stem  cell–derived DCs. PCR with  specific primers to decysin (Fig.  3 A) and β actin was performed  on reverse transcribed RNA  from the cell lines, stem cell– derived DCs harvested at days 6  and 12 of cell culture (6) as well  as GCDC library subtracted (Fig. 1, lane 7) and nonsubtracted (Fig. 1, lane 5). In the subtracted library, β actin could not be amplified. (B) A low level of  cDNA is seen in PBMCs, monocytes and B cells. GCDCs were activated for 24 h by αCD40 antibody G28-5 in complete medium. Peripheral blood  mononuclear cells, monocytes, and T lymphocytes were obtained from human peripheral blood, and B lymphocytes from human tonsils.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199019&req=5

Figure 2: Restricted expression profile of decysin by RT-PCR. (A) Decysin is not detected in PMA/ionomycin-activated human cell lines TF1 (myeloid precursor cell), JURKAT (T cell), CHA, MRC5 (kidney epithelial and lung fibroblastic cells), and JY (B cell), but is expressed in PMA/ionomycin-activated stem cell–derived DCs. PCR with specific primers to decysin (Fig. 3 A) and β actin was performed on reverse transcribed RNA from the cell lines, stem cell– derived DCs harvested at days 6 and 12 of cell culture (6) as well as GCDC library subtracted (Fig. 1, lane 7) and nonsubtracted (Fig. 1, lane 5). In the subtracted library, β actin could not be amplified. (B) A low level of cDNA is seen in PBMCs, monocytes and B cells. GCDCs were activated for 24 h by αCD40 antibody G28-5 in complete medium. Peripheral blood mononuclear cells, monocytes, and T lymphocytes were obtained from human peripheral blood, and B lymphocytes from human tonsils.
Mentions: In the case of the human cell lines and stem cell-derived DCs (Fig. 2 A) 50 μg of total RNA was treated with 20 units DNAse (Boehringer Mannheim GmbH, Mannheim, Germany) in the presence of RNAse inhibitor (RNAsin; Promega, Madison, WI) in standard reaction conditions. After phenol/chloroform extraction, the RNA was reverse transcribed using Superscript™II (GIBCO BRL, Gaithersburg, MD) and a dT(12-18) oligonucleotide, according to instructions (GIBCO BRL). The reaction was stopped, and nucleic acids were ethanol precipitated and resuspended in water. About 20 ng cDNA was used in one PCR reaction. For all other cells, total RNA was prepared from ∼100,000 cells, reverse transcribed using SuperscriptTMII (GIBCO BRL) and a random hexaoligonucleotide (pd[N]6; Pharmacia) in a 20 μl reaction. 1 μl was used in a PCR reaction. The 50 μl PCR reaction contained 100 ng primers, 200 μM dNTP (Pharmacia, Uppsala, Sweden), and 0.5 units AmpliTaq™ polymerase (Roche Molecular Systems Inc., Branchburg, NJ) in standard PCR buffer (Roche Molecular Systems) and was subjected to 28 or 35 cycles of denaturing (30 s, 94°C), annealing (30 s, 60°C), and extension (2 min, 72°C) on the Perkin-Elmer thermal cycler 480. Decysin primers U137 and L677 are shown in Fig. 3 A, β actin primers were purchased (Stratagene, La Jolla, CA). The Marathon™ kit (Clontech) was used for RACE PCR and performed as recommended by the supplier. The outward primers were 5′-CCCATCAGACCAGATTTCCATACCTACC (upstream) and 5′-CCCATCTTCGGTTGCTGTTATTGAGGCT (downstream), and were used with the Marathon™ recommended cycling program 1. The two distinct PCR products were cloned into the T/A-vector and sequenced.

Bottom Line: It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction.In situ hybridization showed distinct expression of this gene in the germinal center.This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough Laboratory for Immunological Research, 69571 Dardilly, France.

ABSTRACT
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

Show MeSH