Modulation of chemokine gene expression in CD133+ cord blood-derived human mast cells by cyclosporin A and dexamethasone.
We have recently developed a protocol for generating huge numbers of mature and functional mast cells from in vitro differentiated umbilical cord blood cells. Using CD133 as a positive selection marker to isolate haematopoietic progenitors we routinely expand the number of recovered cells at least 150-fold, which vastly exceeds the yields of conventional protocols using CD34+ cells as a source of progenitors. Taking advantage of the large quantities of in vitro differentiated mast cells, here we assess at the levels of transcription and translation the kinetics of chemokine gene induction following receptor mediated mast cell activation or following pharmacological activation of specific signal transduction cascades that become activated upon classical FcepsilonRI receptor crosslinking. We demonstrate that chemokine genes encoding IL-8, MCP-1, MIP-1alpha, and MIP-1beta are induced with different kinetics and with different amplitudes in a receptor activation dependent manner, and that these events can be mimicked using pharmacological agents which activate distinct signal transduction pathways. These findings were corroborated by adding immunomodulators such as cyclosporin A and dexamethasone prior to mast cell activation. Finally, we demonstrate that the same modulators added after mast cell activation can differentially quench ongoing chemokine gene induction. Thus, considering the vast yields of mast cells, our protocol is valuable not only for studying regulation of gene expression in mast cells in general, but also as an experimental tool to develop better and more balanced treatments of mast cell related disorders.
Holm
M
M
Department of Human Genetics, University of Aarhus, Aarhus C, Denmark.
Kvistgaard
H
H
Dahl
C
C
Andersen
H B
HB
Hansen
T K
TK
Schiøtz
P O
PO
Junker
S
S
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000097:blood*; D000954:Antigens, Surface / Q000032:analysis; D018925:Chemokines / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology*; D005312:Fetal Blood; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000097:blood*; D019718:Receptors, CXCR4 / Q000378:metabolism; D017455:Receptors, IgE / Q000378:metabolism; D015533:Transcriptional Activation
D000071916:AC133 Antigen; D015703:Antigens, CD; D000954:Antigens, Surface; D018925:Chemokines; C475664:FCER1A protein, human; D006023:Glycoproteins; D007166:Immunosuppressive Agents; C000606023:PROM1 protein, human; D010455:Peptides; D019718:Receptors, CXCR4; D017455:Receptors, IgE; D003907:Dexamethasone; D016572:Cyclosporine