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Materials and Methods
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Umbilical
Cord Blood-Derived Human Mast Cells.
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In vitro-derived
human mast cells were obtained essentially as described in Kempuraj et al.
(1). Heparinized cord blood (from Brigham and Women's Hospital, Boston, MA or
Stanford University Medical Center, Stanford, CA) was overlaid on Histopaque
1077 (Sigma, St. Louis, MO) and centrifuged at room temperature for 30 min at
400 x g. Mononuclear cells at the interface between plasma and Histopaque 1077
were collected and CD34+ cells were isolated from the mononuclear cells using a
magnetic separation column and CD34+ cell isolation kit (Miltenyi Biotec,
Auburn, CA) according to the manufacturer's instructions. The CD34+ cells were
maintained in culture medium consisting of Iscove's modified Dulbecco's medium (IMDM;
BioWhittaker, Walkersville, MD) supplemented with 0.1% bovine serum albumin (BSA;
Sigma); 80 ng/ml recombinant human stem cell factor164 (rhSCF; Amgen,
Thousand Oaks, CA); 50 ng/ml recombinant human interleukin-6 (rhIL-6; Amgen);
and from Gibco BRL (Gaithersburg, MD): 1% Insulin-Transferrin-Seleniumm, 10 mM
HEPES, 2 mM L-glutamine, antibiotics (100 U/ml penicillin, 100
mg/ml
streptomycin, and 10
mg/ml
gentamicin), 1x MEM vitamin solution, 50
mM
2-ME, and from BioWhittaker: 1x MEM amino acids and 1mM sodium pyruvate. One ng/ml
recombinant human IL-3 (rhIL-3; PeproTech, Rocky Hill, NJ) was added only once,
at the beginning of the culture; thereafter, half of the volume of the culture
medium (but without rhIL-3) was changed weekly. After 9 wk, 0.1% BSA was
replaced with 10% FCS (Hyclone, Logan, UT). Some populations of mast cells used
in this study were generated with some slight modifications of the method listed
above; 10% FCS was added to the culture medium for the first two weeks of
culture and then for the whole duration of the culture after 6 weeks of culture.
Cells from 11-19 wk cultures were used for experiments, at which time mast cells
accounted for > 99% of the total cells according to Kimura's stain and 98% (1
population) or >99% (all the others) of the cells by immunohistochemistry for
mast cell-associated tryptase (2).
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Stimulation of Mast Cells via the FceRI.
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Mast cells were incubated for 4 days with
purified human myeloma IgE (5
mg/ml,
Biodesign International, Kennebunk, ME), rhIL-4 (10 ng/ml, PeproTech, Rocky
Hill, NJ) to enhance surface expression of FceRI
(3). The cells then were stimulated with 10 or 1
mg/ml
of anti-human IgE antibody (Sigma). Cells were harvested immediately before or
at 1 or 2 h after stimulation with anti-IgE for microarray analyses. Histamine
release was assessed using the histamine enzyme immunoassay kit (Immunotech,
Westbrook, ME) and IL-11 secreted into the medium was measured with a human
IL-11 immunoassay kit (R&D Systems, Minneapolis, MN).
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cDNA microarray procedures. |
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The cDNA microarray used in these experiments
represents a set of ~ 14,000 genes related to immune function (4). All analyses
were performed using poly-(A)+ mRNA (extracted by Fast Track, Invitrogen,
Carlsbad, CA) (5). The experimental samples were our human mast cell
preparations; the control "reference" samples represented a pool of mRNA
isolated from a human mast cell line (HMC-1, ref. 6), a T cell line (Jurkat),
and a B cell line (Raji). For each cell type, the mRNA used for the pool
consisted of that derived from unstimulated cells (80% of the total) and
stimulated cells (20% of the total); the control cell lines were stimulated with
1 mM
Ionomycin (Sigma) and 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) for
2 h at 37ºC. The use of common control cDNA probe allows the relative expression
of each gene to be compared across all samples.
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References:
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- Kuhn C, III, Homer RJ, Zhu Z, et al. Airway hyperresponsiveness and
airway obstruction in transgenic mice. Morphologic correlates in mice
overexpressing interleukin (IL)-11 and IL-6 in the lung. Am J Respir Cell Mol
Biol. 2000;22:289-295.
- Busse W, Elias J, Sheppard D,
Banks-Schlegel S. Airway remodeling and repair Am J Respir Crit Care Med.
1999;160:1035-1042.
- Yamaguchi M, Sayama K, Yano K, et al. IgE
enhances Fc epsilon receptor I expression and IgE-dependent release of histamine
and lipid mediators from human umbilical cord blood-derived mast cells:
synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I
expression and mediator release. J Immunol. 1999;162:5455-5465.
- Alizadeh AA,
Elsen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma
identified by gene expression profiling. Nature. 2000;403:503-511.
- Eisen
MB, Brown PO. DNA arrays for analysis of gene expression. Methods Enzymol.
1999;303:179-205.
- Butterfield JH, Weiler D, Dewald G, Gleich GJ.
Establishment of an immature mast cell line from a patient with mast cell
leukemia. Leuk Res. 1988;12:345-355.
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