Journal of Leukocyte Biology eBioscience full spectrum cell analysis
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Originally published online as doi:10.1189/jlb.0308172 on July 14, 2008

Published online before print July 14, 2008
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(Journal of Leukocyte Biology. 2008;84:1151-1158.)
© 2008 by Society for Leukocyte Biology

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models

Jacqueline M. Langdon, John T. Schroeder, Becky M. Vonakis, Anja P. Bieneman, Kristin Chichester and Susan M. MacDonald1

The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland, USA

1 Correspondence: The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA. E-mail: smacdona{at}jhmi.edu

Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6–8 weeks and basophils with IL-3 (6.7 ng/ml) for 2–3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 ± 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 ± 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.

Key Words: siRNA • releasability






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