Co-cultivation of mast cells and Fc epsilon RI alpha+ dendritic-like cells from human hip bone marrow.
BACKGROUND
Mast cells contribute to the pathogenesis of asthma and allergy through the release of a plethora of pro-inflammatory mediators and cytokines. Their study is hampered by the difficult access to human tissue samples. Human mast cells have been cultured from CD34+ progenitors in the bone marrow of normal volunteers following iliac crest bone marrow biopsy but this is invasive. Hip bone marrow could provide a more convenient less invasive source of mast cell progenitors.


OBJECTIVE
To characterize mast cells cultured from human bone marrow obtained at routine hip surgery.


METHODS
Mononuclear cells were isolated from the bone marrow reamings of patients undergoing routine hip replacement surgery and were cultured with recombinant stem cell factor (SCF), IL-6 and IL-10. Cell surface markers were examined using flow cytometry, protease expression monitored using immunohistochemistry, histamine measured by radioenzymic assay, Ca2+ responses analysed using ratiometric Ca2+ imaging, and ion currents recorded via the patch-clamp technique.


RESULTS
Mast cells were absent at baseline, but accounted for 65 +/- 7% of cells after 8-12 weeks of culture, equating to a mean 0.6 +/- 0.14 x 10(6) mast cells per culture. Fifty-three percent of tryptase+ cells also contained chymase. The remaining cells comprised a population of large CD1a+ HLA-DR+ and Fc epsilon RI alpha+ cells, most likely dendritic cells. All mast cells expressed CD117 and the high-affinity IgE receptor alpha-chain (Fc epsilon RI alpha) constitutively, and developed a Ca2+ response following IgE-dependent activation. These cells exhibited 7.8 +/- 2.9% net IgE-dependent histamine release, and demonstrated a similar ion channel profile to human lung mast cells. In particular, the intermediate conductance Ca(2+)-activated K+ channel opened following IgE-dependent activation.


CONCLUSIONS
Mast cells grown from human hip marrow provide a rich non-invasive source of functionally mature mast cells. In addition, this culture system may be useful for the generation of Fc epsilon RI alpha+ dendritic cells.
Kaur
D
D
Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester and Warwick Medical School, University Hospitals of Leicester, Glenfield General Hospital, Leicester, UK. dk24@leicester.ac.uk
Berger
P
P
Duffy
S M
SM
Brightling
C E
CE
Bradding
P
P
D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000276:immunology*; D004594:Electrophysiology; D005434:Flow Cytometry; D006615:Hip; D006636:Histamine Release; D006801:Humans; D008407:Mast Cells / Q000276:immunology*; D017455:Receptors, IgE / Q000276:immunology*; D013048:Specimen Handling
D017455:Receptors, IgE