The human neutrophil NB1 glycoprotein (NB1 GP, HNA-2a, CD177) has gained clinical importance for being involved in pulmonary transfusion reactions and immune neutropenias. The NB1 GP shows the unique feature of being expressed only on a neutrophil subpopulation. Recently, we identified splicing defects responsible for an NB1 GP deficiency. In this study, we have investigated the molecular basis of the heterogeneous expression of NB1 GP by separating the 2 neutrophil subpopulations using immunofluorescence followed by single-cell picking or by fluorescence-activated cell sorter. We found a lack of NB1 mRNA in the NB1 GP ؊ cells that remained constant even after granulocyte colony-stimulating factor (G-CSF) administration. Comparing the cDNA sequences of donors with a large (> 60%) and those with a small (< 40%) NB1 GP-expressing subpopulation, we found 6 polymorphisms. Of the 6, 3 were significantly associated with a small NB1 GP-expressing subpopulation, indicating a genetic basis for NB1 GP nonexpression. (Blood. 2003;102:731-733)

Bone lining cells cover > 80% of endosteal surfaces of human cancellous bone. Current research assigns to them a dual role: (1) as a biological membrane regulating exchange of substrates between the bone fluid compartment and the extracellular fluid of bone marrow and (2) as a signaling link between the osteocytic network as mechanical receptor and the osteoclastic cell pool for local induction of bone resorption. Furthermore, a catabolic role has been considered. We therefore examined the presence of matrix-metalloproteinases (MMPs) and their physiological tissue inhibitors (TIMPs) as putative proteolytic elements. Firstly, human cancellous bone from 60 patients was examined by immunofluorescence with antibodies against MMPs and TIMPs. Secondly, we applied laser-assisted microdissection (LMD) to isolate bone lining cells from frozen sections of human trabecular bone. mRNA analysis was performed using a single-cell PCR protocol. Three laser microdissection systems were tested: the new generation of Leica LMD and P.A.L.M. laser pressure catapulting (LPC) were compared to P.A.L.M. laser microdissection and micromanipulation (LMM). In a few pooled cell profiles, mRNA of MMP13, MMP14, TIMP1, and CBFA-1 was clearly detected. By immunofluorescence MMP13 and -14 as well as TIMP1 and -2 were strongly present in lining cells, while MMP2, TIMP3, and TIMP4 showed weak or negative signals. Although the functional impact of these enzymatic components remains open, there is additional evidence for a catabolic function of lining cells. The new diode-laser microdissection with LMD and LPC proved to be especially suitable to gain new insights into the properties of bone lining cells.