CYTOKINE RELEASE BY HUMAN HONE MARROW-CELLS - ANALYSIS AT THE SINGLE-CELL LEVEL

Regulation of haemopoiesis is closely mediated by a number of growth f actors in the marrow microenvironment. The identification of the cell type secreting these regulatory polypeptides is difficult due to the h eterogeneity of bone marrow cells. To analyse the release of haemopoie tic growth factors by normal human bone marrow cells at the single cel l level, we employed the reverse haemolytic plaque assay (RHPA). Fresh ly isolated human marrow cells were examined for the release of interl eukin-la (IL-1 alpha), IL-3, IL-6 and granulocyte-monocyte colony stim ulating factor (GM-CSF). In order to identify various cytokine-secreti ng cell types, the RHPA was combined with immunocytochemical or enzyma tic staining. The total of secreting marrow cells as well as the amoun t of several secretory haemopoietic subpopulations could be determined with this technique under various conditions. Following incubation wi th pure serum-free medium without addition of any mediator, only few c ells secreting either IL-1 alpha, IL-3, IL-6 or GM-CSF could be observ ed. After 2 h incubation with recombinant human-IL-1 alpha (rhIL-1 alp ha) (10.0 ng/ml) or rhGM-CSF (10.0 pg/ml) the number of cytokine-secre ting cells significantly increased for all secretory products tested. Using cytochemical staining reactions, we were able to identify 55% of all cells secreting a specific cytokine. Glycophorin C-positive eryth ropoietic cells turned out to be the largest fraction (up to 89%) of c ytokine-releasing haemopoietic cells, followed by neutrophil granulocy tes (between 6 and 48%), and monocytes/macrophages (between 4 and 23%) . Only few CD 61-positive cytokine-secreting megakaryocytes could be d etected. Dose- and time-dependent kinetics after stimulation with rhGM -CSF revealed that the bulk of secretory activity originates from haem opoietic or rather from erythropoietic cells following low level stimu lation and after short stimulation time. Thus, our data are in keeping with the assumption, that especially erythropoietic cells are produci ng a repertoire of cytokines that is thought to exhibit regulatory fun ctions within marrow microenvironment. In the present study the RHPA i s presented as an appropriate tool for measuring cytokine release not only of cells of the haematopoietic system but also of other tissues, for example solid tumours or malignant lymphomas.