TGF-a, transforming growth factor a, is a potent growth factor belonging to the family of EGF (epidermal growth factor) -related proteins. Binding to the same receptor as EGF it has a ubiquitous repertoire of target cells, including, mesenchymal, epithelial and neuronal cells. The occurrence and role of TGF-a in hematopoietic cells has not been elucidated. Therefore the present work was initiated to (I) examine the expression of TGF-a and the EGF receptor in nmmal circulating human blood cells; (2) study the effects of specific cytokines on TGFa mRNA levels and protein release in mature blood cells; (3) examine the occurrence of TGF-a and the EGF receptor in normal human bone marrow cells; (4) determine TGF-a gene expression and protein production during granulocyte and monocyte/macrophage differentiation in vitro.
TGF-a mRNA was consistently found in white blood cells from normalhealthy donors as shown by Northern blot analysis. In situ hybridizationexperiments assigned the TGF-a gene expression to the eosinophils; no other cell types were recognized by the complementary TGF-a RNA probe. Incubation of white blood cells led to liberation of TGF-a to the culture medium, as determined by ELISA.
White blood cells exposed in vitro to the cytokines GM-CSF (granulocytemacrophage colony stimulating factor) or interleukin (IL)-3 showed increased levels of TGF-a mRNA. The effect of GM-CSF was different from that of IL-3, since only GM-CSF augmented the release of TGF-a protein from the cells.
Immunohistochemical examination of human normal bone marrow cells, using a monoclonal TGF-a antibody, revealed TGF-a-reactive material on erythroid cells, at all stages of differentiation. The nature of the stain, in conjunction with the fact that the cells could be pulled out by immunomagnetic cell sorting would seem to indicate a membrane-bound, extracellular configuration of TGF-a, rather than an intracellular one. Using a different antibody a different staining pattern was obtained, indicating the presence of TGF-a in eosinophilic precursor cells and in promyelocytes and neutrophilic myelocytes.
Attempts were made to identify target cells for TGF-a, i.e. EGF receptorcarrying cells. Intron-differential reverse transcriptase PCR was used to detect the EGF receptor signal. Immunohistochemistry revealed the EGF receptor protein in a small but distinct population of immature, blast-like cells of myelomonocytic appearance.
The expression of TGF-a was monitored, at the mRNA and protein level, in human leukemic cells induced to differentiate in culture. Differentiation of the promyelocytic cell line, HL-60, along the granulocytic pathway was accompanied by increased TGF-a mRNA levels and TGF-a protein release.
When tbe HL-60 cells were brought towards monocytes/macrophages tbe effect on TGF-a expression depended on the inducing agent used, irrespective of a number of differentiation criteria.
Differentiation of tbe monocytoid cell line, U-937, witb different inducers had different effects on TGF-a mRNA levels as well as TGF-a release. This supports tbe idea of phenotypic heterogeneity in tbe differentiated cells.
Linköping: Linköpings universitet , 1994. , 41 p.
1994-04-15, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Papers, included in the Ph.D. thesis, are not registered and included in the posts from 1999 and backwards.