To test the role of Fc gamma RIIa in IgG anti-RhD-mediated phagocytosis three IgG1 and two IgG3 human monoclonal anti-D antibodies were tested for ability to mediate binding/phagocytosis of cDE/cde and -D-/-D- red cells by Fc gamma RIIa-R131 and Fc gamma RIIa-H131 cDNA-transfected 3T6 fibroblasts. Both IgG3 monoclonal antibodies brought about -D-/-D- cell interaction with IIa-transfected fibroblasts, while only one of them, Og3, mediated binding of cDE/cde targets. Although Fc gamma RIIa expression was three times greater on IIa-R131 than on IIa-H131 fibroblasts, the latter bound significantly more Og3-coated cDE/cde- and IgG3 anti-D-sensitized -D-/-D- cells, respectively, than the former effectors and showed some phagocytosis of the -D-/-D- targets. IgG1 anti-D antibodies were inactive in mediating red cell interaction with the fibroblasts. Moreover, monoclonal anti-Fc gamma RII IV.3 partially inhibited the phagocytosis by adult or fetal monocytes of Og3-sensitized cDE/cde cells. Fc gamma RIIa-H/H131 monocytes exhibited higher phagocytic indices towards these targets than monocytes of other IIa allotypes. The results indicate that Fc gamma RIIa can participate in the phagocytosis of red cells coated with IgG3 anti-D; in this case the allotype of the receptor will modify the extent of red cell destruction.

Aims-To determine the role of insulinlike growth factors (IGF) in the proliferation of tumour cells, by studying the mitogenic response to IGFs of three cell lines of differing phenotype established from both malignant rhabdoid and Wilms tumour, representing a range of cell types (GOS 4, G401, and T3/73). Methods-Production ofIGF-II and IGF-I was measured by radioimmunoassay, and the presence of IGF binding protein complexes was observed by gel exclusion chromatography. Following growth analyses in serum-free media to ascertain the dependence of the cell lines on exogenous IGFs, the generation of autocrine growth was measured by a density dependence assay ofproliferation in culture. Receptors were measured by radioligand cross linking and autocrine growth through these receptors assayed by the use of blocking antibodies. Results-While GOS 4 and G401 were able to proliferate in serum-free medium over a period of 5 d, T3/73 showed an absolute dependence on IGFs added daily at 1-10 ng/ml. Plating at clonal density showed that cell growth was directly density dependent in serum-free medium. The serum independent proliferation of G401 and GOS 4 was blocked by the addition of an antibody to the type 1 IGF receptor (a-IR3) suggesting that the effects of autocrine factors are mediated through type 1 IGF receptors. SI nuclease protection analysis indicated that all three cell lines produced significant amounts of mRNA derived mainly from the P3 IGF-II promoter, but transcripts for IGF-I were undetectable. Radioimmunoassay of IGFs from conditioned media showed that all the lines made assayable , and 6-1 ng/mll24 h/106 cells for GOS 4, G401, and T3/73 respectively). The presence of species consistent with both type 1 and type II IGF receptors was demonstrated using radioligand binding to cell membranes followed by cross linking. Conclusions-Autocrine IGF-II may contribute to the serum independence of GOS 4 and G401 cells, whereas T3/73 may depend on exogenous IGF-II for proliferation. (7 Clin Pathol: Mol Pathol 1995;48:M333-M341)

Although insulin-like growth factor (IGF-1) stimulated 3H-thymidine incorporation upon addition to the interleukin-3 (IL-3)-dependent cell line FDC-P1, IGF-1 did not relieve IL-3 dependency for growth. To further examine the effects of IGF-1 on hematopoietic cells, FDC-P1 cells were infected with a retroviral construct (LISN) containing the human IGF-1 receptor (hIGF-1R) and neo genes. IL-3-independent cells were readily isolated after LISN infection when either IGF-1 or supraphysiologic concentrations of insulin were included in the culture medium. These cells were transformed to IL-3 independence by a ligand- dependent mechanism because their growth was dependent on the presence of either IGF-1 or insulin and growth factors capable of supporting autocrine growth were not detected. Furthermore, a monoclonal antibody (MoAb) directed against the human IGF-1R (alpha IR-3) inhibited IGF-1 but not IL-3-induced proliferation and these cells contained 20- to 200- fold more IGF-1 receptors than uninfected FDC-P1 cells. In contrast, when LISN-infected cells were plated in medium without exogenously supplied IGF-1 or insulin, factor-independent cells were rarely isolated. Growth of these cells was also inhibited by the alpha IR-3 MoAb and they expressed 100- to 400-fold more IGF-1 receptors than uninfected FDC-P1 cells. The endogenous IGF-1 and/or insulin present in the calf serum may have enabled their growth because these cells, unlike the parental cells, would proliferate in serum-free defined media and their growth was again inhibited by the alpha IR-3 MoAb. These results demonstrate that IGF-1 can replace IL-3 for growth when FDC-P1 cells overexpress the IGF-1R. Given the fairly ubiquitous expression of the IGF-1 receptor, these and additional experiments might help to determine whether increased expression of endogenous receptors by cells can lead to leukemogenesis and tumorigenesis. Moreover, hIGF-1R-infected cells will be useful in investigating the mechanisms of IGF1-mediated signal transduction because they are now known to proliferate in response to IGF-1.