463 rhGM-CSF promotes an antigen-presenting phenotype in human macrophages

Abstract

461 Distribution o f Plasma Cell Markers and Intracellular IgE in t h e Cell L i n e U266. E Lagging BS, M van HageHamsten MD. B H~rfast PhD, G Halld~n PhD, Stockholm, SWEDEN We made use of the IgE producing plasma cell line U266 in order to be able to stud)' intracellular IgE and the distribution of the plasma cell markers CD38 and PCA-I. The cell were penneabilized according to the FOG method (G Hallddn, J lmmunol Methods 124. 1989) and analysed with flow ~'tometric technique. Further cell cycle analyses were performed by means ofpropidium iodide (PI) staining. Interestingly we found two separate populations of U266 based on the differences in their light scatter properties in the floxx~'tometer. One population had high scatter signals (HIGH SS) and the other low scatter signals (LOW SS). Most (75%) of the cells in the LOW SS cluster were positive for intracellular IgE while the expression of surface IgE was lower than that of the HIGH SS cluster. We further confirmed the immcellular location of IgE in the FOG treated U266 cells by means of immuuofluoresccnce microscopy analyses. In comparing the two clusters for expression of the plasma cell antigens CD38 and PCA-1 different patterus were observed. While the LOW SS cluster displayed a high (76%) expression of PCA-I and a low (5%) CD38 expression, the expression of the HIGH SS cluster was 21% and 42%, respectively. About 2/3 of the cells in the LOW SS cluster were in the G~ phase of the cell ~,cle while 1/3 were in the S/G2 phase. As to the HIGH SS cluster the opposite distribution (2/3 in S/G:) was observed. Taken together our results implicate an association between cell cycle stages, expression ofintraccllular IgE. and plasma cell markers. 463 rhGM-CSF promotes an antigen-presenting phenotype in human macrophages JJ Caulfield BSe. DM Kemenv PhD. 31-1 Lee MD UMDS, Guy's Hospital, London, UK Asthmatic airways are charaeterised by the presence of enhanced levels of GM-CSF and increased numbers of macrophages. Ja order to assess whether GM-CSF can modulate macrophage function, we have developed an in vitro assay in which monocytes isolated from human peripheral blood were cultured for seven days in RPMI + 50% AB serum in the presence or absence of rhGM-CSF (50 U/ml). The cultured monocyte-dedved macrophages (MDMs) increased in size and phagocytic ability and expressed antigens involved in co-stimulation and antigen presentation (CDIIa, CDIIb, CDIIc, CD54 and HLADR). After 7 days, monocytes and CD4s were isolated from the same donor. Proliferation assays were then performed in triplicate with lx104 freshly isolated monocytes or MDMs and lx105 CD4 cells/well with either HDM or PPD as antigen, for 6 days. CD4 proliferation was determined by [3H]-thymidine incorporation. In the absence of GM-CSF, CD4 proliferation was reduced when HDM or PPD was presented by MDMs, compared to the values obtained with freshly isolated monocytes. In the presence of GM-CSF, CD4 proliferation was comparable to that observed with monocytes when the respective antigen was presented by MDMs. This data suggests that GM-CSF may direct macrophage differentiation into an antigen-presenting, rather than a suppressive, phenotype in the asthmatic lung.