Differential expression of CD32 isoforms following alloactivation of human T cells.

Receptors for the Fc region of immunoglobulin G (IgG) (Fc gamma Rs) exist in three main forms: membrane bound, soluble and cytoplasmic. The function of cytoplasmic Fc gamma Rs is poorly understood. We have previously demonstrated cytoplasmic Fc gamma RII (cCD32) within most normal human peripheral blood lymphocytes (PBL), including T cells. In this study we have investigated the hypothesis that following lymphocyte activation, up-regulation of cCD32 occurs, resulting in increased expression at the cell surface. Normal PBL were activated in vitro using a two-way mixed lymphocyte reaction (MLR) and expression of CD32 monitored by flow cytometry and by immunoperoxidase staining using specific monoclonal antibodies and aggregated mouse IgG subclasses. Furthermore, we designed oligonucleotide probes specific for the three main isoforms of CD32 and looked for changes in mRNA expression throughout the MLR using an in situ hybridization technique. Increased surface expression of CD32 was found on both activated human T and B lymphocytes, but this was found only in the early stages of the MLR, on days 3 and 4, and was virtually absent by day 7. An inverse relationship between cell surface expression of CD32 and mRNA for the IIb isoforms was noted with strong mRNA expression for IIb isoforms occurring in the later stages of the MLR (days 6-7) when interleukin-2R (IL-2R)-positive T cells were predominant. A soluble IgG binding factor (soluble CD32?) was also detected in the MLR culture supernatant. These observations provide support for the hypothesis that synthesis of IIb isoforms of CD32 occurs following alloantigen activation of human T lymphocytes.

Demonstration of cytoplasmic CD32 (Fc gamma RII) within human lymphocytes following microwave treatment.

We have recently described a cytoplasmic from of CD32 (Fc gamma RII) within the vast majority of normal human peripheral blood lymphocytes (PBL) including T cells. The function of cytoplasmic CD32 is not known. These flow cytometric studies were conducted using single cell suspensions of PBL that had been pre-fixed and permeabilized using methanol/triton-X-100. In this study we have attempted to visualize cytoplasmic CD32 by immunocytochemistry using normal PBL processed in various ways and have also looked for CD32 within tissue lymphocytes. Weak cytoplasmic CD32 staining was observed in paraffin sections of normal lymphocytes but only when sections were microwave treated. The intensity of staining for CD32 did however, appear to be much stronger within infiltrating lymphocytes found in autoimmune diseases or in rejecting allografts: an observation that suggests that up-regulation of cytoplasmic CD32 may occur when T cells become activated in vivo. Microwave treatment of PBL suspensions was shown to disrupt the outer cell membrane, thus effectively permeabilizing the cell and allowing for the detection of cytoplasmic components, like CD32, by flow cytometry. Microwave treatment may, therefore, afford an alternative method for cell permeabilization and may prove to be a useful method for the study of cytoplasmic molecules in cell suspensions and in paraffin-embedded tissues.