The mouse mammary gland is an excellent model system with which to study both the regulation of development and the functional differentiation of an organ. Most of the development occurs postnatally, when the gland undergoes a highly regulated cascade of invasive growth, branching, differentiation, secretion, apoptosis and remodelling during each pregnancy cycle [1,2]. Terminal differentiation of the alveolar epithelium is completed at the end of gestation with the onset of milk secretion (lactation). APR = acute-phase response; C/EBP = CCAAT/enhancer-binding protein; H&E = haematoxylin and eosin; IHC = immunohistochemistry; IL = interleukin; LIF = leukaemia inhibitory factor; LPS = lipopolysaccharide; OSM = oncostatin M; RT-PCR = reverse transcriptase polymerase chain reaction; SOM = self-organising map; TNF = tumour necrosis factor; WAP = whey acidic protein. Abstract Introduction: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process.

Summary We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic ‘stores’ of three key molecules normally associated with antigen presentation and T‐cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7‐1) and CD86 (B7‐2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross‐linking (X‐L) of Mac‐1: an early neutrophil activation signal. In this study we have compared X‐L of Mac −1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N‐formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G–Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow‐cytometry measurements of size, granularity and phenotype. Significant up‐regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual‐staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.

Summary Recent studies suggest that neutrophils may play a role in antigen presentation. In support of this hypothesis it has been shown that these cells appear to contain cytoplasmic stores of molecules required for this function, i.e. major histocompatibility complex class II (DR) antigen, CD80 and CD86. In this study we have considered a mechanism for the translocation of these preformed molecules onto the cell surface which does not require active synthesis. Cross‐linking of the Mac‐1 molecule (CD18 + CD11b) was shown to result in rapid cell surface expression of CD80, CD86 and DR antigen on the surface of normal human peripheral blood neutrophils. A distinct subpopulation (approximately 20%) of neutrophils appeared to be enlarged and were found to express significantly elevated levels of these molecules on the cell surface following cross‐linking of CD11b when compared with control cells. The level of expression of CD80, CD86 and DR antigen on these large cells was comparable to, and in some cases greater than, the levels found expressed on the surface of monocytes obtained from the same donors. In addition, these cytoplasmic molecules were shown by confocal laser microscopy and by immunoelectron microscopy to be located within secretory vesicles. Following rapid translocation onto the cell surface, CD80 and CD86 appeared to be colocalized within large clusters reminiscent of the supramolecular antigen clusters previously found on conventional antigen‐presenting cells. These findings therefore lend further support for the hypothesis that neutrophils may have a role to play in antigen presentation and/or T‐cell activation.

SUMMARYReceptors for the Fc region of immunoglobulin G ( IgG) (FccRs) exist in three main forms: membrane bound, soluble and cytoplasmic. The function of cytoplasmic FccRs is poorly understood. We have previously demonstrated cytoplasmic FccRII (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.

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