Gerald D. Johnson

Germinal centres in T-cell-dependent antibody responses

For more than a century follicles have been recognized as a site of intense cell proliferation and cell death. At last the significance of this activity is beginning to emerge: antigen-driven B-cell proliferation, somatic mutation, positive and negative selection, and memory and plasma cell development all appear to take place within the follicle.

Maturation and dispersal of B-cell clones during T cell-dependent antibody responses.

Germinal centers develop in follicles during TD antibody responses in the first 3 wk following each immunization. In primary responses to protein-based antigens, T-cell help is limiting, follicles develop towards the end of the 1st wk from immunization and the size of the follicular response in relatively small. When T-cell help is provided the primary B-cell response in follicles is much larger, B cells start to proliferate in follicles within a few hours of immunization and reach peak size 3-4 d later. Available evidence suggests that virgin B cells that colonize follicles to form germinal centers must first be activated by antigen outside follicles, probably in T zones. Memory B cells also proliferate in follicles and they can do so without first being activated outside follicles. Germinal center formation consists of an initial phase of exponential proliferation of B cells within the follicular dendritic cell network. After a single immunization the follicular response is oligoclonal and on average only 3 cells colonize each follicle. In responses to hapten-protein in rats primed previously with the carrier protein these 3 cells increase to around 10(4) cells in 3 d with a cell cycle time of about 6 h. At the end of the period of exponential growth of B blasts, the classical structure of germinal centers emerges. The B blasts become centroblasts in the dark zone of the germinal center which develops at that pole of the FDC network nearer the T zones. The centroblasts are still in rapid cell cycle but do not more than sustain their numbers. The rest of their progeny move to the heart of the FDC network where they come out of cell cycle as centrocytes. Evidence is cited which indicates that somatic mutation occurs in the Ig V-region genes of centroblasts and that centrocytes are selected on the basis of their ability to respond to antigen held on FDC. Centrocytes not receiving this antigen-dependent signal kill themselves by apoptosis. Centrocytes positively selected by interaction with antigen on FDC receive further signals which induce the cells to differentiate to become either plasma cells or memory B cells. The nature of some of these differentiation signals is described. It is shown that proliferation, selection and differentiation occur within germinal centers in distinct micro-environments.(ABSTRACT TRUNCATED AT 400 WORDS)

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Immunodetection of 11β-Hydroxysteroid Dehydrogenase Type 2 in Human Mineralocorticoid Target Tissues: Evidence for Nuclear Localization1

11β-Hydroxysteroid dehydrogenase (11βHSD) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11βHSD2 gene, suggest that it is the 11βHSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11βHSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11βHSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the ...

Alkaline phosphatase-fast red, a new fluorescent label: Application in double labelling for cell surface antigen and cell cycle analysis

We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that obtained with other commonly used red fluorochromes or by inspection of the reaction product under bright field illumination. Of particular value, the reaction product is unaffected by the denaturing conditions required for the detection of incorporated nuclear BrdU with FITC conjugated anti-BrdU antibody and provides a simple and robust method for the simultaneous detection of cell proliferation and cell surface markers.

Effect of oxidative stress and disruption of Ca2+ homeostasis on hepatocyte canalicular function in vitro

Isolated rat hepatocyte couplets were used to study the effects of menadione and a rise in the intracellular concentration of calcium on biliary canalicular function. Canalicular function was assessed by counting the percentage of couplets which were able to accumulate the fluorescent cholephile, cholyl lysyl fluorescein (CLF) into the canalicular vacuole between the two cells. Menadione induced a concentration-dependent inhibition of the canalicular vacuole accumulation (CVA) of CLF reaching 7.6 +/- 1.8% of control at 100 microM menadione. This disruption was not prevented by blocking receptor-operated calcium channels with Ni2+ (300 microM). The concentration range of menadione used did not deplete cellular ATP content. In contrast glutathione content was reduced to 52% of its control value by 100 microM menadione. A rise in cytosolic calcium induced by the calcium ionophore, A23187 (up to 30 microM) also disrupted CVA in a concentration-dependent manner. Release of endoplasmic reticulum calcium stores by thapsigargin (50 nM) affected the retention of canalicular contents to a much lesser extent, although it was able to stimulate a reduction in canalicular area to 40% of its original value, assumed to be due to canalicular contraction. Menadione (30 and 100 microM) reduced the fluorescence of phalloidin-FITC-labelled F-actin in both the total and pericanalicular cytoskeleton. Canalicular function was therefore disrupted by non-lethal concentrations of menadione via a mechanism which does not appear to involve ATP depletion or the entry of extracellular calcium, but is associated with a depletion of both cellular glutathione and F-actin. An increase in the concentration of intracellular calcium can stimulate canalicular contraction, and at relatively high concentrations calcium can also disrupt canalicular function.

Doppa induces cell death but not differentiation of U937 cells: Evidence for the involvement of PKC-βl in the regulation of apoptosis

Recent reports have claimed that activation of protein kinase C (PKC)-beta is sufficient for both differentiation and apoptosis in promyeloid HL60 cells. Phorbol esters which differentially activate PKC isoenzymes in vitro were used to induce differentiation and apoptosis in U937 cells; TPA and Dopp activate all U937 PKC isoenzymes, except PKC-zeta and Doppa activate only PKC-beta l. At concentrations of Doppa below 50 nM, only PKC-beta l was activated by 2 min and apoptosis was induced, but there was no differentiation of cells towards monocytes. TPA (1-25 nM) and Dopp (5-100 nM) activated PKC-alpha, -beta l and-gamma within 2 min and induced differentiation, but only increased apoptosis at the highest concentrations used. Thus, initial activation of PKC-beta l is insufficient for differentiation of U937 cells, but may lead to the induction of apoptosis.

T‐Cell Proliferation and Expression of MHC Class II Antigens

Two monoclonal antibodies to major histocompatibility complex (MHC) class II antigens, which in combination identify β chains encoded by the SB and DR loci, were used to investigate which of these gene‐ products were expressed at the cell surface of unstimulated T cells and at various stages of mitogen‐induced T‐cell maturation. In tests on blood lymphocytes from healthy donors 12% of Tcells expressed class II antigens, but only SB antigens were expressed. During activation of T lymphocytes, SB‐coded antigens were expressed before DR antigens, and the kinetics of SB expression correlated with the proliferative response of T cells. These results and consideration of recent reports from other laboratories lead us to suggest that 58‐coded class II antigens play a role in T‐cell proliferation.

Rat oligodendrocyte O-2A precursor cells and the CG-4 oligodendrocyte precursor cell line express cadherins, β-catenin and the neural cell adhesion molecule, NCAM

Oligodendrocyte precursor cell migration throughout the developing central nervous system (CNS) and cessation of migration are poorly understood but are likely to involve cell adhesion molecules. The expression and distribution of neural cell adhesion molecule (NCAM), cadherins and beta-catenin were investigated in the CG-4 cell line and primary rat oligodendrocyte progenitor cells (O-2A) by immunofluorescence and Western blotting. NCAM was expressed by both cell types and was found all over the surface of both CG-4 cells and O-2A progenitor glia. The presence of a cadherin was detected in both CG-4 cells and O-2A progenitor glia, and this molecule was distributed all over the cell body and cell processes at different stages of differentiation. Beta-catenin showed a very similar distribution to that of the cadherin. We conclude that CG-4 cells are a valid model system to study cell adhesion molecule expression and function in oligodendrocyte progenitor cells.

Origin and properties of soluble CD21 (CR2) in human blood

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L‐cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR‐LON‐HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130 kD found by SDS–PAGE analysis. This may be partly accounted for by the non‐globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.