Stimulation of tyrosine phosphorylation without inositol lipid hydrolysis in human B lymphocytes on engaging CD72.

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co-stimulus to immobilized anti-mu for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5-10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross-linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.

Effects of red light-emitting diode irradiation on dental pulp cells.

Light irradiation activates a range of cellular processes in a variety of cell types, including stem cells, and can promote tissue repair. This study investigated the effects of light-emitting diode (LED) exposure on dental pulp cells (DPCs). Dose response analysis at 20-second intervals up to 120 seconds demonstrated that a LED array emitting 653-nm red light stimulated significantly increased cell growth at 3 and 7 days post-irradiation with 40 (149 mJ/cm(2)) and 60 (224 mJ/cm(2)) seconds of radiant exposure. Double-dosing cells at days 1 and 4 of a 7-day culture period with 60-second (224 mJ/cm(2)) LED exposure significantly increased cell growth compared with a single dosing regime. BrdU analysis demonstrated significantly increased proliferation rates associated with significantly increased ATP, nitric oxide (NO), and mitochondrial metabolic activity. LED-stimulated NO levels were not reduced by inhibition of NO-synthase activity. Light exposure also rescued the inhibition of mitochondrial dysfunction and increased levels of in vitro mineralization compared with control. Media exchange experiments indicated that autocrine signaling was not likely responsible for red-light-induced DPC activity. In conclusion, data analysis indicated that 653-nm LED irradiation promoted DPC responses relevant to tissue repair, and this is likely mediated by increased mitochondrial activity.

Species identification and authentification of human and rodent cell cultures using polymerase chain reaction analysis of vomeronasal receptor genes.

Cell culture and the use of cell lines are routinely used in basic scientific research. It is therefore imperative for researchers to ensure the origin of the cell lines used and that they are routinely re-analysed for contamination and misidentification. Inter-species contamination is relatively frequent, and the most commonly used cell lines are of human, mouse and rat derivation. We have developed simple species specific primer assays based on genomic sequence differences in vomeronasal receptor gene family members to discriminate between human, mouse and rat DNA using standard agarose gel electrophoresis. Furthermore, these PCR assays are able to identify the species composition within an inter-species mixed population. This approach therefore provides a valuable tool to enable a rapid, simple and relatively inexpensive determination of the authentication and contamination of cell cultures.

Factors modifying survival pathways of germinal center B cells. Glucocorticoids and transforming growth factor-beta, but not cyclosporin A or anti-CD19, block surface immunoglobulin-mediated rescue from apoptosis.

The tendency for germinal center (GC) B cells to enter apoptosis is suppressed on engaging antigen receptor with immobilized anti-immunoglobulin; cross-linking of surface CD40 by monoclonal antibodies provides an additional signal for rescuing GC cells from programmed death. These observations are believed to reflect events that, in vivo, would allow for the selection of centrocytes which have undergone somatic mutation on Ig V-region genes to generate antigen receptor of high affinity. The purpose of the present study was to identify factors capable of modifying the survival pathways of GC cells. Transforming growth factor-beta, at an optimal concentration of 1 ng/ml, was found to inhibit surface immunoglobulin (sIg)-mediated rescue of GC cells but had no influence on survival promoted through CD40. Both routes of rescue were blocked by the glucocorticoid prednisolone at pharmacological concentrations (ID50 = 10(-7) M). Cyclosporin A, an antagonist of sIg-mediated signaling in resting B cells, failed to block rescue of GC cells through either of the receptor-activated pathways. Antibody to CD19--which also suppresses the activation of resting B cells--not only left GC cell rescue undiminished, but rather provided a modest survival signal of its own; interferon-alpha behaved similarly while interferon-gamma failed to influence GC cell survival in either direction.

A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis.

Germinal center cells (GCC) are programmed to die by apoptosis unless they receive a positive signal for rescue. The primary signal in vivo is believed to be dependent on interaction with antigen held as immune complexes on follicular dendritic cells (FDC), a subset of which express large amounts of CD23, a low-affinity receptor for IgE. Recombinant soluble CD23 (sCD23) and interleukin-1 have been found to potentiate the survival of GCC in vitro. Recently, CD23 was shown to interact specifically with a ligand other than IgE, namely CD21 (CR2/Epstein-Barr virus receptor). In the present study, we show that a subset of anti-CD21 monoclonal antibodies behave similarly to soluble CD23 in their effect on GCC inasmuch as they: (i) diminish the occurrence of apoptosis; (ii) promote a plasmacytoid appearance in rescued cells; (iii) up-regulate expression of the Bcl-2 proto-oncogene. These findings indicate that FDC-derived CD23 exerts its effects on GCC via CD21.

Growth factor requirements for the stimulation of germinal center B cells: evidence for an IL-2-dependent pathway of development.

Germinal center (GC) B cells readily undergo apoptosis, a tendency which can be suppressed in vitro by immobilized anti-Ig; mAb to CD40 and soluble CD23 (in synergy with IL-1 alpha) also effect rescue of GC cells from programmed cell death. In the present study, the signals which stimulate rescued GC populations to DNA synthesis have been examined and compared to those established for the activation of follicular mantle (FM) B cells. On co-culture with anti-Ig, optimal responses in FM B cells can be achieved with a combination of IL-4 and CD40 antibody; these activities also provided a modest stimulus to GC cells but, for this population, anti-Ig was ineffective at augmenting the response further. Stimulations of GC B cells were enhanced, however, when performed on a support of primary fetal lung fibroblasts; a major influence of stroma was to promote, by direct cell-cell contact, the CD40-dependent survival of GC B cells. FM B cells were relatively independent of such stromal support. In marked contrast to FM cells, GC B cells were found to respond by enhanced DNA synthesis to IL-2 even when quite low concentrations of the factor were present (IC50 = 2 U/ml). Stimulation of GC cells via this pathway was augmented almost 2-fold on the inclusion of anti-Ig whereas neither fibroblasts, IL-4, nor CD40 antibody made any additional contribution to the IL-2-dependent response. The requirements found for stimulating GC cells in vitro are discussed with reference to the signals that this population may encounter in appropriate microenvironments in vivo: the variety of options apparently available could reflect changing priorities at different stages of a developing GC response.

Recombinant 25-kDa CD23 and interleukin 1 alpha promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis.

Germinal centers contain a proliferating pool of centroblasts which give rise to non-dividing centrocyte. Centrocytes are programmed to die by apoptosis unless they receive a positive signal for rescue. Rescue, in vivo, is likely to be dependent, initially, on interaction with antigen held on follicular dendritic cells (FDC). A subset of FDC located in that part of the germinal center furthest from centroblasts is particularly rich in CD23. Supernatants containing high levels of soluble CD23 were found not only to encourage the survival of germinal center B cells but also to promote their differentiation toward a plasmacytoid morphology; these activities were diminished following removal of CD23 from the supernatants. Recombinant 25-kDa CD23 was initially found to be incapable of providing the signal for germinal center cell development but on the addition of interleukin 1 alpha which, by itself, was inactive, rescue and differentiation of germinal center B cells were now achieved. Apoptosis in germinal center cells could also be prevented by the ligation of surface CD40 with monoclonal antibody: however, rescue via this pathway was not accompanied by plasmacytoid differentiation. These findings provide a functional rationale to the high level expression of CD23 found within a discrete subset of FDC and indicate a bifurcation in the development of germinal center B cells following their rescue from apoptosis.

The extrafollicular-to-follicular transition of human B lymphocytes: induction of functional globotriaosylceramide (CD77) on high threshold occupancy of CD40.

Amongst lymphocytes, expression of CD77 (globotriaosylceramide, Gb3) is exclusive to B cells of the germinal center (GC). Its acquisition by extrafollicular B cells may thus herald their commitment to a follicular response. Here we show that high threshold occupancy of CD40 by its cognate ligand (CD40L) promotes rapid induction of CD77 expression in non-GC (CD38(lo)) B cells. The kinetics of CD77 acquisition mirrored those of GC-related markers CD95 and CD86 but contrasted with the more delayed increase in CD38 expression. Induction of CD77 was not a simple consequence of cell cycle entry: other conditions of stimulation equally capable of driving proliferation failed to promote CD77 expression. CD77 was functional in that cells were now sensitive to Verotoxin-1, an Escherichia coli-derived ligand of Gb3. These data indicate that acquisition by extrafollicular B cells of CD77 results from high threshold occupancy of CD40, a situation that should be reached physiologically only once a critical level of T cell priming has been achieved.

IL-2 expands and maintains IgM plasmablasts from a CD5+ subset contained within the germinal centre cell-enriched (surface IgD-/CD39- buoyant) fraction of human tonsil.

IL-2 was found to promote the rapid growth of a minority population contained within the germinal centre (GC) cell-enriched (CD39- and/or IgD- buoyant) fraction of human tonsillar B lymphocytes. The cells emerging in response to IL-2 had a high mitotic index and morphologically resembled plasmablasts. Cultures could be maintained in the absence of feeder cells for up to 3 weeks in IL-2 and were characterized by large amounts of IgM in their supernatants: approximately 40% of the cells contained readily detectable cytoplasmic IgM by day 10 of culture. Negligible quantities of IgG and IgA were found. The target population for IL-2-driven expansion and IgM secretion was smIg+/CD38+ and was subject to suppression by anti-IgM antibody. While only 8% of cells within the GC cell-enriched fraction were CD5+ (compared with 15% of high density resting B cells), their removal led to an 83% reduction in the amount of IgM produced in response to IL-2, IL2 selectively expanded this minor CD5+ subset such that by day 6 of culture they comprised 57% of all viable cells. Cultures established with IL-2 showed increasing expression of cytoplasmic Bcl-2 and withdrawal of growth factor resulted in cell death via apoptosis. We discuss these results in relation to CD5+ B cells and their potential role in antibody responses to TD antigens.

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bc1-2 induction.

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37 degrees C, > 80% of cells remained viable in the presence of CD40L as compared to < 1% in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bc1-2 protein, the expression of which has been linked to hemopoietic cell survival. Bc1-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bc1-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.

Minimal cross-linking and epitope requirements for CD40-dependent suppression of apoptosis contrast with those for promotion of the cell cycle and homotypic adhesions in human B cells.

Eight different CD40 mAb shared with soluble trimeric CD40 ligand (sCD40LT) the capacity to rescue germinal center (GC) B cells from spontaneous apoptosis and to suppress antigen receptor-driven apoptosis in group I Burkitt's lymphoma cells. Three mAb (G28-5, M2 and M3) mimicked sCD40LT in its ability to promote strong homotypic adhesion in resting B cells, whereas others (EA5, BL-OGY/C4 and 5C3) failed to stimulate strong clustering. Binding studies revealed that only those mAb that promoted strong B cell clustering bound at, or near to, the CD40L binding site. While all eight mAb and sCD40LT were capable of synergizing with IL-4 or phorbol ester for promoting DNA synthesis in resting B cells, co-stimulus-independent activation of the cells into cycle through CD40 related directly to the extent of receptor cross-linking. Thus, mAb which bound outside the CD40L binding site synergized with sCD40LT for promoting DNA synthesis; maximal levels of stimulation were achieved by presenting any of the mAb on CD32 transfectants in the absence of sCD40LT or by cross-linking bound sCD40LT with a second antibody. Monomeric sCD40L, which was able to promote rescue of GC B cells from apoptosis, was unable to drive resting B cells into cycle. These studies demonstrate that CD40-dependent rescue of human B cells from apoptosis requires minimal cross-linking and is essentially epitope independent, whereas the requirements for promoting cell cycle progression and homotypic adhesion are more stringent. Possible mechanisms underlying these differences and their physiological significance are discussed.