Sixteen years have passed since the description of the nuclear factor-кB (NF-кB) as a regulator of к light-chain gene expression in murine B lymphocytes (Sen & Baltimore, 1986a) During that time, over 4,000 publications have appeared, characterizing the family of Rel/NF-кB transcription factors involved in the control of a large number of normal and pathological cellular processes The physiological functions of NF-кB proteins include immunological and inflammatory responses, developmental processes, cellular growth and modulating effects on apoptosis In addition, these factors are activated in a number of diseases, including cancer, arthritis, acute and chronic inflammatory states, asthma, as well as neurodegenerative and heart diseases

Activators and target genes of Rel/NF-kappaB transcription factors.

The classical type of programmed cell death is characterized by its dependence on de novo RNA and protein synthesis and morphological features of apoptosis. We confirmed that stimulated 2B4.11 (a murine T-cell hybridoma) and interleukin-3 (IL-3)-deprived LyD9 (a murine haematopoietic progenitor cell line) died by the classical type of programmed cell death. Assuming that common biochemical pathways might be involved in the deaths of 2B4.11 and LyD9, we isolated the PD-1 gene, a novel member of the immunoglobulin gene superfamily, by using subtractive hybridization technique. The predicted PD-1 protein has a variant form of the consensus sequence found in cytoplasmic tails of signal transducing polypeptides associated with immune recognition receptors. The PD-1 gene was activated in both stimulated 2B4.11 and IL-3-deprived LyD9 cells, but not in other death-induced cell lines that did not show the characteristic features of the classical programmed cell death. Expression of the PD-1 mRNA in mouse was restricted to the thymus and increased when thymocyte death was augmented by in vivo injection of anti-CD3 antibody. These results suggest that activation of the PD-1 gene may be involved in the classical type of programmed cell death.

https://www.embopress.org/doi/epdf/10.1002/j.1460-2075.1992.tb05481.x

Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.

I. Introduction DEATH, along with growth and differentiation, is a critical part of the life cycle of a cell. Homeostatic control of cell number is thought to be the result of the dynamic balance between cell proliferation and cell death. It is only in the past few years, however, that attention has been focused on the physiological occurrence of cell death and its role in homeostasis. Researchers have become increasingly more aware during this time that this type of “natural” death, which is now called apoptosis or programmed cell death, is a widespread phenomenon that plays a crucial role in a myriad of physiological and pathological processes. This review first briefly covers some historical perspective on the discovery of apoptotic cell death, the characteristic morphology that accompanies this process, and the numerous cell types and mediators with which programmed cell death is associated. The bulk of the review discusses our current understanding of the biochemical and molecular mechanisms by which...

Apoptosis: the biochemistry and molecular biology of programmed cell death.

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay Camptothecin, teniposide, and 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle The method of detection of DNA strand breaks (3′-hydroxyl termini) individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker The protein-associated DNA breaks in the “cleavable” DNA-topoisoerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods

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Detection of DNA Strand Breaks in Individual Apoptotic Cells by the in Situ Terminal Deoxynucleotidyl Transferase and Nick Translation Assays

Defective B cell development and function in Btk-deficient mice

WEHI-231 and CH31 are phenotypically immature sIgM+ murine B cell lymphomas whose growth is inhibited by anti-immunoglobulin (Ig) antibodies. These lines have therefore been used as models for studying the role of surface Ig receptors in the induction of B cell tolerance. We show here that anti-mu antibodies induce DNA cleavage into oligonucleosomal fragments characteristic of programmed cell death (apoptosis) in both cell lines, although WEHI-231 cells are less susceptible than CH31. This effect was reversed by lipopolysaccharide, in agreement with the known effects of lipopolysaccharide on anti-Ig-induced growth inhibition. These results therefore indicate that these lymphomas afford a potentially interesting model to study the mechanisms of programmed cell death induced by ligation of the antigen receptors on normal B cells.

Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas.

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45-50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5-10% of both CD4+ and CD8+ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40+, whereas CD40+ cells were not detectable in the thymus, even following collagenase digestion. Some 35-40% of lymphoid cells in the bone marrow of adult mice are CD40+ and virtually all of these are B220+, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-mu and anti-delta mAb, and moderately enhanced by co-stimulation with interleukin-4. Hypercross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.

Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.

It is widely accepted that extensive cross-linking of surface immunoglobulin (sIg) receptors on mature B cells promotes their activation and progression through the cell cycle. A commonly employed method to maximize receptor cross-linking via anti-receptor antibodies is to immobilize them on tissue culture plastic. We show here that immobilizing monoclonal anti-mu or anti-delta antibodies, which are mitogenic in solution, on plastic abrogates their capacity to induce DNA synthesis in mature murine B cells, even in the presence of interleukin-4 (IL-4). The cells do become abortively activated, as evidenced by up-regulation of major histocompatibility complex class II antigen levels, but subsequently virtually all of them die, manifesting DNA fragmentation characteristic of apoptosis. The induction of apoptosis is abrogated by the inclusion of either IL-4 or anti-CD40 antibodies in the cultures, with the two stimuli acting in concert. We believe that the system represents a polyclonal model of clonal deletion tolerance in mature B cells, such as may be induced under physiological conditions by antigens with repeating epitopes.

Plastic‐immobilized anti‐μ or anti‐δ antibodies induce apoptosis in mature murine B lymphocytes

The CBA/N mouse carries the X-linked immunodeficiency xid, resulting in defective B cell development. B cells from these animals cannot mount antibody responses to type 2 T-independent antigens, and do not synthesize DNA when stimulated with anti-immunoglobulin (Ig) antibodies which are mitogenic for normal B cells. The primary antibody responses of CBA/N mice to T-dependent antigens have also been reported to be abnormal. Here we describe the results of experiments which demonstrate that the B cells from these animals respond abnormally to ligation of CD40, a B cell surface molecule now known to play a key role during T cell-B cell interactions, via its interaction with the counterligand (CD40L) expressed by activated T cells. Hence, xid B cells fail to proliferate when cultured with preactivated T helper type 2 (Th2)T cells (known to express CD40L), with a soluble CD40L-CD8 fusion protein, or in response to monoclonal antibodies to CD40, even in the presence of IL-4 and/or anti-Ig reagents. However, xid B cells do receive abortive activation signals following ligation of CD40, as manifested by up-regulation of class II major histocompatibility complex and CD23 antigens. Since the xid defect has now been identified as a point mutation in the protein tyrosine kinase Btk, our results point to an important role for this kinase in the downstream signaling cascade elicited in response to ligation of either surface Ig or CD40.

B cells from CBA/N mice do not proliferate following ligation of CD40

Stimulation of human B cells via CD40 is known to induce their homotypic aggregation. We show here that anti-mouse CD40 monoclonal antibodies (mAb) also induce B cells to form large, spherical, extremely stable clusters. This clustering is markedly enhanced by co-stimulation with either interleukin-4 (IL-4) or anti-immunoglobulin (Ig). The aggregation is slow in onset, and is largely (but not completely) abrogated by anti-LFA-1 mAb, but not by mAb directed against other potentially important adhesion molecules on B cells. Anti-LFA-1 mAb also partially suppressed DNA synthesis induced by anti-CD40, but not by other B cell mitogens, suggesting that clustering is an important component of B cell activation via CD40. This concept is supported by analyses of the phenotype of clustered B cells: the cells within clusters express higher levels of various activation markers, and also more of them are in cell cycle than non-clustered cells. These results therefore suggest that CD40 stimulation may either induce B cells to secrete soluble factors which act in an autocrine way to promote B cell activation, or that clustering generates cell contact-mediated signals which are important in the activation cascade.

Jhagvaral Hasbold

Papers

Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas.

Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.

Plastic‐immobilized anti‐μ or anti‐δ antibodies induce apoptosis in mature murine B lymphocytes

B cells from CBA/N mice do not proliferate following ligation of CD40

Properties of mouse CD40: the role of homotypic adhesion in the activation of B cells via CD40.