Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter.

Programmed death-1 ligand 2 (PD-L2) expression extends beyond macrophages/dendritic cells to B-1 B cells, a distinct B-cell lineage that is responsible for natural immunoglobulin and which is repertoire skewed toward autoreactive specificities. PD-L2 expression is constitutive in B-1 cells, whereas it is inducible in other cell types, suggesting that PD-L2 is regulated differently in the former versus the latter, and this proved to be the case, both in transcription and promotion. B-1 cells express a PD-L2 transcript that lacks exon 1, in contrast to macrophages/dendritic cells for which exon1 is included, reflecting a unique start site upstream of exon 2. PD-L2 transcription in B-1 cells is regulated by a novel intronic promoter located between exons 1 and 2. This intronic promoter binds Octamer binding protein 1 (Oct1) and Oct2, and although these transcription factors are present in all B cells, Oct2 binding is found in vivo only in B-1 cells and not PD-L2-negative B-2 cells. Moreover, the proximal promoter upstream of exon 1 that is active in macrophages is inactive in B-1 cells. Thus, PD-L2 expression is regulated by two different promoters that function in a lineage-specific manner, with the B-1-specific promoter being constitutively active as a result of Oct1 and Oct2 binding.

Abnormal transcription factor induction through the surface immunoglobulin M receptor of B-1 lymphocytes.

Populations of murine peritoneal B-1 and splenic B-2 cells, highly purified by negative selection techniques, were used to demonstrate that B-1 cells completely fail to enter cell cycle in response to surface immunoglobulin M (sIgM) crosslinking without any decrease in cell number or viability. This failure of B-1 cell responsiveness appears to represent a specific defect in sIgM-derived signaling inasmuch as stimulation to enter S phase occurs normally in response to activated and fixed T cells, and to lipopolysaccharide (LPS). The level at which sIgM signaling fails was determined by evaluating the nuclear expression of the transcription factor complex, NF-kappa B, whose sIgM-mediated induction in B-2 cells is dependent on protein kinase C (PKC) activation but is independent of protein synthesis. There was no induction of nuclear NF-kappa B in B-1 cells stimulated by sIgM crosslinking, although NF-kappa B was stimulated by phorbol myristate acetate and by LPS. In contrast, NF-kappa B was induced in B-2 cells by all three stimuli. Thus, in B-1 cells, the sIgM-mediated induction of a transcription factor that is substantially stimulated by anti-IgM in B-2 cells is blocked. However, all sIgM-derived signaling in B-1 cells was not impaired inasmuch as anti-IgM increased I-A antigen expression. These results strongly suggest that sIgM receptor-mediated signaling in B-1 cells is interrupted early in the signal transduction pathway, at a point proximal to the activation of PKC. These results further demonstrate that transcription factor induction can be used to analyze the level at which receptor-mediated signaling is blocked.

Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with specific antigen unless a surface immunoglobulin threshold is exceeded.

Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)-specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.

T cell-dependent induction of NF-kappa B in B cells.

In comparison to B cell stimulation mediated by surface immunoglobulin (Ig) antigen receptor ligation, little is known about the intracellular events associated with T cell-dependent B cell responses. A model for the efferent phase of T cell-B cell interaction was used to examine the capacity of activated T cells to trigger nuclear expression of the trans-acting transcription factor, NF-kappa B, in B cells. Fixed, activated, but not fixed, resting Th2 cells were found to induce increased binding activity for a kappa B site-containing oligonucleotide in a time-dependent manner. This induction of NF-kappa B was eliminated by an antibody directed against a 39-kD cell interaction protein on activated T cells as well as by a soluble form of B cell CD40. Of particular relevance to intracellular signaling, NF-kappa B induction was not diminished by prior depletion of B cell protein kinase C (PKC) with phorbol myristate acetate. These results strongly suggest that T cell-dependent B cell stimulation is associated with NF-kappa B induction via p39-CD40 interaction and that this is brought about by non-PKC dependent signaling, in marked contrast to the previously documented requirement for PKC in sIg receptor-mediated stimulation. This suggest that NF-kappa B responds to more than one receptor-mediated intracellular signaling pathway in B cells and may be part of a "final common pathway" for B cell stimulation.

Fine specificity of idiotope suppression in the A/J anti-azophenylarsonate response.

Two hapten-inhibitable murine monoclonal antiidiotopic antibodies identified two idiotopes expressed by the heavy chain of hybridoma protein 36-65, whose amino acid sequence is encoded in the germ line of A/J mice. Among cross-reactive idiotype-positive hybridoma proteins and p-azophenylarsonate-immune antibodies, the two idiotopes were not always expressed together; some diversified antibodies expressed one idiotope without the other. Suppression that was induced by the two antiidiotopes was idiotope specific and corresponded to the fine specificities of these two reagents.

A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes.

The sensitivity of primary splenic B cells to Fas-mediated apoptosis is modulated in a receptor-specific fashion. Here we used a differential display strategy to detect cDNAs present in B cells rendered Fas resistant but absent in those rendered Fas sensitive. This led to the cloning and characterization of a novel 1.2-kb gene that encodes a Fas apoptosis inhibitory molecule (FAIM). faim-transfected BAL-17 B lymphoma cells were less sensitive by half or more to Fas-mediated apoptosis than were vector-transfected controls, using Fas ligand-bearing T cells or a cytotoxic anti-Fas antibody to trigger Fas, and this was associated with inhibition of Fas- induced poly-ADP ribose polymerase (PARP) cleavage. In primary B cells, the time course of faim mRNA and FAIM protein expression correlated with the induction of Fas resistance by surface (s)Ig engagement. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced by sIg engagement in B cells. However, faim is broadly expressed in various tissues and the faim sequence is highly conserved evolutionarily, suggesting that its role extends beyond lymphocyte homeostasis. As FAIM has no significant regions of homology to other gene products that modulate Fas killing, it appears to represent a distinct, new class of antiapoptotic protein.

Induction of idiotope suppression in the anti-azophenylarsonate response of T-depleted A/J mice.

The homologous, monoclonal antiidiotope, MB, induced idiotope suppression that was remarkably stable and could be transferred by B lymphocytes. Marked depletion of T cell function, confirmed by limiting diluting analysis, did not affect the ability of MB to suppress the corresponding idiotope. Suppression induced by MB appears to result from direct interaction with idiotope-positive B cells, without the intervention of idiotope-specific T suppressor cells.

Early induction of cyclin D2 expression in phorbol ester-responsive B-1 lymphocytes.

B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2-4 h, in phorbol ester-stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester- stimulated B-2 cells. Phorbol ester-stimulated cyclin D2 expression was accompanied by the formation of cyclin D2-Cdk4, and, to a lesser extent, cyclin D2-Cdk6, complexes; cyclin D2- containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset.

Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.

Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

Receptor crosstalk: reprogramming B cell receptor signalling to an alternate pathway results in expression and secretion of the autoimmunity-associated cytokine, osteopontin.

Receptor crosstalk: reprogramming B cell receptor signalling to an alternate pathway results in expression and secretion of the autoimmunity-associated cytokine, osteopontin (Review). J Intern Med 2009; 265: 632-643.Intracellular signalling emanating from the B-cell antigen receptor is considered to follow a discrete course that requires participation by a set of mediators, grouped together as the signalosome, in order for downstream events to occur. Recent work indicates that this paradigm is true only for naïve B cells. Following engagement of the IL-4 receptor, a new, alternate pathway for B-cell receptor (BCR)-triggered intracellular signalling is established that bypasses the need for signalosome elements and operates in parallel with the classical, signalosome-dependent pathway. Reliance on Lyn and sensitivity to rottlerin by the former, but not the latter, distinguishes these two pathways. The advent of alternate pathway signalling leads to production and secretion by B cells of osteopontin (Opn). As Opn is a polyclonal B-cell activator that is strongly associated with a number of autoimmune diseases including lupus and rheumatoid arthritis, this novel finding is likely to be clinically relevant. Our results highlight the potential role of B-cell-derived Opn in immunity and autoimmunity and suggest that stress-related IL-4 expression might act to strengthen immunoglobulin secretion at the risk of autoantibody formation. Further, these results illustrate receptor crosstalk in the form of reprogramming, whereby engagement of one receptor (IL-4R) produces an effect that persists after the original ligand (IL-4) is removed and results in alteration of the pathway, and outcome, of signalling via a second receptor (BCR) following its activation.

Lysosomal physiology in Tetrahymena. 3. Pharmacological studies on acid hydrolase release and the ingestion and egestion of dimethylbenzanthracene particles.

The ingestion of (14)C-labeled 9,10-dimethyl-1,2-benzanthracene particles, the extracellular release of acid phosphatase, ribonuclease, and alpha-glucosidase, and the egestion of preingested dimethylbenzanthracene particles by Tetrahymena taken from logarithmically growing cultures and resuspended in a dilute salt solution were followed in the presence of several pharmacologic agents. Serotonin, caffeine, and, to a lesser extent, dibutyryl cyclic AMP increased the rate of particle ingestion, but did not alter the rate of release of the three acid hydrolases studied. Added catecholamines did not affect either particle ingestion or acid hydrolase release, but particle ingestion was inhibited by the catecholamine antagonists, dichloroisoproterenol, desmethylimipramine, reserpine, and phenoxybenzamine. These drugs also increased the release of acid phosphatase and ribonuclease in 5-h incubations. Desmethylimipramine acted within 1 h to increase acid hydrolase release, but the effect of dichloroisoproterenol developed more slowly and was secondary to a change in cellular content of the hydrolases. Desmethylimipramine increased the energy of activation for the release of acid phosphatase, while dichloroisoproterenol did not. Both of these drugs enhanced the egestion of preingested dimethylbenzanthracene particles, supporting the view that acid hydrolase release occurs through a cytoproct egestion mechanism. Particle ingestion was also inhibited by colchicine, vinblastine, and cytochalasin B, but these agents had no effect on acid hydrolase release, thus further differentiating the properties of the ingestion mechanism from those of the egestion mechanism. It appears that both microtubules and microfilaments play a role in the ingestion process and that this process may be controlled in part by a cyclic AMP-mediated serotoninergic and adrenergic system.

Lysosomal physiology in Tetrahymena. I. Effect of glucose, acetate, pyruvate, and carmine on intracellular content and extracellular release of three acid hydrolases.

Log-phase Tetrahymena were washed and resuspended in a dilute salt solution supplemented with glucose, acetate, pyruvate, or carmine, as desired, and then incubated for 5 h. Intra- and extracellular activities of acid phosphatase, alpha-glucosidase, and ribonuclease were assayed. Extracellular activities were corrected for proteolytic degradation. The three nutritive substrates affected both the amount and pattern of extracellular enzyme release, but carmine had no effect. Intracellular activities declined early in the starvation period, but partially recovered with time, particularly alpha-glucosidase activity. Acetate reduced the decline in acid phosphatase activity; acetate and glucose enhanced the recovery of alpha-glucosidase activity; carmine had no effect on intracellular enzyme activities. Protein content changed little and was unaffected by the addition of substrates. Glycogen content increased during incubation; acetate and glucose enhanced the increase.

Signals and susceptibility to programmed death in b cells.

Programmed death in B cells is a highly regulated process. During the past year it has become increasingly apparent that specific receptor signals influence B cell apoptosis in distinct ways as a function of developmental stage and/or apoptotic trigger. Studies making use of opposing signals for programmed death have begun to reveal molecular correlates of sensitivity and resistance to apoptosis.

Two-step stimulation of B lymphocytes to enter DNA synthesis: synergy between anti-immunoglobulin antibody and cytochalasin on expression of c-myc and a G1-specific gene.

Previously we demonstrated that stimulation of resting murine splenic B lymphocytes with goat anti-mouse immunoglobulin antibody (GaMIg) plus cytochalasin D (CD) led to DNA synthesis; GaMIg and CD added simultaneously, or GaMIg added before CD, induced this response (T. L. Rothstein, J. Immunol. 136:813-816, 1986). Cells similarly treated with GaMIg or CD alone did not enter S phase. Here we have measured the effects of this two-signal stimulation on the c-myc, 2F1, and gamma-actin genes. The expression of these growth-related genes is known to change either during the G0-to-G1 transition or in the G1 phase of the cell cycle. For the 2F1 and c-myc genes, neither the GaMIg nor CD stimulus alone led to a prolonged increase in mRNA levels, whereas GaMIg plus CD allowed for continuous elevated expression of these genes. Furthermore, GaMIg pretreatment rendered expression of the c-myc and 2F1 genes susceptible to subsequent action by CD. In contrast, CD alone was sufficient to produce changes in gamma-actin gene expression. Thus there are synergistic effects of competence- and progressionlike factors on the expression of the c-myc and 2F1 genes, and these effects correlate with the progression of B lymphocytes to DNA synthesis.

Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease.

Affinity analysis of idiotype-positive and idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera.

The possibility that idiotype dominance may be associated with increased affinity for hapten was investigated in the murine A/J anti-p-azophenylarsonate (Ars) response. Fluorescence quenching of 14 Ars-binding hybridoma proteins by Ars-tyrosine was measured and Ka calculated using computer-assisted curve fitting. There was a 200-fold range in Ka for idiotype-positive hybridoma proteins, with 2 IgM hybridoma proteins being near the median. No clear difference in Ka was apparent between idiotype-positive (Id+) and idiotype-negative (Id-) hybridoma proteins. Ka was measured by fluorescence quenching on affinity-purified anti-Ars antibodies from 6 conventional antisera; there was no difference between Id+ and Id- (idiotype suppressed) sera. The affinities of the hybridoma proteins were correlated with the ratio of binding to Ars36-BSA and Ars10-BSA by direct radioimmunoassay. With this calibration, functional affinities of Ars-immune sera could be determined from relative binding ratios without the need for prior affinity purification. This was done for 18 Ars-immune sera, and again there was no clear difference between Id+ and Id- sera. Studies from this laboratory have identified the amino acid sequence of a hybridoma protein which corresponds to the germ line DNA sequence for the cross-reactive idiotype family. The present study shows that the protein directly encoded by the germ line gene has low affinity for hapten suggesting that somatic diversification operating on the germ line sequence can produce antibodies with increased affinity for hapten within the cross-reactive idiotype family. The present study also suggests that affinity is not the driving force behind idiotype dominance of the Ars-immune response.

Abnormal kappa B-binding protein in the cytoplasm of a plasmacytoma cell line that lacks nuclear expression of NF-kappa B.

The transcription factor NF-kappa B appears to play an important role in immunoglobulin gene expression and lymphokine production, and may play a role in primary B cell activation. Constitutive nuclear expression of NF-kappa B has been found in all mature B cell lines with the notable exception of the murine plasmacytoma, S107. We report herein that S107 cells express cytoplasmic kappa B-binding material detected by electrophoretic mobility shift assay that by several criteria represents authentic NF-kappa B. Despite the presence of cytoplasmic NF-kappa B, several stimuli known to induce nuclear translocation of NF-kappa B failed to do so in S107 cells, including: the PKC agonist, PMA; the protein synthesis inhibitor, cycloheximide; and LPS. Transfection of S107 cells with a kappa B-CAT reporter gene construct confirmed the absence of functional activity. Importantly, a global failure of nuclear transcription factor expression was ruled out by the ability of PMA to induce nuclear expression of another trans-acting factor, AP-1. Thus, rather than lacking NF-kappa B altogether, S107 cells manifest disordered regulation of NF-kappa B in which cytoplasmic material is incapable of translocation to the nucleus. While Northern analysis failed to reveal a gross defect in the mRNA coding for the DNA binding subunit of NF-kappa B, UV-photo-cross-linking followed by denaturing gel electrophoresis demonstrated the presence of a cytoplasmic kappa B-binding protein of abnormally elevated molecular size. This finding suggests that the abnormal regulation of NF-kappa B in S107 cells is associated with the appearance of an unusual kappa B-binding molecule.

An alternatively spliced long form of Fas apoptosis inhibitory molecule (FAIM) with tissue-specific expression in the brain.

The gene encoding Fas apoptosis inhibitory molecule (FAIM) was cloned by differential display using RNA obtained from Fas-resistant and Fas-sensitive primary murine B lymphocytes. FAIM is highly evolutionarily conserved and broadly expressed, suggesting that its gene product plays a key role in cellular physiology. Here we report the identification of a new, longer form of FAIM (FAIM-L) and characterization of the genomic locus that clarifies its origin. The murine FAIM gene is located at chromosome 9f1, a region syntenic to the corresponding location of the human FAIM gene. The gene consists of six exons and contains putative translation initiation sites within exons II and III. The long form of FAIM is generated by all six exons, whereas the originally cloned form of FAIM, now termed FAIM-Short (FAIM-S) is generated from five exons by alternative splicing. FAIM-L is dominantly expressed in the brain whereas FAIM-S is widely expressed in many tissues.

C-terminal sequence of the NF-kappa B p50 precursor from primary murine B-lymphocytes.

We report a DNA sequence encoding the nuclear factor NF-kappa B p50 precursor from primary murine B-lymphocytes that differs from that previously published for the murine 22D6 B-cell line. The variation is located in one of the ankyrin-like repeats at the C terminus of the molecule.

Cranial neuropathy, myeloradiculopathy, and myositis: complications of Mycoplasma pneumoniae infection.

Polymyositis, transverse myelitis, ascending polyneuritis, bilateral optic neuritis, and hearing loss developed in a patient with high complement-fixing antibody titers to Mycoplasma pneumoniae. Each of her three children had primary atypical pneumonia with isolation of the organism. The neurologic disturbance is thought to represent a postinfectious complication of M pneumoniae infection.