Laser-generated waves and wakes in rotating ion crystals.

Locally excited plasma waves are generated in a Coulomb crystal by "pushing" with radiation pressure on a rotating cloud of laser-cooled 9Be+ ions. The waves form a stationary wake that is directly imaged through the dependence of the ion fluorescence on Doppler shifts, and theoretical calculations in a slab geometry are shown to accurately reproduce these images. The technique demonstrates a new method of exciting and studying waves in cold ion clouds.

Immunobiology of the immature B cell: plasticity in the B-cell antigen receptor-induced response fine tunes negative selection.

The immature and transitional immature B-cell stages define an important window in B-cell development, as it is at this point that cells committed to the B-cell lineage first express the clonotypic B-cell antigen receptor (BCR) and cells expressing self-reactive specificities may be identified and eliminated. The intrinsic susceptibility of the immature B cell to negative selection following BCR engagement distinguishes these cells functionally from mature-stage B cells in which BCR cross-linking leads to activation. Our laboratory has been interested in determining the molecular events responsible for the distinct and disparate responses of immature and mature B cells to antigen receptor signaling in order to understand the molecular basis of negative selection of developing B cells. These studies have indicated that developmentally regulated mechanisms, intrinsic to the B cell, regulate the differential responsiveness of the immature and mature stage B cell to antigen. However, the "hard-wired" BCR-induced apoptotic response of the immature B cell can be modified by the microenvironmental context in which the antigen is encountered. This plasticity fine tunes the BCR-induced response of the immature B cell by regulating the mechanism of negative selection and, under defined circumstances, allowing for recruitment into an immune response.

A dominant-negative mutant of c-Jun inhibits cell cycle progression during the transition of CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes.

While Jun/Fos-containing transcription factors are known to be necessary for many TCR-mediated events in mature T cells, relatively little is known about their roles in thymocyte development. We have generated transgenic mice that express a trans-dominant-negative mutant of c-Jun (TAM-67) specifically in thymocytes. Expression of TAM-67 inhibited the up-regulation of AP-1-responsive genes such as c-jun and IL-2 in stimulated thymocytes from transgenic mice. In addition, altered thymocyte development in TAM-67-expressing mice was revealed by a decrease in thymic cellularity ( approximately 50%) which could be accounted for primarily by a reduction in the number of CD4(+)CD8(+) thymocytes, a large percentage of which retained CD25. The decrease in the number of CD4(+)CD8(+) thymocytes did not appear to be due to an enhanced rate of apoptosis but rather to a decrease in the number of CD4(-)CD8(-)CD25(-) cells in the S + G(2)/M stages of the cell cycle. These results indicate that Jun/Fos-containing transcription factors promote the proliferative burst that accompanies the transition from the CD4(-)CD8(-) to the CD4(+)CD8(+) stage of thymocyte development.

Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells.

Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells.

Effects of carbaryl, permethrin, 4-nonylphenol, and copper on muscarinic cholinergic receptors in brain of surrogate and listed fish species.

We investigated the regulation of the muscarinic cholinergic receptor (MChR) in brain from seven species of fish, two surrogates and five threatened or endangered species exposed to a series of chemicals as a measure of compensatory response among species. Fish were classified as either cold water (rainbow trout-surrogate, apache trout, lahanton trout) or warm water (fathead minnow-surrogate, razorback sucker, bonytail chub, colorado squawfish) and were exposed to chemicals shown to affect cholinergic pathways (carbaryl and permethrin) and two chemicals whose relationships to the cholinergic system is less clear (4-nonylphenol and copper). Downregulation of MChR occurred in all warm water species, except colorado squawfish, and at carbaryl concentrations similar to those causing downregulation observed in rainbow trout. Permethrin exposure resulted in downregulation in fathead minnow and razorback sucker, but the concentrations required for observation of this phenomenon were much greater than observed in cold water species. Copper exposure caused a decrease in brain MChR in rainbow trout and apache trout, whereas 4-nonylphenol exposure resulted in a decrease in brain MChR in all three cold water species. Our results indicate that surrogates are useful in assessing sublethal physiological responses to chemicals with a known mechanism of action such as carbaryl and support use of surrogates for assessing physiological responses to chemicals with diverse, less clear mechanisms of action.

A positive role for thymus-derived steroids in formation of the T-cell repertoire.

T cells undergo rigorous selection processes in the thymus that are necessary to prevent T cells with either autoreactive or nonfunctional T-cell receptors (TCRs) from entering the periphery. Although both positive and negative selection depend on TCR-mediated signals, the means by which a thymocyte interprets these signals to result in survival or death is not understood. Glucocorticoids are known to induce thymocyte apoptosis at high concentrations, but at lower concentrations glucocorticoids can antagonize TCR-mediated deletional signals and allow survival of thymocytes and T cell hybridomas. Interestingly, transgenic mice in which the expression of the glucocorticoid receptor has been downmodulated specifically in thymocytes have abnormal thymocyte differentiation, indicating that glucocorticoids play a significant role in T-cell development. Furthermore, we have demonstrated the presence of steroidogenic enzymes in the thymic epithelium and can show that, in vitro, these cells readily synthesize pregnenolone, the first product in the steroidogenic pathway, and deoxycorticosterone. Inhibition of local glucocorticoid biosynthesis in thymi from TCR transgenic mice during fetal thymic organ culture (FTOC) revealed significant alterations in the process of thymocyte selection. These data suggest that glucocorticoids do not simply suppress the immune system but rather are necessary for thymocyte survival and differentiation.

Thymocyte glucocorticoid resistance alters positive selection and inhibits autoimmunity and lymphoproliferative disease in MRL-lpr/lpr mice.

Thymus-derived glucocorticoids antagonize T cell receptor (TCR)-induced thymocyte apoptosis, allowing the survival (positive selection) of cells bearing TCRs that recognize self antigens with low-to-moderate avidity. Here we demonstrate that expression of an antisense glucocorticoid receptor transgene in thymocytes of spontaneously autoimmune MRL-lpr/lpr mice causes the loss of specific TCR Vbeta-bearing T cells that are normally positively selected in this strain. These transgenic mice had lower autoantibody production and milder symptoms of autoimmune disease than MRL-lpr/lpr controls and had markedly reduced accumulation of the TCR+Thy-1+CD4-CD8-B220+ T cells that are the hallmark of the lpr mutation. Thus, decreased glucocorticoid signaling in thymocytes alters the T cell repertoire and greatly diminishes autoimmunity in MRL-lpr/lpr autoimmune mice.

Lack of a role for Jun kinase and AP-1 in Fas-induced apoptosis.

Cross-linking of Fas (CD95) induces apoptosis, a response that has been reported to depend upon the Ras activation pathway. Since many examples of apoptosis have been reported to involve AP-1 and/or the AP-1-activation pathway. Since many examples of apoptosis have been reported to involve AP-1 and/or the AP-1-activating enzyme Jun kinase (JNK), downstream effectors of Ras or Ras-like small GTP-binding proteins, we evaluated the role of these molecules in Fas-mediated apoptosis. Although cross-linking of Fas on Jurkat T cells did result in JNK activation, increased activity was observed relatively late, being detectable only after 60 min of stimulation. Expression of a dominant negative form of SEK1 that blocked Fas-mediated induction of JNK activity had no effect on Fas-mediated apoptosis. Furthermore, maximally effective concentrations of anti-Fas did not cause JNK activation if apoptosis was blocked by a cysteine protease inhibitor, suggesting that under these conditions, activation of JNK may be secondary to the stress of apoptosis rather than a direct result of Fas engagement. Despite the activation of JNK, there was no induction of AP-1 activity as determined by gel shift assay or induction of an AP-1-responsive reporter. The lack of a requirement for AP-1 induction in Fas-mediated death was further substantiated with Jurkat cells that were stably transfected with a dominant negative cJun, TAM-67. While TAM-67 effectively prevented AP-1-dependent transcription of both the interleukin-2 and cJun genes, it had no effect on Fas-induced cell death, even at limiting levels of Fas signaling. Thus, induction of JNK activity in Jurkat cells by ligation of Fas at levels sufficient to cause cell death is likely a result, rather than a cause, of the apoptotic response, and AP-1 function is not required for Fas-induced apoptosis.

Regulation of thymocyte development by glucocorticoids.

It is generally believed that the avidity of the T cell receptor for self antigen/MHC determines the fate of a thymocyte. However, it is not understood how the thymocyte distinguishes a survival signal (positive selection) from a death signal (negative selection). Recent studies from our laboratory have explored the role that thymus-produced glucocorticoids may play in influencing thymocyte development. It appears that glucocorticoids are important and necessary at several points during thymocyte differentiation and that they may regulate antigen-specific T cell development.

Cross-talk between the T cell antigen receptor and the glucocorticoid receptor regulates thymocyte development.

The fate of an immature thymocyte, life or death, is largely determined by the ligand-specificity of its T cell antigen receptor (TCR). The default pathway for thymocytes bearing TCRs with subthreshold avidity for self-antigens is death (death by neglect). Thymocytes bearing TCRs with high avidity for self also undergo apoptosis (negative selection). Those thymocytes with intermediate avidities, or that perhaps recognize self-peptides that have partial agonist or antagonist properties, survive and differentiate into mature immunocompetent T cells (positive selection). How TCR avidity is interpreted as a "rescue" signal or a death signal is unknown. Based upon a T cell hybridoma model, our laboratory proposed that glucocorticoids, which themselves are potent inducers of thymocyte apoptosis, antagonize TCR-mediated thymocyte deletion and allow positive selection to occur. In fact, epithelial cells in the thymus proved to be a source of steroid production, and interference with steroid synthesis in fetal thymic organ culture resulted in a greatly enhanced sensitivity of thymocytes to TCR-mediated apoptosis. Transgenic mice with reduced glucocorticoid receptor (GR) levels were produced by tissue-specific expression of GR antisense. Thymocytes in these mice had high levels of spontaneous apoptosis, and were exquisitely sensitive to deletion induced by cross-linking the TCR. Moreover, there was a very large (> or = 90%) loss of CD4+CD8+ thymocytes, signifying a block at the CD4-CD8- to CD4+CD8+ transition, perhaps due to apoptosis of cells upon engagement of the pre-TCR in the absence of an antagonizing glucocorticoid stimulus. The molecular mechanism of the antagonism is currently being investigated. These data indicate that there is cross-talk in thymocytes between the TCR and glucocorticoid signaling pathways resulting in apoptosis, and that locally produced steroids, in a paracrine fashion, participate in setting the TCR avidity thresholds that determine whether developing thymocytes survive or die, and therefore help to mold the antigen-specific T cell repertoire.

A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development.

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

Muscarinic cholinergic receptors in brain and atrial membranes of adult brook trout (Salvelinus fontinalis) measured by radioligand binding techniques.

Muscarinic cholinergic receptors were measured by radioligand binding techniques in crude membrane particulate preparations of brain and atrial tissues from laboratory reared brook trout (Salvelinus fontinalis). The radioligand [3H]N-methyl scopolamine was used to determine number and affinity of receptors in saturation experiments. The affinity of the radioligand did not differ in brain and atrial preparations (96 +/- 8 and 60 +/- 4 pM, respectively). However, the number of binding sites was greater in atrium compared with brain (269 +/- 19 and 166 +/- 7 fmol/mg protein, respectively). The rank order of potency of competing drugs in inhibition experiments was similar for antagonists with atropine > or = scopolamine > pirenzepine. Pirenzepine, an M1-selective drug had a 3-fold higher affinity in brain than atrium. The agonists oxotremorine and carbachol each bound to two sites in both tissues. In contrast, pilocarpine bound to only one site in brain and two in atrial tissue. These results are compared with those observed in other nonmammalian species and discussed with reference to conservation of proteins that serve important cellular roles.

To be or not to be: mutually antagonistic death signals regulate thymocyte apoptosis.

Recognition of self-antigens by immature thymocytes results in either activation-induced apoptosis (negative selection) or survival (positive selection). While it is believed that T cell receptor avidity plays a role in determining the outcome, the mechanisms responsible for this life or death decision are not known. Recent data concerning the mutual antagonism between activation- and glucocorticoid-induced apoptosis have prompted an examination of the potential interaction of these two signaling pathways in the regulation of antigen-specific selection.

Role of endogenous retroviruses in autoimmune diseases.

Retroviruses have been implicated in the pathogenesis of murine and human lupus; however, many positive findings have been followed by alternative explanations. Initial findings implicating xenotropic retroviruses were subsequently invalidated. The first solid demonstration that endogenous retroviruses mediate disease was the study of SL/Ni mice. Here budding ecotropic retroviral particles from arterial smooth muscle cells caused an antibody response to the particles with subsequent complement deposition. Our laboratory has focused on derangements in endogenous MCF retroviral expression. We found that lupus-prone NZB, BXSB and MRL strains have a marked increase in expression of Mpmv RNA in their thymuses while bone marrow expression did not differ from normal strains. Sequence analysis demonstrated mutations in the NZB endogenous retroviruses which could alter expression. A phosphorothioate antisense oligonucleotide to the initiation sequence of Mpmv caused lymphocyte activation in vivo in normal mice, providing further evidence for in vivo effects of Mpmv and potential for pathological abnormalities in lupus-prone strains.

Constitutive expression of interferon gamma-inducible protein 10 in lymphoid organs and inducible expression in T cells and thymocytes.

Interferon gamma-inducible protein 10 (IP-10), a member of a family of small proinflammatory chemotactic polypeptides, is expressed in interferon gamma-stimulated keratinocytes, macrophages, fibroblasts, and endothelial cells. Here we report that IP-10 is also expressed by activated but not resting T hybridoma cells, normal T cells, and thymocytes. Although resting lymphocytes did not synthesize IP-10, surprisingly high levels of IP-10 transcripts were found in lymphoid organs (spleen, thymus, and lymph nodes). Thymic and splenic stromal cells were found to express constitutively high levels of both IP-10 mRNA and protein, accounting for the high level of spontaneous expression in lymphoid tissue. Therefore, in addition to its role as a proinflammatory cytokine, IP-10 may participate in T cell effector function and perhaps T cell development.

Thymocyte and T cell apoptosis: is all death created equal?

During thymocyte development, potentially autoreactive thymocytes are eliminated by a process known as apoptosis or programmed cell death. While it has long been known that this clonal elimination or negative selection of thymocytes expressing T cell receptors with high affinity for self antigens plays a major role in preserving self tolerance, it is now apparent that apoptosis may also play an active role in maintaining peripheral T cell tolerance. Although it is clear that apoptosis plays a major role in shaping the immune response, the mechanisms responsible for its induction and the regulatory mechanisms that influence susceptibility to cell death are not well characterized. In this article, we will concentrate on some of the most recent findings in this area. In particular, we will emphasize the protective 'rheostat' mechanism exemplified by the Bcl-2 family members, and the role of Fas in activation-induced apoptosis. In addition, we will compare the physiologic signals that trigger apoptosis in thymocytes and peripheral T cells, and discuss whether central and peripheral deletion are regulated by similar or distinct mechanisms.

Signaling for death of lymphoid cells.

The induction of programmed cell death in lymphocytes is a common response to a wide variety of physiological and pharmacological stimuli. While there is still much to be learned about the transmembrane signals that lead to programmed cell death, progress has been made in identifying new cell surface molecules (e.g. APO-1/Fas) that may regulate the physiological induction of lymphocyte death, molecules whose expression inhibits apoptosis (e.g. Bcl-2), and the antagonism of activation-induced cell death in T-cell hybridomas and thymocytes by members of the steroid receptor superfamily.

Mouse mammary tumor proviral gene expression in cells of the B lineage.

Mouse mammary tumor proviruses (MMTV) use a common enhancer/promoter region to accommodate their transcription in two different cellular environments. In mammary tissue, transcription is regulated through the hormone response element located in the 5' LTR. In B cells, transcription is hormone independent, can be stimulated following B cell activation, and is distinct from the transcription of other known inducible genes, including immunoglobulin. The open reading frame (ORF) in the viral 3' LTR has at least two functions. Its gene product(s) acts as a viral superantigen, but also has autoregulatory properties, leading to MMTV transactivation. We propose a scheme suggesting that MMTV evolved to use the B cell as an intermediary in its viral life cycle.

Molecular aspects of systemic lupus erythematosus: murine endogenous retroviral expression.

Systemic lupus erythematosus is an immune-mediated disease in which the etiology is unknown. Full-length (8.4 kb), type C, modified polytropic (Mpmv) retroviral transcripts from the thymus are characteristic of murine lupus. Reciprocal bone marrow transplantation studies determined that this thymic expression maps to the pre-T bone marrow stem cell. In vitro and in vivo oligonucleotide antisense work suggest that type C retroviruses play a role in immune activation. This paper summarizes our studies of endogenous retroviruses in murine lupus.