Leukemia inhibitory factor (LIF) potentiates antinociception activity and inhibits tolerance induction of opioids.

BACKGROUND: The efficacy of opioids typically decreases after long-term use owing to the development of tolerance. Glial activation and the upregulation of proinflammatory cytokines are related to the induction of tolerance. We investigated the effect of leukemia inhibitory factor (LIF) on morphine analgesia and tolerance. METHODS: LIF concentrations in rat spinal cords were measured by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) after morphine administration. LIF distribution was examined using confocal microscopy. To evaluate the effects of LIF on morphine analgesia and tolerance, LIF was intrathecally administered 30 min before morphine injection. The analgesic effect of morphine was evaluated by measuring tail-flick latency. Human LIF concentrations from the cerebrospinal fluid (CSF) of opioid tolerant patients were also determined by specific ELISA. RESULTS: Chronic morphine administration upregulated LIF concentrations in rat spinal cords. Intrathecal injection of LIF potentiated the analgesic action of morphine. Patch clamp recording of spinal cord slices showed that LIF enhanced DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin)-induced outward potassium current. The development of tolerance was markedly suppressed by exogenous LIF, whereas neutralizing the endogenously released LIF with anti-LIF antibodies accelerated the tolerance induction. Moreover, LIF concentrations in the CSF of opioid-tolerant patients were higher than those in the opioid-naive controls. CONCLUSIONS: Intrathecal administration of LIF potentiated morphine antinociceptive activity and attenuated the development of morphine tolerance. Upregulation of endogenously released LIF by long-term use of opioids might counterbalance the tolerance induction effects of other proinflammatory cytokines. LIF might be a novel drug candidate for inhibiting opioid tolerance induction.

NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity.

Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.

Survival of leukemic B cells promoted by engagement of the antigen receptor.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia characterized by the slow but progressive accumulation of cells in a CD5+ B-cell clone. Like the nonmalignant counterparts, B-1 cells, CLL cells often express surface immunoglobulin with the capacity to bind autologous structures. Previously there has been no established link between antigen-receptor binding and inhibition of apoptosis in CLL. In this work, using primary CLL cells from untreated patients with this disease, it is demonstrated that engagement of surface IgM elicits a powerful survival program. The response includes inhibition of caspase activity, activation of NF-kappaB, and expression of mcl-1, bcl-2, and bfl-1 in the tumor cells. Blocking phosphatidylinositol 3-kinase (PI3-K), a critical mediator of signals through the antigen receptor, completely abrogated mcl-1 induction and impaired survival in the stimulated cells. These data support the contention that CLL cell survival is promoted by antigen for which the malignant clone has affinity, and suggest that pharmacologic interference with antigen-receptor-derived signals has potential for therapy in patients with CLL.

c-Rel is required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis.

The NF-kappaB/Rel transcription factor family has been shown to protect many cell types from apoptotic signals. However, it is not known whether NF-kappaB is required for all survival pathways and whether each NF-kappaB member plays a unique or a redundant role. Here we describe the results of studies on the role of c-Rel in survival. Mature B cells from c-Rel(-/-) mice exhibit defects in survival, including sensitivity to Ag receptor-mediated apoptosis as well as increased sensitivity to ionizing radiation and glucocorticoids. Transgene expression of Bcl-x(L), a c-Rel target gene, rescues c-Rel(-/-) B cells from their survival defects. Thus, c-Rel-dependent survival pathways are crucial for protection from apoptotic signals that target the mitochondrial pathway. Despite a lack of Bcl-x(L), c-Rel(-/-) B cells can still be rescued from Fas-mediated apoptosis via B cell receptor signaling. The Fas apoptosis inhibitor molecule and FLICE inhibitory protein (c-FLIP) proteins are up-regulated normally in c-Rel(-/-) B cells, and these two molecules may play a more physiological role in the Fas pathway. Furthermore, unlike the TNF sensitivity of RelA(-/-) fibroblasts, c-Rel-deficient fibroblasts are refractory to TNF-mediated cell death. Thus, c-Rel is dispensable for protection against death receptor-mediated apoptosis. Taken together, our data suggest that distinct NF-kappaB/Rel members are required for protecting cells from different types of apoptotic signals.

Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

Nucleosome remodeling at the IL-12 p40 promoter is a TLR-dependent, Rel-independent event.

Lipopolysaccharide (LPS) induction of the gene encoding interleukin 12 p40 requires remodeling of a promoter-encompassing nucleosome and the Toll-like receptor (TLR)-mediated activation of a c-Rel-containing complex. Analysis of TLR4-mutant mice revealed that remodeling requires TLR signaling. However, Rel proteins and other proteins required for transcription of an integrated p40 promoter were insufficient for remodeling. c-Rel was also unnecessary for remodeling, as remodeling was observed in c-Rel-/- macrophages, which lack p40 transcripts. These results suggest that remodeling requires TLR signaling pathways that diverge from the c-Rel activation pathways. The factors that stimulate remodeling may represent, therefore, newly identified targets of TLR signaling and of agents that regulate inflammatory responses and TH1 development.

Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages.

A major challenge in the study of gene regulation by NF-kappaB/Rel transcription factors is to understand, at the biological and mechanistic levels, the selective functions of individual Rel family members. To study selectivity, we have examined the NF-kappaB/Rel protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 cell development. In nuclear extracts from lipopolysaccharideactivated macrophages, the predominant Rel dimers capable of binding the IL-12 p40 Rel site were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The two heterodimers bound the site with comparable affinities and exhibited comparable transactivation activities. In striking contrast, p40 mRNA and protein concentrations were reduced dramatically in c-Rel(-/-) macrophages and only modestly in p65(-/-) macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel(-/-) macrophages. These results reveal that a c-Rel-containing complex is an essential and selective activator of p40 transcription, which may reflect unique regulatory mechanisms or biological functions of IL-12. Furthermore, because selectivity was not observed in vitro or in transient transactivation experiments, these findings suggest that an understanding of the selectivity mechanism may require an analysis of the endogenous p40 locus.

Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.

CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-kappaB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-kappaB/Rel activation. This is achieved via activation of AKT and the degradation of IkappaBalpha. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-kappaB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-kappaB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-kappaB/Rel and PI-3K are crucial for CD40-induced proliferation.

Nuclear factor kappa B is required for the development of marginal zone B lymphocytes.

Although immunoglobulin (Ig)M(hi)IgD(lo/-)CD21(hi) marginal zone B cells represent a significant proportion of naive peripheral splenic B lymphocytes, few of the genes that regulate their development have been identified. This subset of peripheral B cells fails to emerge in mice that lack nuclear factor (NF)-kappa Bp50. Less drastic reductions in marginal zone B cell numbers are also seen in the spleens of recombination activating gene (Rag)-2(-/-) mice reconstituted with NF-kappa Bp65(-/-) fetal liver cells and in c-Rel(-/-) mice. In contrast, steady-state levels of IgD(hi) splenic follicular B cells are not significantly reduced in the absence of NF-kappa Bp50, NF-kappa Bp65, or c-Rel. Reconstitution of B cells in Rag-2(-/-) mice with a mixture of p50(-/-)/p65(-/-) fetal liver cells and Rag-2(-/-) bone marrow cells revealed that the generation of marginal zone B cells requires the expression of NF-kappa B in developing B cells, as opposed to supporting cells.

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction.

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.

Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-kappaB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-kappaB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-kappaB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-kappaB activity and prolonged CLL cell survival in vitro. The principle NF-kappaB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-kappaB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-kappaB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.

NF-kappa B/Rel transcription factors: c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation.

The NF-kappa B/Rel family of transcription factors induces many genes involved in immune and inflammatory responses. Mice with germline deletions of individual NF-kappa B/Rel subunits have different phenotypes, suggesting that the NF-kappa B/Rel transcription factors have different functions. We tested whether c-Rel promotes allergic asthma using a murine model of allergen-induced pulmonary inflammation and airway hyperresponsiveness. Our investigation focused on c-Rel, which is expressed in lymphoid cells and is important for lymphocyte activation. In response to allergen sensitization and challenge, c-Rel-deficient mice did not develop increases in pulmonary inflammation, bronchoalveolar lavage fluid eosinophilia, or total serum IgE. c-Rel deficiency also prevented the induction of airway hyperresponsiveness. Allergen-treated wild-type mice had increased DNA binding to an NF-kappa B consensus site. Chemokine expression was altered in allergen-treated c-Rel-deficient mice. Monocyte chemoattractant protein-1, which is regulated by NF-kappa B, was decreased in allergen-treated c-Rel-deficient mice relative to wild-type controls. The increase in NF-kappa B/Rel transcription factors after allergen challenge in wild-type mice and the decrease in allergen reactivity found in c-Rel-deficient mice indicate that c-Rel promotes allergic inflammation. Alteration of pulmonary chemokine expression in c-Rel-deficient mice may inhibit allergen-induced pulmonary inflammation and airway hyperresponsiveness.

The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis.

The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins. By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1. Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells. In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line. Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS. Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD. Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector.

c-Rel is crucial for lymphocyte proliferation but dispensable for T cell effector function.

The TCR signals are essential for T cell activation and proliferation, primarily through the induction of cytokine and cytokine receptors. Several transcription factor families, including NF-kappaB/Rel, have been implicated in the regulation of cytokine gene expression in T cells in response to antigen, cytokine and mitogenic stimulation. In this study, we show that the mice with a null mutation in the lymphoid-specific c-Rel gene have normal development of lymphoid tissues and T cell compartment. However, T cells derived from the c-Rel knockout mice have several functional abnormalities. The c-Rel-deficient T lymphocytes fail to respond to activation and proliferation signals mediated by the TCR and mitogens in vitro. This is attributed to an impaired production of cytokines IL-2, IL-3 and granulocyte macrophage colony stimulating factor. In addition, the induction of IL-2R alpha chain is impaired in the c-Rel(-/-) T cells. The poor expression of cytokines and IL-2R alpha chain correlates with a reduced nuclear translocation of NF-kappaB components in c-Rel(-/-) T cells. Since activation is prerequisite for differentiation into effector cells, c-Rel(-/-) T cells failed to differentiate into cytotoxic T cells or Th cells without rescuing cytokines. However, upon supplement with exogenous IL-2, the c-Rel(-/-) cytotoxic T lymphocytes are able to execute cytotoxicity and the c-Rel(-/-) Th cells are capable of providing help to normal B cells. These data suggest that c-Rel is important for inducible cytokine and cytokine receptor expression, and a key regulator of early activation and proliferation in T cells.

c-Rel is essential for B lymphocyte survival and cell cycle progression.

c-Rel is a lymphoid-specific member of the NF-kappaB/Rel family of transcriptional factors. To investigate the role of c-Rel in B lymphocyte function, we generated a c-Rel(-/-) mouse via a gene targeting approach. Although early lymphocyte development is normal in c-Rel(-/-) mice, there are significantly fewer B cells displaying a memory (IgM/IgD-) phenotype. Upon immunization, c-Rel(-/-) mice generate fewer B cells with a germinal center (PNAhi) phenotype. In vitro, c-Rel(-/-) B cells proliferate poorly upon ligation of their surface IgM or CD40 receptors or when stimulated with either lipopolysaccharide (LPS) or T cell help. Early molecular events that precede proliferation, such as increases in RNA synthesis as well as IL-2 receptor alpha chain expression, are greatly diminished in c-Rel(-/-) B cells. Furthermore, c-Rel(-/-) B cells are impaired in the ability to receive survival signals generated by anti-IgM or LPS. In contrast, CD40-mediated cell survival is normal in c-Rel(-/-) B cells, suggesting the involvement of a survival-signaling pathway that is independent of c-Rel. When c-Rel (-/-) B cells are co-stimulated with either anti-IgM and CD40 or LPS and CD40, they are rendered capable of progressing through the cell cycle. Finally, co-culture experiments suggest that the defects observed in c-Rel(-/-) B cells are intrinsic to the cell and can not be rescued through either cell-cell contact or addition of soluble factors. Thus, c-Rel is requisite for differentiation to the germinal center and memory B cells in vivo and is required for the transduction of survival and cell cycle progression signals mediated by anti-IgM and LPS in vitro. Furthermore, while c-Rel is involved in CD40-induced proliferation, it is apparently dispensable for the survival signals transduced by CD40.

Nerve terminal currents induced by autoreception of acetylcholine release.

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.

CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells.

We used our monoclonal model of germinal center maturation, CL-01 B cells, to investigate the role of CD30 in human B cell differentiation. CL-01 cells are IgM+ IgD+ CD30+ and switch to IgG, IgA, and IgE when exposed to CD40L and IL-4. Switching is hampered by CD30 coengagement, possibly through interference with the CD40-mediated NF-kappaB-dependent transcriptional activation of downstream C(H) genes. The physiological relevance of this phenomenon is emphasized by similar CD30-mediated effects in naive B cells. Expression of CD30 by these cells is induced by CD40L but is inhibited by B cell receptor coengagement and/or exposure to IL-6 and IL-12. Our data suggest that CD30 critically regulates the CD40-mediated differentiation of non-antigen-selected human B cells.

Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD30 is expressed on normal activated lymphocytes, on several virally transformed T- or B-cell lines and on neoplastic cells of Hodgkin's lymphoma. The interaction of CD30 with its ligand induces pleiotropic effects on cells resulting in proliferation, differentiation, or death. The CD30 cytoplasmic tail interacts with TNF receptor-associated factors (TRAFs), which have been shown to transduce signals mediated by TNF-R2 and CD40. We demonstrate here that TRAF2 also plays an important role in CD30-induced NF-kappa B activation. We also show that TRAF2-mediated activation of NF-kappa B plays a role in the activation of HIV transcription induced by CD30 cross-linking. Detailed site-directed mutagenesis of the CD30 cytoplasmic tail reveals that there are two independent binding sites for TRAF, each interacting with a different domain of TRAF. Furthermore, we localized the TRAF-C binding site in CD30 to a 5-7 amino acid stretch.