Biomedical subjects

R P Ryseck

Publications and source records attributed to R P Ryseck.

At least 19 recordsLinked to original sources

PubMed2001

Inhibition of NF-kappaB in T cells blocks lymphoproliferation and partially rescues autoimmune disease in gld/gld mice.

The Fas ligand (FasL)/Fas pathway is crucial for the maintenance of homeostasis of the peripheral immune system. Its importance is illustrated by the spontaneous mouse mutants gld andlpr which lack functional FasL and Fas receptor, respectively. These animals develop lymphadenopathy, splenomegaly, increased serum Ig and autoantibodies, leading to an autoimmune syndromeand premature death. The Rel/NF-kappaB family of transcription factors plays an important role in peripheral lymphocyte proliferation and survival. In this report, we studied the consequences of T cell-specific inhibition of NF-kappaB on the development of the gld phenotype. Transgenic gld/gld mice expressing a non-degradable form of IkappaBalpha under the control of T cell-specific regulatory elements show dramatically reduced lymphadenopathy, splenomegaly, and an almost complete elimination of Thy-1(+)B220(+)CD4(-)CD8(-) abnormal T cells, correlating with reduced proliferative responses and increased apoptosis of peripheral T cells upon TCR triggering. Interestingly, the B cell abnormalities that are characteristic of gld/gld mice, such as the production of autoantibodies, high levels of serum Ig, and the development of glomerulonephritis, are partially corrected. These results suggest that the T cell-specific inhibition of NF-kappaB opens apoptotic pathways distinct from FasL/Fas which, along with a diminished proliferative response, blocks splenomegaly and lymphadenopathy and partially rescues autoimmune disease in gld/gld mice.

Animals↗

PubMed1999

Peptides corresponding to the N and C termini of IkappaB-alpha, -beta, and -epsilon as probes of the two catalytic subunits of IkappaB kinase, IKK-1 and IKK-2.

The signal-inducible phosphorylation of serines 32 and 36 of IkappaB-alpha is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaB-alpha, which then releases NF-kappaB to promote gene transcription. The multisubunit IkappaB kinase (msIKK) responsible for this phosphorylation contains two catalytic subunits, termed IKK-1 and IKK-2. Using recombinant IKK-2, a kinetic pattern consistent with a random, sequential binding mechanism was observed with the use of a peptide corresponding to amino acids 26-42 of IkappaB-alpha. Values of 313 microM, 15.5 microM, and 1.7 min(-1) were obtained for K(peptide), K(ATP), and k(cat), respectively. The value of alpha, a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.2. Interestingly, the recombinant IKK-1 subunit gave similar values for alpha and K(ATP), but values of 1950 microM and 0.016 min(-1) were calculated for K(peptide) and k(cat), respectively. This suggests that the IKK-2 catalytic subunit provides nearly all of the catalytic activity of the msIKK complex with the IKK-1 subunit providing little contribution to catalysis. Using peptides corresponding to different regions of IkappaB-alpha within amino acids 21-47, it was shown that amino acids 31-37 provide most binding interactions (-4.7 kcal/mol of binding free energy) of the full-length IkappaB-alpha (-7.9 kcal/mol) with the IKK-2. This is consistent with the observation that IKK-2 is able to phosphorylate the IkappaB-beta and IkappaB-epsilon proteins, which have consensus phosphorylation sites nearly identical to that of amino acids 31-37 of IkappaB-alpha. A peptide corresponding to amino acids 279-303 in the C-terminal domain of IkappaB-alpha was unable to activate IKK-2 to phosphorylate an N-terminal peptide, which is in contrast to the results observed with the msIKK. Moreover, the IKK-2 catalyzes the phosphorylation of the full-length IkappaB-alpha and the amino acid 26-42 peptide with nearly equal efficiency, while the msIKK catalyzes the phosphorylation of the full-length IkappaB-alpha 25,000 times more efficiently than the 26-42 peptide. Therefore, the C terminus of IkappaB-alpha is important in activating the msIKK through interactions with subunits other than the IKK-2.

Amino Acid Sequence↗

PubMed1998

Antigen-induced eosinophilic lung inflammation develops in mice deficient in chemokine eotaxin.

The mechanisms that regulate the selective infiltration of eosinophils in certain allergic diseases are still poorly understood. The CC chemokine eotaxin is a potent chemoattractant, highly specific for eosinophils. Recent studies have implicated that eotaxin plays an important role in the recruitment of eosinophils in different inflammation processes. A number of other chemokines, cytokines, and chemoattractants also have chemotactic activities for eosinophils and some of them present high selectivity for eosinophils. To further study the role of eotaxin in inflammation, we generated mutant mice with the eotaxin gene disrupted and replaced by the Escherichia coli beta-galactosidase gene. These mice developed normally and had no histologic or hematopoietic abnormalities. Furthermore, our studies showed that the lack of eotaxin did not affect the recruitment of eosinophils in the inflammation models induced by Sephadex beads and thioglycollate, as well as in an experimental lung eosinophilia model induced by ovalbumin aerosol challenge, even at the onset of the inflammatory response. The replacement of the eotaxin gene by the beta-galactosidase gene provided a useful marker to monitor the activity of the eotaxin promoter under normal conditions and after antigen challenges. Immunohistochemical staining suggested that endothelial cells were the major sources of eotaxin expression.

Animals↗

PubMed1998

The Drosophila ebony gene is closely related to microbial peptide synthetases and shows specific cuticle and nervous system expression.

The previously detected ebony (e) locus (Caizzi et al., 1987) consists of a complex gene structure that is divided into seven exons. An open reading frame encoding the putative Ebony protein of 98.5 kDa exhibits homology to a family of peptide synthetases (Stachelhaus and Marahiel, 1995), in good correlation with the proposed function as beta-alanyl-dopamine synthetase. Multiple ebony transcripts are detected throughout development. P-factor mediated transformation of genomic DNA rescues the cuticle, electrophysiological and behavioural phenotypes. Fusion of the ebony reading frame with that of beta-galactosidase of E. coli reveals expression in cuticle and nervous system. Strong staining in the first and, to a lesser extent, in the second optic neuropile may reflect the pronounced visual defect observed in ebony mutants. In addition, weak central brain and thoracic ganglion expression is detected in flies. Conservation of a multidomain protein structure known from peptide synthetases should have functional implications on the putative reaction mechanism of peptide bond formation.

Amino Acid Sequence↗

PubMed1998

Functional redundancy of the nuclear factor kappa B inhibitors I kappa B alpha and I kappa B beta.

The transcription factor NF-kappaB is sequestered in the cytoplasm by the inhibitor proteins of the IkappaB family. Each member of the IkappaB exhibits structural and biochemical similarities as well as differences. In an effort to address the functional redundancy of two closely related IkappaB molecules, IkappaBalpha and IkappaBbeta, we generated knock-in mice by replacing the IkappaBalpha gene with the IkappaBbeta gene. The knock-in mice do not express IkappaBalpha, but express a T7-tagged IkappaBbeta under the promoter and regulatory sequence of ikba. Unlike the IkappaBalpha-deficient mice, which display severe postnatal developmental defects and die by postnatal day 8, homozygous knock-in mice survive to adulthood, are fertile, and exhibit no apparent abnormalities. Furthermore, thymocytes and embryonic fibroblasts from the knock-in animals exhibit an inducible NF-kappaB response similar to that of wild-type animals. These results indicate that IkappaBalpha and IkappaBbeta share significant similarities in their biochemical activity, and that they acquired their different functions from divergent expression patterns during evolution.

Animals↗

PubMed1998

Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor.

The CC-chemokine receptor CCR5 has been shown to be the major coreceptor for HIV-1 entry into cells, and humans with homozygous mutation in the ccr5 gene are highly resistant to HIV-1 infection, despite the existence of many other HIV-1 coreceptors. To investigate the physiologic function of CCR5 and to understand the cellular mechanisms of these clinical observations, we generated a CCR5-deficient mouse model (ccr5[-/-]) by targeted deletion of the ccr5 gene. We found that although developed normally in a pathogen-free environment, CCR5-deficient mice showed reduced efficiency in clearance of Listeria infection and exert a protective effect against LPS-induced endotoxemia, reflecting a partial defect in macrophage function. In addition, CCR5-deficient mice had an enhanced delayed-type hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge, indicating a novel role of CCR5 in down-modulating T cell-dependent immune response.

Animals↗

PubMed1997

Gastric hyperplasia and increased proliferative responses of lymphocytes in mice lacking the COOH-terminal ankyrin domain of NF-kappaB2.

The nfkb2 gene encodes the p100 precursor which produces the p52 protein after proteolytic cleavage of its COOH-terminal domain. Although the p52 product can act as an alternative subunit of NF-kappaB, the p100 precursor is believed to function as an inhibitor of Rel/NF-kappaB activity by cytoplasmic retention of Rel/NF-kappaB complexes, like other members of the IkappaB family. However, the physiological relevance of the p100 precursor as an IkappaB molecule has not been understood. To assess the role of the precursor in vivo, we generated, by gene targeting, mice lacking p100 but still containing a functional p52 protein. Mice with a homozygous deletion of the COOH-terminal ankyrin repeats of NF-kappaB2 (p100(-/-)) had marked gastric hyperplasia, resulting in early postnatal death. p100(-/-) animals also presented histopathological alterations of hematopoietic tissues, enlarged lymph nodes, increased lymphocyte proliferation in response to several stimuli, and enhanced cytokine production in activated T cells. Dramatic induction of nuclear kappaB-binding activity composed of p52-containing complexes was found in all tissues examined and also in stimulated lymphocytes. Thus, the p100 precursor is essential for the proper regulation of p52-containing Rel/NF-kappaB complexes in various cell types and its absence cannot be efficiently compensated for by other IkappaB proteins.

Animals↗

PubMed1997

Genetic approaches to study Rel/NF-kappa B/I kappa B function in mice.

The generation of animal models in which individual members of a gene family are genetically altered is a particularly attractive way to elucidate their function-Members of the Rel/NF-kappa B/I kappa B family constitute an important network of transcription factors and regulatory proteins that control the expression of numerous cellular and viral genes crucial for a variety of processes. A few examples are developmental pattern formation and immune response in Drosophila, viral replication, and immune, inflammatory, acute phase and stress responses in vertebrates. The findings from knockout and transgenic mice developed to study Rel/NF-kappa B/I kappa B function in vivo are reviewed here. In general, these studies point to the essential role of these factors in the development and function of the vertebrate immune system.

Animals↗

PubMed1996

Characterization of ES cells deficient for the p105 precursor (NF-kappa B1): role of p50 NLS.

Proteolytic processing of the p105 precursor (NF-kappa B1) generates the p50 subunit of NF-kappa B. To address the function of p105 precursor, we have generated embryonic stem (ES) cells which lack the C-terminal inhibiting ankyrin-containing domain of p105 (I kappa B gamma) but contain a functional p50 by homologous recombination. Two forms of p50 were found in ES cells lacking p105. While one was a p50 molecule with the expected size of 415 amino acids, the other was an isoform of 358 amino acids generated by alternative splicing. This isoform lacked the C-terminal 62 amino acids of p50 including the putative nuclear localization signal (NLS), but included five unrelated amino acids. This p50 molecule lacking the NLS behaved similar to wild type p50. Importantly, the p50 isoform-RelA complex interacted efficiently with I kappa B alpha, implying that the NLS of p50 is not essential for these functions. Also, p50 isoform dimers were present in the nucleus. Loss of p105 and the augment of total p50 produced an increase in the nuclear kappa B-binding activity of p50-RelA and p50 dimers. In wild type ES cells, a significant proportion of total p50 is associated with p105. Therefore, the mutation introduced in NF-kappa B1 eliminates both the processing to generate p50 and the trapping of p50 by p105. In contrast to p50, only a small fraction of RelA is associated with p105. These findings indicate that p105 plays a unique role in controlling p50 dimer activity.

Alternative Splicing↗

PubMed1996

Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.

Animals↗

PubMed1996

RelB, a member of the Rel/NF-kappa B family of transcription factors.

RelB, originally identified as an immediate early gene product, is a member of the Rel/NF-kappa B family of transcription factors important for the regulation of genes involved in immune and inflammatory processes. RelB by itself is inactive due to its inability to homodimerize and to bind to kappa B sequences. However, in the presence of the Rel/NF-kappa B proteins p50 or p52, RelB is a potent transactivator. Transcriptional activation domains were identified in the NH2 and COOH termini of RelB separated by the approximately 300 amino acids spanning the Rel homogy domain (RHD). The last 120 amino acids of this domain are necessary for the dimerization of RelB and were analyzed in detail by in vitro mutagenesis. RelB forms complexes with p50 and p52 but not with RelA and c-Rel. In contrast to RelA-containing complexes, RelB-containing complexes are only weakly inhibited in their activity by I kappa B alpha. Furthermore, in lymphoid tissues RelB is not associated with I kappa B alpha. In contrast to other members of the Rel/NF-kappa B family, high expression of RelB is limited to interdigitating dendritic cells. Mice with a targeted disrupted relB locus show phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells.

Animals↗

PubMed1995

Specific inhibition of RelB/p52 transcriptional activity by the C-terminal domain of p100.

The Rel/NF-kappa B family transcriptional factors plays an important role in the regulation of immune and acute phase responses. The activity of Rel/NF-kappa B complexes is regulated by their interactions with members of the I kappa B family of inhibitors. We have previously shown that the RelB/p52 heterodimer is not effectively inhibited by any of the known I kappa B molecules: I kappa B alpha, I kappa B gamma and Bcl3. Here we report that the C-terminal domain of p100 (the putative I kappa B delta) functions as a strong inhibitor of RelB/p52 transcriptional activity. In vivo interaction with I kappa B delta leads to the cytoplasmic retention and decreased DNA binding activity of RelB/p52 complexes. Thus, I kappa B delta is the only I kappa B molecule able to efficiently modulate the activity of RelB/p52 heterodimer. In Daudi cells, a 46 kD protein, probably representing the C-terminal product of the proteolytic processing of p100, remains associated with Rel/NF-kappa B complexes and might have a transient regulatory function. Our results indicate a specific role for the putative I kappa B delta and suggest a possible mechanism of how the truncation of the ankyrin domain of p100, found in a number of lymphoid neoplasias, might contribute to tumorigenesis.

Cell Line↗

PubMed1995

Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family.

RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Aging↗

PubMed1995

Characterization of elements determining the dimerization properties of RelB and p50.

Members of the Rel/NF-kappa B family of transcription factors share a region of approximately 300 amino acids which mediates dimerization and sequence-specific binding to DNA. Here we report a detailed characterization of the dimerization domain of RelB. The structural core sufficient to form stable Rel/NF-kappa B dimeric complexes consists of about 110 residues. The dimerization and DNA binding properties of more than 50 RelB mutants were analyzed by using p50 and p52 as partners. We present evidence that amino acids of a conserved element in the dimerization domain play a role in the recognition of a kappa B DNA target sequence. The analysis of hybrid molecules with dimerization domains containing different parts of p50 and RelB allowed us to identify some important structural elements determining homo- and heterodimerization properties. Furthermore, we were able to rescue the dimerization-defective mutant RelB-N287D by the introduction of a counteracting mutation intramolecularly (cis), and also intermolecularly (trans) by a mutation in the NF-kappa B dimerization partner p50. Correspondingly, a dimerization defective p50 mutant was effectively rescued by RelB-N287D.

Amino Acid Sequence↗

PubMed1994

Differential interactions of Rel-NF-kappa B complexes with I kappa B alpha determine pools of constitutive and inducible NF-kappa B activity.

The Rel-NF-kappa B family of transcription factors plays a crucial role in the regulation of genes involved in inflammatory and immune responses. We demonstrate that in vivo, in contrast to the other members of the family, RelB associates efficiently only with NF-kappa B1 (p105-p50) and NF-kappa B2 (p100-p52), but not with cRel or p65. The RelB-p52 heterodimers display a much lower affinity for I kappa B alpha than RelB-p50 heterodimers or p65 complexes. However, similarly to the other Rel-NF-kappa B complexes, RelB-p52 can upregulate the synthesis of I kappa B alpha leading to the cytoplasmic trapping of dimers which have a higher affinity for the inhibitor. We suggest that a hierarchy of interactions between I kappa B alpha and the different Rel-NF-kappa B complexes governs their cellular distribution. This results in the presence of two distinct pools of NF-kappa B activity which differ in their composition: one a constitutive nuclear and the other an inducible cytoplasmic activity.

B-Lymphocytes↗

PubMed1993

Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

Amino Acid Sequence↗

PubMed1993

The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine.

We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.

Amino Acid Sequence↗

PubMed1993

Expression of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells.

We have studied the expression of the relB gene during mouse development using in situ hybridization and immunocytochemical analysis. The results show that the expression of the relB gene is highly restricted to a subpopulation of cells that colonize the lymphoid tissues and that appear very late during the process of hematopoietic diversification. RNA transcripts of relB are very low or undetectable in early and late embryos. Low relB expression is observed in the thymus at late stages of embryogenesis but rapidly increases after birth. In adult lymphoid tissues, relB is detected in the medullary region of the thymus, the periarterial lymphatic sheaths of the spleen, and the deep cortex of the lymph nodes, which correspond to the regions where T cells of mature phenotype and interdigitating dendritic cells are present. Using double immunofluorescent labeling of thymic cell suspensions, we have identified the interdigitating dendritic cells as the target of RelB expression. These cells are part of a system of antigen-presenting cells that function in the induction of several immune responses, such as, tolerance, sensitization of MHC-restricted T cells, rejection of organ transplants and formation of T-dependent antibodies. Our observations indicate that RelB may play a particular role in the signal transduction pathway that regulate dendritic cell differentiation and its cellular responses.

Animals↗