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Biomedical subjects

M Dy

Publications and source records attributed to M Dy.

At least 19 recordsLinked to original sources
PubMed2001

Differential expression and tissue distribution of parkin isoforms during mouse development.

Mutations of the parkin gene are a cause of autosomal recessive juvenile parkinsonism. Although the parkin gene has been isolated from mouse, rat, and human, little is known about its expression in neural and nonneural tissues during development. In this study, we used a polyclonal antibody to a peptide downstream of the parkin ubiquitin domain to investigate (1) the differential expression of parkin isoforms in protein extracts from fetal and adult mouse tissues, and (2) the distribution of parkin in mouse fetal tissues at different developmental stages and in adult CNS tissues. By Western blot analyses, at least three isoforms of parkin of 22, 50, and 55 kDa were differentially expressed in mouse tissues. The p22 and p50 isoforms were found in fetal and adult mouse CNS tissues, while the p55 isoform was found only in adult tissues. The p50 isoform is the predominant form in both fetal and adult tissues. Immunolocalization in mouse fetuses showed that parkin was expressed only after neuronal differentiation. Although parkin was localized throughout the cytoplasm, the highest level of parkin was found in the neurites of both fetal and adult neurons.

Age Factors↗
PubMed2001

Thrombopoietin induces histidine decarboxylase gene expression in c-mpl transfected UT7 cells.

The leukemic cell line UT7 is endowed with both megakaryocyte and basophil differentiation potential, as judged by its capacity to respond to PMA by displaying megakaryocytic and basophilic markers and to produce histamine by neosynthesis. Herein, we addressed the question whether the biological activities characteristic of basophil differentiation were still induced when c-mpl-transfected UT7 cells received a specific megakaryocytic differentiation signal delivered by thrombopoietin (TPO). Surprisingly, we found that histamine synthesis did effectively occur in response to the growth factor. This activity was not associated with megakaryopoiesis since it was not detected in megakaryocytes generated from CD34(+) cells cultured in the presence of TPO. Comparing different c-mpl-transfected cell lines, we found that the amount of histamine generated in response to TPO correlated with their responsiveness to PMA, but not with their level of c-mpl expression, thus revealing an intrinsic basophil differentiation potential. Both PMA- and TPO-induced histamine synthesis was reduced by PKC and MEKs inhibitors, indicating that the induction occurred through a common signalling pathway.

Basophils↗
PubMed2001

Natural killer cell-dependent apoptosis of peripheral murine hematopoietic progenitor cells in response to Fas cross-linking: involvement of tumor necrosis factor-alpha.

Recently, a marked extramedullary myelopoiesis in Fas/CD95- or FasL/CD95L-deficient mice has been reported. In the present in vitro study, the mechanisms underlying Fas-induced apoptosis of normal peripheral colony-forming unit-C (CFU-C) progenitors in the spleen were analyzed. Surprisingly, it was found that clonogenic progenitors were protected from gammaIFN plus Fas-induced programmed cell death when Lin(+) cells were removed from cultured splenocytes. The cells that rendered CFU-C sensitive to the activation of the Fas pathway did not belong to the T or the myelocytic-monocytic lineage but comprised a non-B-cell subset expressing the activation marker B220. Among CD19(-) B220(+) splenocytes, nearly half were natural killer (NK) 1.1(+) cells whose in vivo depletion or deficiency in RAG2-gamma(c)(-/-) mice abrogated the effect of Fas cross-linking. NK cells exerted their accessory function, at least in part, through tumor necrosis factor-alpha (TNF-alpha), which they readily produced during pretreatment with the anti-Fas/CD95 monoclonal antibody and IFN-gamma and whose addition could compensate for the loss of sensitivity. In conclusion, this study provides evidence that peripheral clonogenic progenitors are not directly responsive to Fas cross-linking, even in the presence of IFN-gamma, but require NK cells as a source of TNF-alpha to make them susceptible to this death pathway.

Animals↗
PubMed2001

IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells.

NKT cells are a remarkably versatile population whose functional capacities are determined by cytokines present in their microenvironment. In this study, we provide evidence for a new immunoregulatory effect of the proinflammatory cytokine IL-18 on NKT cells. We found that IL-18, mainly known for its involvement in NK cell activation and in Th 1 immune responses, substantially enhanced IL-4 production as well as the percentage of IL-4(+) cells among NKT lymphocytes activated by their specific ligand alpha-galactosylceramide (alpha-GalCer). The effect of IL-18 on IL-4 production by activated NKT cells took place both in vivo and in vitro and was not affected by IL-12 which increased IFN-gamma secretion in the same conditions. We show that NKT cells are the main targets for IL-18-induced IL-4 production since it occurred neither in NKT-deficient mice nor after stimulation of Th2 lymphocytes. Finally, we provide evidence that the IL-4 promptly generated by NKT cells in response to IL-18 plus alpha-galactosylceramide in vivo can effectively contribute to the adaptive Th2 immune response by up-regulating the early activation marker CD69 on B cells. Our data support the notion that, in contrast to the exclusive IFN-gamma inducer IL-12, IL-18 acts in a more subtle manner as a costimulatory factor in both pro-Th1 and pro-Th2 responses depending on the nature of the stimulation and the target cells.

Adjuvants, Immunologic↗
PubMed2000

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes.

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.

Animals↗
PubMed1999

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement.

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.

Animals↗
PubMed1999

Increased fetal and extramedullary hematopoiesis in Fas-deficient C57BL/6-lpr/lpr mice.

In this study, we examined the consequences of Fas deficiency on hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in day-18 fetal livers of the lpr phenotype. Three weeks after birth, CFU-S increased in peripheral blood and spleen and colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later. Extramedullary myelopoiesis augmented progressively in Fas-deficient mice, reaching a maximum within 6 months. By then, mature and immature myeloid cells had infiltrated the spleen, the liver, and the peritoneal cavity. Similar changes occurred in C57BL/6-gld/gld mice, indicating that they resulted from Fas/FasL interactions. Medullary hematopoiesis was not significantly modified in adult mice of either strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon gamma, consistent with a regulatory function of Fas/FasL interactions in early progenitor cell development. These data provide evidence that Fas deficiency can affect hematopoiesis both during adult and fetal life and that these modifications occur independently from other pathologies associated with the lpr phenotype.

Animals↗
PubMed1999

TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells.

TGF-beta 1 is critical for differentiation of epithelial-associated dendritic Langerhans cells (LC). In accordance with the characteristics of in vivo LC, we show that LC obtained from human monocytes in vitro in the presence of TGF-beta 1 1) express almost exclusively intracellular class II Ags, low CD80, and no CD83 and CD86 Ags and 2) down-regulate TNF-RI (p55) and do not produce IL-10 after stimulation, in contrast to dermal dendritic cells and monocyte-derived dendritic cells. Surprisingly, while LC exhibit E-cadherin down-regulation upon exposure to TNF-alpha and IL-1, TGF-beta 1 prevents the final LC maturation in response to TNF-alpha, IL-1, and LPS with respect to Class II CD80, CD86, and CD83 Ag expression, loss of FITC-dextran uptake, production of IL-12, and Ag presentation. In sharp contrast, CD40 ligand cognate signal induces full maturation of LC and is not inhibited by TGF-beta 1. The presence of emigrated immature LCs in human reactive skin-draining lymph nodes provides in vivo evidence that LC migration and final maturation may be differentially regulated. Therefore, due to the effects of TGF-beta 1, inflammatory stimuli may not be sufficient to induce full maturation of LC, thus avoiding potentially harmful immune responses. We conclude that TGF-beta 1 appears to be responsible for both the acquisition of LC phenotype, cytokine production pattern, and prevention of noncognate maturation.

Antigens, CD↗
PubMed1999

Functional requirement of p23 and Hsp90 in telomerase complexes.

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

Adenosine Triphosphate↗
PubMed1999

Modulation of histidine decarboxylase activity and cytokine synthesis in human leukemic cell lines: relationship with basophilic and/or megakaryocytic differentiation.

In the present study, we show that UT7D1 cells, derived from the pluripotent cell line UT7, express high levels of histidine decarboxylase (HDC) mRNA spontaneously. These cells conserve the ability to differentiate into megakaryocytes upon stimulation with PMA, while greatly increasing their HDC activity. We provide evidence that enhanced HDC activity reflects the basophil rather than the megakaryocytic differentiation potential of UT7DI cells. Indeed, in addition to HDC mRNA, they express spontaneously several other mRNA coding for molecules present in basophils (FcepsilonRI, CCR3, IL-4Ralpha, IL-5Ralpha). Furthermore, the basophil antigen Bsp-1 is displayed on the surface of some UT7D1 cells in response to PMA concomitantly with increased histamine synthesis and mRNA expression of typical basophil-derived cytokines (IL-6, IL-4, and IL-13). Nevertheless, PMA cannot sustain the differentiation of this lineage, because mRNAs for basophil markers gradually diminish during long-term culture, whereas molecules associated with the megakaryocytic lineage remain prominent. In support of the notion that HDC activity is not related with megakaryopoiesis, we show that PMA-induced CD41 expression and PDGF transcription occurs in the K562 cells, though neither HDC mRNA nor any known basophil marker are expressed in these conditions. In contrast, all these markers are expressed in the basophilic leukemia cell line KU812F. Interestingly, the megakaryocytic cell line HEL produces also substantial amounts of histamine and expresses FcepsilonRI, thus revealing its basophil differentiation potential. HEL as well as KU812F need not be stimulated with PMA to react with Bsp-1 mAb, suggesting that they are more engaged into the basophil differentiation scheme than UT7D1. Other leukemic cell lines unrelated to the megakaryocyte or basophil lineage, like HL60 and U937 do neither synthesize histamine nor express basophil markers before or after PMA stimulation. To our knowledge, this is the first evidence for a factor-dependent cell line with megakaryocyte/basophil bipotentiality with which early stages of basophil commitment can be analyzed.

Basophils↗
PubMed1998

Transforming growth factor beta1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells.

Langerhans cells (LCs) are dendritic cells (DCs) that are present in the epidermis, bronchi, and mucosae. Although LCs originate in bone marrow, little is known about their lineage of origin. In this study, we demonstrate that in vitro LCs may originate from monocytes. Adult peripheral blood CD14+ monocytes differentiate into LCs (CD1a+, E-cadherin+, cutaneous lymphocyte-associated antigen+, Birbeck granules+, Lag+) in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4, and transforming growth factor beta1 (TGF-beta1). This process occurs with virtually no cell proliferation and is not impaired by 30 Gy irradiation. Selection of monocyte subpopulations is ruled out since monocyte-derived DCs can further differentiate into LCs. Our data suggest that in vivo LC differentiation may be induced peripherally, from a nonproliferating myeloid precursor, i.e., the monocyte, in response to a TGF-beta1-rich microenvironment, as found in the skin and epithelia. Therefore, the monocyte may represent a circulating precursor critical to the immune response in vivo.

Cell Differentiation↗
PubMed1998

IL-4-producing NK T cells are biased towards IFN-gamma production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells.

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the microenvironment, NK T lymphocytes can preferentially produce either IL-4 or IFN-gamma. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4-CD8-TCRalphabeta+ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-gamma in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-gamma is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-gamma production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-gamma secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant upregulation of the capacity of NK T cells to produce IFN-gamma after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.

Animals↗
PubMed1998

Fas cross-linking mimics the inhibitory effect of anti-CD3 on IL-3-induced histamine and cytokine production by murine myeloid spleen cell precursors.

In the present study we investigated the effect of anti-CD3 stimulation on IL-3-induced histamine, IL-6, and IL-4 synthesis by murine hematopoietic precursor cells. These activities were strikingly decreased in splenocytes from mice that had received a single intravenous injection of 10 microg of anti-CD3 monoclonal antibody (mAb) 24 hours previously. A similar inhibition occurred after 24-hour in vitro stimulation of normal spleen cells with 1 microg/mL of anti-CD3 mAb. In both situations the inhibitory effect depended on T cell activation in that treatment with F(ab')2 fragments of anti-CD3 did not diminish secretion of histamine and cytokines. Cross-linking of Fas antigen on spleen cells mimicked the action of anti-CD3, provided that interferon (IFN)-gamma was present during the incubation period. Substantial amounts of this cytokine were detected in spleen cell supernatants, which were able to replace recombinant IFN-gamma during Fas receptor cross-linking. This effect was entirely mediated by IFN-gamma, as assessed by its neutralization in the presence of anti-IFN-gamma mAbs. In contrast to splenocytes, bone marrow cells responded normally to IL-3 after in vivo or in vitro stimulation with anti-CD3. They were also not affected by combined treatment with anti-Fas mAb and IFN-gamma. Together, our data support the notion that the decrease in IL-3-induced histamine and IL-6 production by splenocytes pretreated with anti-CD3 is mediated, at least in part, by Fas/FasL interactions, suggesting that the activity of extramedullary myeloid precursor cells can be modulated by molecules involved in apoptosis.

Animals↗
PubMed1997

T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity.

We found that T cells recognizing viral superantigen (vSAG) can be subdivided into two distinct functional subsets based on IL-2R alpha (CD25) expression. CD4+Vbeta6+CD25- and CD4+Vbeta6+CD25+ T cells were sensitive to vSAG activation. When obtained from BALB/c(SW) mice, both subsets were infected and capable to induce the tolerance process when transferred into noninfected recipients. However, in contrast to CD4+Vbeta6+CD25- cells, which were gradually deleted in MMTV(SW)-infected mice, the pool of CD4+Vbeta6+CD25+ lymphocytes was constant even at the end of the deletion process, and maintained a limited reactivity to vSAG-induced activation. The constant number of Vbeta6+CD25+ observed in infected mice could not be explained by their rapid turnover (deletion and renewal), as their proliferative rate measured by BrdU incorporation was similar in infected and naive mice, as well as in virus-nonspecific (Vbeta8.2+) cells. Neither was the Vbeta6+CD25+ subset dependent on vSAG activation since it was also present in MMTV-free mice and was not generated from Vbeta6+CD25- cells upon in vivo vSAG stimulation. Vbeta6+CD25+ T cells constitutively expressed IL-4 and IL-10 mRNA. IL-10 has been shown to be associated with viral, bacterial, and parasitic infections. This permanent CD25+ subpopulation may play a role in the control of viral infection and tolerance induction via vSAG recognition and IL-10 production.

Animals↗
PubMed1997

Binding of histamine H3-receptor antagonists to hematopoietic progenitor cells. Evidence for a histamine transporter unrelated to histamine H3 receptors.

Hematopoietic progenitor cells can take up histamine or release IL-3-induced histamine through a bi-directional transport system that is blocked by H3-receptor antagonists. In the present study we demonstrate a correlation between the affinity of various H3-receptor antagonists and their potency as inhibitors of histamine uptake. All compounds that blocked histamine uptake also inhibited IL-3-induced histamine release. Yet, classical H3 receptors are not involved in this biological activity, since highly specific histamine H3-receptor agonists neither alter histamine uptake nor affect the release of endogenous histamine synthesized in response to IL-3. Furthermore, the inhibitory effect of H3-receptor antagonists on histamine uptake was not reversed by the agonists. Unlike H3-receptor antagonists, the agonists did not displace the binding of the labeled antagonist iodoproxyfan.

Animals↗
PubMed1997

Hematopoietic changes induced by a single injection of anti-CD3 monoclonal antibody into normal mice.

The present study evaluates hematopoietic modifications consecutive to in vivo treatment of mice with anti-CD3 monoclonal antibodies (mAb). The hamster mAb 145-2C11, administered in a single i.v. injection of 10 micrograms, induced the release of both interleukin 3 (IL-3) and GM-CSF into the circulation. IL-3 could be detected in the serum within 1 h, attained maximal levels after 4 h and had disappeared after 24 h. Three days later, treated mice exhibited a two- to threefold rise in blood neutrophil levels and increased spleen cell counts. Concomitantly, the incidence of nucleated erythroid cells in these spleens increased around 10-fold, relative to controls having received hamster Ig. At the same time point, clonogenic progenitor frequencies were 10-fold higher in spleens from treated mice than in those from control mice. Furthermore, the responsiveness of these splenocytes to IL-3, in terms of histamine synthesis, was enhanced. In contrast, bone marrow cell populations were only slightly affected by anti-CD3 injection. All hematopoietic changes required multivalent crosslinking of the mAb for induction, since F(ab')2 fragments lacked this activity. A return to normal occurred 7-10 days after treatment. Two i.v. injections of recombinant murine IL-3 together with recombinant murine GM-CSF on a single day had a less pronounced effect on progenitor cell frequencies in the spleen than treatment with anti-CD3. This difference is probably due to the amplification of growth factor-induced hematopoiesis by the interaction with other cytokines generated in response to anti-CD3.

Animals↗
PubMed1997

Requirement of IL-7 for IL-4-producing potential of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes.

IL-7 plays an important role in the growth and differentiation of T cells. We have previously reported that IL-7 induces preferential expansion of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes which express a skewed V beta repertoire and are potent IL-4 producers. In this report, we provide evidence that IL-1 in combination with granulocyte macrophage colony stimulating factor can also expand this population. Yet, these cells do not share the functional characteristics of those obtained in the presence of IL-7, i.e. cytotoxic activity and high IL-4 production. These functional capacities can be acquired by adding IL-7. In conclusion, our findings demonstrate that the capacity of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes to produce IL-4 as well as to kill target cells is IL-7 dependent.

Animals↗