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D Lo

Publications and source records attributed to D Lo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Th2-induced eotaxin expression and eosinophilia coexist with Th1 responses at the effector stage of lung inflammation.

The T cell-mediated lung inflammation that is associated with allergic asthma is characterized mainly by massive eosinophil infiltration, which induces airway injury and the subsequent late-phase reactivity. Because Th2 cells are often isolated from asthmatic subjects, these cells are postulated to play a role in asthma pathogenesis. We report that adoptively transferred, influenza hemagglutinin-specific Th1 and Th2 cells induced different patterns of chemokines leading to different types of cellular infiltration. Th2 cells were sufficient to induce dramatic Ag-dependent lung eosinophilia and eotaxin expression; by contrast, Th1 transfer primarily induced neutrophil recruitment with little eotaxin production. To determine whether Th1 cells show inhibitory effects on Th2 cell-mediated responses, Th1 and Th2 cells were cotransferred. Hemagglutinin-specific Th1 cells did not inhibit Ag-induced lung eosinophilia, nor did they inhibit eotaxin expression. Furthermore, influenza virus infection of the lung in mice receiving hemagglutinin-specific Th2 cells also induced eotaxin expression and eosinophilia that could not be inhibited by the cotransfer of Th1 cells. Our results show that Th2-mediated allergic lung inflammation coexists with the Th1-mediated responses that are stimulated by diverse forms of Ags.

Adoptive Transfer↗
PubMed1998

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Animals↗
PubMed1998

Thymic skewing of the CD4/CD8 ratio maps with the T-cell receptor alpha-chain locus.

The thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells or to homeostatic mechanisms. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset. The variable region of each TCR alpha chain (V alpha) studied to date is found to be overrepresented in either CD4+ or CD8+ cells, suggesting that each V alpha element can interact more favourably with either MHC class I or class II molecules. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules, and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V alpha element can be responsible for determining MHC specificity. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains and in human populations. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR alpha-chain complex, Tcra. This was confirmed by comparing Tcra(b) with Tcra(a) or Tcra(c) haplotypes in congenic mice. We propose that the array of V alpha genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

Animals↗
PubMed1998

Protection against diabetes by MHC heterozygosity and reversal by cyclophosphamide.

In type I diabetes in both rodents and humans, genetic susceptibility to disease is strongly linked to MHC class II alleles. In some cases, however, certain class II alleles provide resistance to disease. To examine this effect in a well-defined system, we studied double transgenic mice expressing influenza hemagglutinin (HA) on pancreatic islet beta cells and an HA-specific TCR on CD4 T cells. On a susceptible B10.D2 background, 70% of double transgenic mice develop an early-onset spontaneous autoimmune diabetes. MHC heterozygosity induced variable protection from diabetes, depending on the specific nonpermissive allele, but insulitis was invariably present. Autoreactive T cells retained the ability to induce diabetes because cyclophosphamide treatment induced diabetes in 81% of young MHC(d/b) transgenic mice, although the effect was diminished in older mice. Most importantly, treatment induced higher IFN-gamma/IL-4 ratios among CD4 T cells, suggesting a strong shift toward Th1 development, perhaps through direct effects on patterns of gene expression in CD4 T cells.

Animals↗
PubMed1998

The density of the class II MHC T cell receptor ligand influences IFN-gamma/IL-4 ratios in immune responses in vivo.

Activation of CD4+ T cells depends on T cell receptor recognition of MHC class II/peptide and on costimulation provided by CD28/B7. It has been shown that different levels of costimulation can influence T helper cell differentiation into Th1 versus Th2 phenotypes. Similar arguments have been made for different levels of peptide/MHC density on antigen-presenting cells, but to date supportive evidence has only come from in vitro studies. Here, using transgenic mice with reduced MHC class II expression on both B cells and dendritic cells, we demonstrate that T helper cell differentiation in vivo is also influenced by the density of expression of MHC class II. Although priming and expansion of antigen-specific T cells were normal in these mice, T cell responses were dominated by the Th1-associated cytokine IFN-gamma, with reduced levels of the Th2 cytokine IL-4 compared to controls. These results provide direct evidence that the efficiency of antigen presentation in vivo can determine effector cell phenotype.

Adjuvants, Immunologic↗
PubMed1998

Mature microglia resemble immature antigen-presenting cells.

Owing to the difficulties of isolating adequate numbers of microglia from adult tissue, much of our understanding of their function is based on characterizations of microglia that develop in mixed glial cultures. To learn more about the nature of these cells in vivo, we have compared the phenotypes of murine microglia isolated from adults, neonates, and from mixed glial cultures with spleen cells from fetuses, neonates, and adults. In the adult CNS, the only resident population of cells that express CD45, a protein tyrosine phosphatase, are the F4/80+ and FcR+ cells: the microglia. In contrast to all other differentiated cells of hemopoietic origin, microglial CD45 levels fail to increase from the neonatal period through adulthood. Rather, their levels are indistinguishable from the low levels found on a small population of embryonic day 16 liver cells. Conversely, we find that the F4/80 values of microglia are elevated as compared to splenic macrophages. Strikingly, microglia that develop in mixed glial cultures display a more activated phenotype, with low F4/80 values, weak MHC class II expression, and the appearance of a subset of cells positive for the dendritic cell marker, NLDC145. Additionally, CD45 values are elevated to a level intermediate between that of adult microglia and adult spleen, a level similar to that found on microglia activated in vivo. Consistent with this activated phenotype, indomethacin revealed the ability of mixed glial culture microglia to present a peptide antigen to naive T-cells expressing a defined T-cell receptor. Although adult microglia did express costimulatory molecules, B7.2, ICAM-1, and CD40, and could be induced to express MHC class II, they failed to present antigen in the same assay. Interestingly, these same cells could stimulate T-cell proliferation in a mixed lymphocyte reaction but not in an allogeneic specific manner. Taken together these data suggest that adult microglia remain in a relatively immature and unactivated state of differentiation as compared to other tissue macrophages.

Animals↗
PubMed1998

DNA immunization in relB-deficient mice discloses a role for dendritic cells in IgM-->IgG1 switch in vivo.

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM --> IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to drive IgG1 class switch readily after priming. Since GM-CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM --> IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF-kappaB/Rel family as these mice lack bone marrow-derived mature DC. RelB (-/-) mice and (-/-) bone marrow chimeras inoculated with DNA/GM-CSF did not produce IgG1 antibodies during the primary immune response. Since relB (-/-) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.

Animals↗
PubMed1998

Immunological memory after somatic transgene immunization is positively affected by priming with GM-CSF and does not require bone marrow-derived dendritic cells.

Inoculation of plasmid DNA is a promising vaccination approach but optimal regimes and ways to enhance immunogenicity remain to be established. Among natural immunological adjuvants, granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to increase the potency of immunization against tumor cells and protein antigens. Here we studied the effect of GM-CSF on memory responses against a 12-mer B cell epitope in mice primed with a single DNA inoculation. The results show that GM-CSF given at priming as a DNA/GM-CSF chimeric vaccine enhances the magnitude of the anamnestic response irrespective of the form of antigen used subsequently in the booster immunization. Using mice lacking bone marrow-derived dendritic cells we also determined that the enhancing effect is not strictly dependent on these cells. These results expand our understanding of the activity of GM-CSF in vivo as a modulator of the immune response including immunological memory.

Animals↗
PubMed1998

RelB is essential for the development of myeloid-related CD8alpha- dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells.

The transcription factor RelB had been shown to be important for dendritic cell (DC) development, but the type of DC involved was not clear. Here, we report that RelB mRNA is expressed strongly in CD8alpha- DEC-205- DC but only weakly in CD8alpha+ DEC-205+ DC. In addition, CD8alpha+ DEC-205+ DC are present and functional in RelB null mice, the DC deficiency being mainly in the CD8alpha- DEC-205- population. By constructing bone-marrow chimeric mice, we demonstrate that the partial deficiency in RelB null thymic DC is a secondary effect of disrupted thymic architecture. However, the deficiency in splenic CD8alpha- DEC-205- DC is a direct, stem cell intrinsic effect of the RelB mutation. Thus, RelB selectively regulates a myeloid-related DC lineage.

Animals↗
PubMed1997

Identification of a new mouse beta-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells.

Thymus-derived chemotactic agent 4 (TCA4), a new member of the beta-chemokine family, was cloned from a mouse thymic cDNA library. High levels of TCA4 mRNA are expressed in thymus; lower levels of message are found in spleen, heart, and kidney. Anti-TCA4 antibodies were used to localize sites of TCA4 expression within lymphoid tissues. In the thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained with anti-TCA4. TCA4 was also expressed as a meshlike network in splenic white pulp and in the medullary region of the lymph nodes. In addition, some lymph node and splenic blood vessels stained with anti-TCA4 antibodies. Rel B NFkappaB-deficient mice lack a transcription factor required for the generation of dendritic cells and the development of an organized thymic medulla. Rel B-deficient animals express very low levels of TCA4 in the thymus and little or no TCA4 in the periphery. At subnanomolar concentrations, TCA4 is a chemoattractant of mature T cells; the potential role of this novel chemokine in facilitating normal lymphocyte traffic is discussed. TCA4 is also a chemoattractant of cultured mesangial cells. Neutralizing anti-TCA4 mAb was used to demonstrate the specificity of TCA4-mediated cell migration. Finally, competitive binding studies with a SV40-transformed mouse mesangial cell line demonstrated that other murine beta-chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and thymus-derived chemotactic agent 3) do not compete for TCA4 binding.

Amino Acid Sequence↗
PubMed1997

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.

Animals↗
PubMed1997

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals, respectively, to thymic medullary stromal cells.

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3 epsilon-deficient mice where thymocyte maturation is arrested at the CD4- CD8- stage, few scattered 95+ and 29+ epithelial cells are found. When few mature thymocytes develop as in CD3- zeta/eta mice, expansion and organization of 95+ but not 29+ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29+ epithelial cells are absent and 95+ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1+ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95+ and 29+ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29+ medullary epithelial cells.

Animals↗
PubMed1997

Dendritic-like cells from relB mutant mice.

Mice deficient in the NF-kappa B transcription factor relB appear to have defects in the production of mature dendritic cells, as secondary lymphoid tissues are absent, and spleen cells show a significant loss of antigen presenting function. Moreover, the thymus appears to be impaired in negative selection, and immune responses in vivo are poor. Since dendritic cell precursors such as skin Langerhans cells appear to be normal, we sought information on the nature of the dendritic cell defect in these mice. Cultures of mutant bone marrow in the presence of GM-CSF revealed a delay in the accumulation of cells with dendritic cell features relative to controls; however, these cells were nearly as potent on a per cell basis as wild type cells in the stimulation of allogeneic mixed lymphocyte cultures. Similarly, skin Langerhans cells from mutant mice also showed significant ability to stimulate allogeneic T cells in culture. Since these findings cannot explain the defect in immune responses and the absence of secondary lymphoid tissues, we also looked at the ability of the relB mutant dendritic-like cells to form aggregates in vitro with naive syngeneic T cells. In this case, while wild type dendritic cells generated compact aggregates with T cells, relB mutant cells only formed irregular small aggregates. Thus, while relB mutant dendritic-like cells have some functions of mature dendritic cells, other functions are deficient. Understanding the role of relB in regulation of these functions should lead to a greater understanding of the molecular basis of dendritic cell development and function.

Animals↗
PubMed1997

Thymic stromal cell specialization and the T-cell receptor repertoire.

Ten years ago, we proposed a model for thymus function in which thymic epithelial cells are primarily responsible for imprinting major histocompatibility complex (MHC)-restricted specificity, and bone marrow-derived macrophages or dendritic cells are responsible for the induction of self-tolerance. Since then, transgenic and knockout models have allowed for a dissection of thymic stromal components in vivo, leading to a new understanding of their specialized functions. We have determined that with regard to class II-restricted CD4 T-cell development, two distinct subsets of thymic epithelium help shape the repertoire: Cortical epithelium appears solely responsible for positive selection, whereas a fucose-bearing subset of medullary epithelium is specialized for negative selection. This absolute separation of positive and negative selection into two distinct spatial and temporal compartments leads to a much simpler view of the process of repertoire selection. Finally, a novel view of the function of the thymic medulla is discussed.

Animals↗
PubMed1997

Increment of hFIX expression with endogenous intron 1 in vitro.

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence. hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i'IX, retroviral vector GINaCi'IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKGoi'IX, 151 ng/10(6) cells/24h; PA317/G1NaCi'IX, 308 ng/10(6) cells/24 h; C2C12/G1 NaCi'IX, 188 ng/10(5) cells/24 h; RSF/G1NaCi'IX, 1929 ng/10(5) cells/24 h; HSF/G1NaCi'IX, 1646 ng/10(6) cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production, a retroviral vector G1NaCi'IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/10(6) cells/24 h with 79% of bioactivity. PCR detection of HT/G1NaCi'IXR cells infected with PA317/G1NaCi'IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron1-carrying hFIX expression cassette can be used in directed gene transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.

Animals↗
PubMed1997

RelB regulation of chemokine expression modulates local inflammation.

The resolution of acute inflammation is incompletely understood but presumably requires the elimination of both inflammatory cells and production of inflammatory cytokines. In the case of recruited bone-marrow-derived inflammatory cells such as granulocytes and macrophages, their short life span helps eliminate these cells and the cytokines they produce. By contrast, resident permanent cells such as fibroblasts require other mechanisms to stop the production of chemokines generated in response to inflammatory triggers such as lipopolysaccharide. Here we demonstrate that RelB is an important regulator of chemokine expression in fibroblasts, thereby playing a key role in the resolution of acute inflammation. Activation of normal fibroblasts by lipopolysaccharide induced a transient production of chemokines, closely followed by induction of RelB expression. However, stimulated RelB-/- fibroblasts exhibited dramatic persistent induction of seven chemokines (RANTES, MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, JE/MCP-1, and KC/CINC). The persistent overexpression of chemokines correlated with increased NF- kappa B binding as well as with increased p50, p65/RelA, and I kappa B alpha expression. Transfection of RelB cDNA into RelB-deficient fibroblasts reversed the lipopolysaccharide-induced chemokine overexpression. In vivo, activated RelB-/- fibroblasts dramatically increased recruitment of granulocytes into tissues. In view of the apparent role of RelB in the resolution of acute inflammation in tissues and previous work showing a requirement for RelB in the initiation of immune responses through the differentiation of antigen-presenting cells, RelB may be an important factor regulating the transition from innate to adaptive immunity.

Animals↗
PubMed1996

Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex.

The normal development of T cells in the thymus requires both positive and negative selection. During positive selection, thymocytes mature only if their T-cell receptors react with some specificity to host major histocompatibility complex (MHC) and host peptides. During negative selection, thymocytes die if their T-cell receptors react with too high an affinity to the presenting cell, self MHC, and peptides to which they are exposed. These two processes are important for the development of the T-cell repertoire and the acquisition of self-tolerance, but their precise location and temporal relationship are not known. We have used the keratin 14 (K14) promoter to re-express a class II MHC antigen (I-Ab) in class II-negative mice. The transgenic I-A molecule is expressed only on thymic cortical epithelium; thymic medullary epithelium and bone-marrow-derived cells are I-A negative. CD4+ cells are positively selected in K14 mice, but clonal deletion does not ocur in K14 mice or in relB-negative mice, which lack a thymic medulla. The K14 CD4 cells are autoreactive, as they proliferate extensively to and specifically lyse I-Ab-positive target cells. These autoreactive cells make up 5% of the peripheral CD4 T cells, providing and estimate of the minimal frequency of positively selected cells that must subsequently undergo negative selection for self-tolerance to be preserved. Thus positive and negative selection occur in anatomically distinct sites.

Animals↗