Developmental immunology最新文献

During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cells that provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytes and adult thymocytes have different phenotypic and functional features. The identification of genes expressed in the thymus only during embryogenesis would help to understand the molecular basis underlying these characteristics. We used the mRNA differential display technique to compare gene expression between thymus and kidney from embryo (171/2 days) and adult mice. This technique is the method of choice for comparing gene expression because it is able to display rapidly and simultaneously the mRNA complement from several different types of cells. The major drawback of the method is that it leads to the cloning of many false positives and therefore needs a high throughput method to screen for the truly differentially expressed cDNAs. We combined advantages from previously described methods in order to develop a new version of the mRNA differential display technique that is fast, cheap, and reliable. Instead of oligo dT priming, we used random hexameres for the reverse transcription of total RNA and 10-mer primers for the amplification of internal parts of the cDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easily identify DNAs differentially amplified between embryo and adult tissues (embryo specific; E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryo and adult thymus (embryo thymus specific; E Thy 58.73) cDNA fragments. After reamplification, cloning, and sequencing of these DNA fragments, it appeared that in most cases, one band corresponded to a single DNA sequence. On a northern blot, each of these candidate genes recognized a transcript that is differentially expressed as expected. Thus, we report an optimized, reproducible, and fast mRNA differential display method that overcomes the usual problems met with the originally described technique or its reported modifications.

Interleukin-12 (IL-12) is an important cytokine for maintenance of normal systemic defense and bioregulation. The Japanese herbal medicine Sho-saiko-to (TJ-9) has been administered to 1.5 million Japanese patients with chronic liver diseases. TJ-9 is known to significantly suppress cancer development in the liver and has macrobiotic effects. In the present study, we examined the in vitro production of IL-12 by circulating mononuclear cells from liver cirrhosis patients and the effects of TJ-9 on IL-12 production. The monocyte/macrophage fraction and the lymphocyte fraction of peripheral blood were obtained from 11 HCV-positive liver cirrhosis patients and 12 healthy subjects. Interleukin-12 levels in the supernatants were measured using ELISA kits. The levels of IL-12 produced by the patients' fractions were significantly lower than those produced by healthy subjects (p < 0.01, p < 0.05). However, when TJ-9 was added to the cultures, the IL-12 production levels in both cell fractions increased approximately three fold, and the levels from the monocyte/macrophage fraction were almost the same as those from healthy subjects. This effect of TJ-9 was attributable to two of its seven herb components, that is, scutellaria root and glycyrrhiza root. One possible mechanism for the macrobiotic effects of TJ-9 on liver cirrhosis patients may be the improvement in IL-12 production.

We have reinvestigated an important issue in the amphibian immunology that has not been settled for years since the pioneer work of Triplett, concerning the necessity of being exposed to organ-specific antigens early in development. It was found that syngeneic Lenses were rejected by frogs, Xenopus laevis, that had been enucleated (eye removed) during early larval life. This rejection did not occur in intact frogs or in those enucleated in later larval or adult life. Whereas the splenocytes from intact frogs did not proliferate in response to a co-cultured syngeneic lens, those from frogs that had been enucleated at any of the larval stages, or even after metamorphosis, proliferated intensely. Both of these responses were shown to be thymus-dependent. In conclusion, it was demonstrated that the frog immune system rejected even syngeneic lenses by enucleation in early larval life and that it began to recognize the syngeneic lenses by lymphoid proliferation after enucleation, even in later life.

We have previously reported that reading-frame usage and functional diversification is developmentally regulated, with virtually all TCRB DJ mRNA transcripts using a single reading frame at 8 weeks of gestational age, tapering to 50% by adult life. We used the polymerase chain reaction to create genomic libraries of DJ rearrangements in the TCRB locus from thymuses at 7.7, 10, and 16 weeks of gestational age, and from adult thymuses. Clones were randomly picked and sequenced to determine junctional sequences and reading-frame utilization. The resulting data address the hypothesis that cells bearing genomic joints in reading frame one are preferentially selected during fetal life. This hypothesis predicts that reading-frame bias would also be observed among genomic DJ joints. Instead, we observed random utilization of the three possible D-region reading frames among genomic D1s1 ==> J1s1 joints during fetal life. Similar results were obtained at 7.7 weeks of gestational age in a second thymus in which both RNA and DNA were simultaneously isolated and used to create libraries of TCRBDJ transcripts or rearrangements. We conclude that reading-frame utilization is random among genomic D1s1-JB1s1 rearrangements and that the preferential usage of a single reading frame among mRNA transcripts of TCRB DJ transcripts is the result of preferential transcription of genomic TCRB DJ joints in a single reading frame, or that TCRB DJ transcripts have a longer half-life than transcripts in reading frames two or three.

Female reproduction is the only system subjected to well defined periodic changes. The final stage of the menstrual cycle in mammals is the maturation of the ovum and the preparation of the female organism to support fetal development fertilization. Once pregnancy occurs, both maternal and fetal sites emit regulatory signals to ensure embryo development and maternal protection against a graft versus host (GvH) reaction initiated by the semi-allogeneic fetus. We and others have previously shown that each day of fetal development in mice is characterized by different cytokine production, detected not only at the proximity of the feto-placental unit (decidua, uterus), but also in maternal lymphoid organs (spleen), as well as in the serum. In the present study, we concentrated on the menstrual cycle and the preimplantation stages of pregnancy and defined the levels of GM-CSF, IL-10, IL-6, and IL-3 in the murine uterus during anoestrus, proestrus, oestrus, and second and third day of gestation. We show by immunofluorescence and ELISA techniques that GM-CSF is maintained at high levels during anoestrus, proestus, oestrus, and the second day of pregnancy while dropping on the third day. IL-3 levels are found elevated during proestrus, second and third day of gestation, IL-6 increases essentially during proestrus, whereas the production of IL-10 was detected during oestrus and the early stages of pregnancy. Immunoperoxidase staining on frozen sections of uteri during the early gestational period localize GM-CSF and IL-3 production in the endometrium, IL-10 in the endometrium on the second day of pregnancy, and endometrium/myometrium on the third day. Low levels of IL-6 could be detected in the endometrium/epithelium on the second day and endometrium/myometrium on the third day of gestation. The role of IL-3, IL-10, and, to a lesser degree, IL-6 is fortified by the embryo itself, since these cytokines were found to be produced by blastocysts as well. These results demonstrate the existence of a specific distribution of lymphokines within the uterine tissue, the role of which is being discussed.

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.

Intercellular channels called gap junctions enable multicellular organisms to exchange information rapidly between cells. Though gap junctions are held to be ubiquitous in solid tissues, we have only recently found them in the lymphoid organs. Functional direct cell-cell communication has now been confirmed by us and other groups in bone marrow, thymus, and in secondary lymphoid tissues. What functions do they serve in the lymphoreticular system where, so far, only cytokines/growth factors and adhesion molecules have been considered as regulators? Here we show evidence for and refer to published work about functional direct cell-cell communication through gap junctions in germinal center reactions and make proposals for their role in the immune response. We found a large amount of the connexin43 (Cx43) gap junctions in the germinal centers of secondary lymphoid follicles. Ultrastructurally and immunohistologically, most of the junctions were detected on the processes of follicular dendritic cells (FDC) enveloping nondividing centrocytes in the light zone of germinal centers where B-cell selection is thought to take place. Further support for this finding came by revealing the Cx43 mRNA in situ at the same location as the protein. On antigen challenge, gap junctions appeared on the FDC as they formed meshworks in germinal centers. In order to find out which germinal center cells communicate directly, we separated FDC-rich, low-density, B-cell fractions from human tonsil. In culture, we injected single FDC with the low-molecular-weight fluorescent dye, Lucifer Yellow (M(r) 457 Da), which passed between adjacent FDC and sometimes from FDC to B cells. Based on these findings and their assigned functions in other tissues, gap junctions may contribute to germinal center reactions in the following ways: (1) they may regulate follicle pattern formation by controlling FDC growth, (2) they may be involved in FDC-B-cell signaling contributing to the final rescue of selected B cells from apoptosis, and (3) they may enable FDC to work as a functional syncytium providing a cellular internet for integrating germinal center events. Data supporting these interpretations are briefly discussed.

Tingible body macrophages (TBM), long thought simply as scavengers of apoptotic lymphocytes, are located in the unique microenvironment of germinal centers in close proximity to antigen-retaining follicular dendritic cells (FDC). Observations that TBM endocytose FDC-iccosomal (immune-complex coated bodies) antigen suggested that TBM might present this antigen and help regulate the germinal center reaction. To test for antigen presentation, the ovalbumin (OVA)-specific T(H) hybridoma, 3DO-54.8, which produces IL-2 on receiving effective presentation of OVA, were used as responders to OVA-bearing TBM. Results showed that OVA-bearing TBM failed to induce IL-2 production. Furthermore, addition of TBM to IL-2-inducing positive controls (B cells) not only failed to augment IL-2 production, but rather TBM significantly (55-90%) reduced B-cell induction of IL-2. We found that TBM were rich in prostaglandin by comparison with other nongerminal center lymph node macrophages and that addition of indomethacin to the cultures reversed the inhibitory effect of TBM. Depletion of TBM from enriched preparations, prior to addition to positive control cultures, also abrogated the inhibitory effect on IL-2 production. These data support the concept that TBM, within the unique microenvironment of germinal centers, may be specialized to downregulate the germinal center reaction.

Neonate organisms display an intrinsic disability to mount effective immune responses to infectious agents or conventional vaccines. Whereas low doses of antigens trigger a suboptimal response, higher doses are frequently associated with tolerance induction. We investigated the ability of a plasmid-expressing nucleoprotein of influenza virus to prime a specific cellular immune response when administered to newborn mice. We found that persistent exposure to antigen following plasmid inoculation of neonates leads to a vigorous priming of specific CTLs rather than tolerance induction. The CTLs were cross-reactive against multiple strains of type A influenza viruses and produced IFNgamma but no IL-4. The immunity triggered by plasmid inoculation of neonates was protective in terms of pulmonary virus clearance as well as survival rate following lethal challenge with influenza virus. Whereas the persistence of the plasmid at the site of injection was readily demonstrable in adult mice at 3 months after inoculation, mice immunized as newborns displayed no plasmid at 3 months and very little at 1 month after injection. Thus, DNA-based immunization of neonates may prove an effective and safe vaccination strategy for induction of cellular immunity against microbes that cause serious infectious diseases in the early period of life.

GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from "virgin" B cells in neonates. Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to "activation" of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and "constitutively" cytotoxic T cells.