Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.     

Expression of theta antigen on mouse thymocytes during the cell cycle

Summary Cells from normal mouse thymus were subdivided according to size and analysed for surface antigen expression, protein content and DNA expression and content. It could be shown, that the expression of theta-antigen followed protein accumulation in individual cells. Protein content on the other hand, was correlated with DNA synthesis even if it varied over a greater range.Only one subgroup of small non-DNA-synthesizing cells was calculated to have a higher density indicative of a more condensed state together with an increased surface antigen content. These cells might represent the G₀ state of the remnant proliferating population and most resembled the small cortical cells.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

IL-4 down-regulates the expression of J11d on murine B cells.

T cell-derived IL-4 has many effects on murine B cells, including the up-regulation of class II antigens and the induction of isotype switching. The development of memory B cells and the decreased expression of J11d antigens on these cells are influenced by T cells. In this report, we determined whether the decreased expression of J11d can also be mediated by T cell-derived lymphokines and, in particular, IL-4. We found that IL-4 can down-regulate the expression of J11d on both large and small B cells, that this effect becomes significant after 48 hr of culture and occurs at doses of IL-4 that are similar to those required to up-regulate murine class II MHC antigens encoded by I-region alpha genes (IA). Anti-IL-4 antibody completely blocks this effect but IFN-gamma does not. Other lymphokines (IL-1, IL-2, IL-3, IL-5, IL-6) neither induce a decrease in J11d nor alter the ability of IL-4 to down-regulate J11d expression. The decrease in J11d expression on B cells is not due to a preferential survival of cells expressing lower levels of J11d, although IL-4 has a more pronounced effect on these B cells. Finally, the down-regulation of J11d and up-regulation of IA by IL-4 occurs on all inducible B cells.     

Expression of the proliferation marker Ki-67 during early mouse development

In somatic tissues, the mouse Ki-67 protein (pKi-67) is expressed in proliferating cells only. Depending on the stage of the cell cycle, pKi-67 is associated with different nuclear domains: with euchromatin as part of the perichromosomal layer, with centromeric heterochromatin, and with the nucleolus. In gametes, sex-specific expression is evident. Mature MII oocytes contain pKi-67, whereas pKi-67 is not detectable in mature sperm. We investigated the re-establishment of the cell cycle-dependent distribution of pKi-67 during early mouse development. After fertilization, male and female pronuclei exhibited very little or no pKi-67, while polar bodies were pKi-67 positive. Towards the end of the first cell cycle, prophase chromosomes of male and female pronuclei simultaneously got decorated with pKi-67. In 2-cell embryos, the distribution pattern changed, presumably depending on the progress of development of the embryo, from a distribution all over the nucleus to a preferential location in the nucleolus precursor bodies (NPBs). From the 4-cell stage onwards, pKi-67 showed the regular nuclear relocations known from somatic tissues: during mitosis the protein was found covering the chromosome arms as a constituent of the perichromosomal layer, in early G1 it was distributed in the whole nucleus, and for the rest of the cell cycle it was associated with NPBs or with the nucleolus.     

Evidence for the existence of distinct heterogeneity among the peripheral CD4-CD8- T cells from MRL-lpr/lpr mice based on the expression of the J11d marker, activation requirements, and functional properties

Autoimmune-susceptible, MRL-lpr/lpr (lpr) mice develop a profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in peripheral lymphoid organs. The source and the mechanism of this abnormal accumulation of cells is still unknown. Recently, we reported that a significant number (approximately 35%) of the CD4-CD8- cells expressed J11d, a marker expressed by immature thymocytes but not by mature functional peripheral T cells. In the present study, we investigated the phenotype, growth requirements, and functional properties of purified J11d+ and J11d- subpopulations. Using the mAb, F23.1, which recognizes a TCR determinant encoded by the V beta 8 gene family, it was observed that approximately 30% of the J11d+ and J11d- DN cells expressed this determinant. Further studies on the thymus revealed that J11d+ DN cells from lpr thymus also contained F23.1+ cells (approximately 25%), whereas, similar cells from normal MRL(-)+/+mice were all F23.1-, consistent with earlier reports in other normal strains. Further phenotypic studies revealed that the peripheral J11d+ and J11d- cells from lpr mice were similar in expressing CD3, Ly-5 (B220), and Ly-24 (Pgp-1) determinants. When stimulated with phorbol myristic acetate (PMA) and recombinant IL-2 (rIL-2), only J11d- cells but not J11d+ cells responded by proliferation. However, in the presence of calcium ionophore (A23187) and PMA, both J11d+ and J11d- subpopulations proliferated by producing and responding to endogenous IL-2 but not IL-4. The lymph node T cells from 1-month-old MRL-lpr/lpr mice responded strongly when stimulated with PMA + rIL-4 or PMA + rIL-6. In contrast both J11d+ and J11d- subpopulations failed to respond when similarly stimulated. The J11d+ but not J11d- cells demonstrated spontaneous cytotoxic activity against the NK-sensitive YAC-1 tumor targets. The J11d- cells did not exhibit cytotoxic potential in spite of culture with PMA + rIL-2. Even after repeated culture in vitro with PMA + A23187 or PMA + rIL-2, both J11d+ and J11d- subpopulations failed to express the mature phenotype bearing CD4 and/or CD8 antigens. The present study demonstrates the expansion of unique J11d+, alpha beta-TCR+, DN T cells with cytotoxic potential in lpr mice and further suggests the existence of phenotypic and functional heterogeneity among the abnormal lpr DN cells.     

Expression of CD1d2 on thymocytes is not sufficient for the development of NK T cells in CD1d1-deficient mice

CD1 is an MHC class I-like molecule that has been conserved throughout mammalian evolution. Unlike MHC class I molecules, CD1 can present unique nonprotein antigens to T cells. The murine CD1 locus contains two highly homologous genes, CD1d1 and CD1d2. CD1d1 is essential for the development of a major subset of NK T cells that promptly secrete IL-4 following activation. However, the function of CD1d2 has not yet been demonstrated. In the present study, we examined the expression of CD1d2 in CD1d1-deficient (CD1d1 degrees) mice with the anti-CD1 Ab 3H3. Unlike CD1d1, which is expressed by all lymphocytes, CD1d2 can be detected only on the surface of thymocytes. To determine whether CD1d2 can select a unique subset of NK T cells, we compared the remnant population of NK T cells in CD1d1 degrees and CD1d1, CD1d2-double deficient (CD1d1 degrees CD1d2 degrees) mice. No significant difference in the number of NK T cells and cytokine secretion capacity can be detected between CD1d1 degrees and CD1d1 degrees CD1d2 degrees mice, indicating that CD1d2 cannot substitute for CD1d1 in NK T cell development. The inability of CD1d2 to select NK T cells is not due to the structural constraints of CD1d2 since CD1d2-transfected cells can be recognized by both NK T cell hybridomas and freshly isolated NK T cells. Given the structural similarities, it is possible that the low levels of surface expression and limited tissue distribution of CD1d2 may prevent it from functioning in the selection and expansion of NK T cells.     

Receptors for thymosin alpha 1 on mouse thymocytes

Thymosin alpha 1 is able to act in vitro to stimulate T-cell precursors and to induce surface markers. The initial mechanism by which alpha 1 activates T cells could be the binding of alpha 1 to cell membranes. Using a specific anti-alpha 1 antibody and an indirect immunofluorescence procedure it was found that thymosin alpha 1 binds to the surface of a large portion of murine lymphocytes. Furthermore, thymocytes have been fractionated into immature and mature subpopulations by using the peanut agglutinin (PNA) technique. It was found that PNA+, immature cells showed specific receptors for alpha 1 on the cell membrane.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

Expression of T cell antigen receptor beta-chains on subsets of mouse thymocytes. Analysis by three-color flow cytometry

The expression on adult mouse thymocytes of a T cell antigen receptor beta-chain epitope, recognized by the antibody F23.1, has been studied by three-color flow cytometry. Low density F23.1 staining was found mainly on CD4+8+ thymocytes. High density staining was mainly on CD4+8- and CD4-8+ cells. Variable proportions of CD4-8- cells were also F23.1+. Among CD4-8+ cells, F23.1 was expressed only on the J11d- subset with mature T cell function. We conclude that many subpopulations of thymocytes express antigen receptors and are candidates for the population subject to thymic selection, but at present no single subpopulation makes a convincing claim.     

Genetic mapping of the mouse ferritin light chain gene and 11 pseudogenes on 11 mouse chromosomes

We typed the progeny of two sets of genetic crosses to determine the map locations for loci containing sequences related to the ferritin light chain (Ft11) gene. Twelve loci were positioned on 11 different chromosomes. One of these genes mapped to a position on Chr 7 predicted to contain the expressed gene on the basis of the previously determined position of the human homolog on 19q13.3-q13.4. Received: 23 July 1997 / Accepted: 20 September 1997     

Expression pattern and functional analysis of mouse Stam2 in the olfactory system

Gene trap mutant mice Stam(gt1Gaj) were investigated in order to elucidate in vivo function of Stam2 (signal transducing adaptor molecule 2) gene, which was in vitro implicated in sorting cargo marked by monoubiquitination toward degradation in the lysosomes. The expression analysis showed high Stam2 expression in the brain including the regions related to olfaction, and in the olfactory epithelium, but not in the respiratory part of nasal mucosa. To test mouse olfaction, ability to find chocolate hidden under the sawdust in the cage was examined. When food was given ad libitum before trials, mutants needed more time and failed more frequently to find the chocolate. In contrast, when the mice were fasted overnight before trial, there were no differences between mutants and wild type mice. No changes in morphology of olfactory mucosa were observed. The obtained results showed the existence of phenotype differences between mutants and wild type mice. However, different results of two approaches aimed to test olfaction, with and without food deprivation, currently do not enable to assign the particular function of Stam2 to olfaction. This emphasizes how slight modification of experimental setup in behavioural testing can cause important differences on the obtained results.     

Expression and localization of SerpinB11 in mouse uteri during peri-implantation and the estrous cycle

Serine protease inhibitor (Serpin) B11 has been identified as a novel serine protease inhibitor but the biological functions of SerpinB11 in female reproduction are unknown. Therefore, we investigate the spatiotemporal expression and regulation of SerpinB11 during the peri-implantation period. SerpinB11 mRNA and protein were detected in the uteri of pregnant mice on days 1–8 (day 1?=?presence of a vaginal plug). SerpinB11 protein was localized in the embryonic implantation site on day 5 when embryo implantation occurred and was also strongly expressed in the primary decidual zone on day 6 and secondary decidual zone on days 7 and 8. The expression of SerpinB11 was induced by the activated blastocyst (based on patterns of expression during pseudopregnancy and delayed implantation) and by artificially induced decidualization. Moreover, expression of SerpinB11 was regulated by estradiol and progesterone in ovariectomized mice. The results were further supported by data from the estrous cycle. Thus, SerpinB11 is probably involved in embryo implantation and decidualization.