Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene.

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a-/- mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a-/- mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/- mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a-/- and A1-a+/- animals. On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a-/-, A1-a+/-, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/-, and A1-a-/-. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice.

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin-associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP-1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development.

Requirement for CD8 beta chain in positive selection of CD8-lineage T cells.

CD8 is either an alpha alpha homodimer or an alpha beta heterodimer, although most peripheral CD8-lineage T cells express only the CD8 alpha beta heterodimer. The physiological function of CD8 beta was elucidated with mice that were chimeric for the homozygous disruption of the CD8 beta gene. The CD8 beta-1- T cells developed normally to CD4+CD8+ stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8 beta gene into CD8 beta-deficient T cells. Thus, CD8 beta is necessary for the maturation of CD8+ T cells.

Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.

The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.

Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.

bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

Mouse model of dermal fibrosis induced by one-time injection of bleomycin-poly(L-lactic acid) microspheres.

OBJECTIVE: Animal models are useful tools to study various aspects of human diseases. Bleomycin (BLM)-induced scleroderma mouse has been widely investigated as an animal model of scleroderma. Repeated injections of BLM, either daily or every other day, for 3-4 weeks are required to induce scleroderma in mice. Poly(L-lactic acid) (PLA) is a biodegradable, biocompatible and bioabsorbable device that has been widely investigated for controlled drug release. In this study, we fabricated BLM-containing PLA microspheres and subcutaneously injected them into C3H mice for only one time. METHODS: Treated skins were harvested at days 7 and 21. Then, histological examination and collagen content measurement assay were performed. The mRNA expression of alpha1(I) collagen (COL1A1), monocyte chemoattractant protein-1 (MCP-1), TGF-beta(1) and connective tissue growth factor (CTGF) were quantified by real-time PCR. RESULTS: Dermal fibrosis was histologically observed at day 7 after injection and remained present at day 21. Tissue responses against BLM-PLA microspheres alone were mild. Soluble collagen content and expression level of alpha1(I) collagen mRNA were significantly elevated at day 21. Expression levels of MCP-1 mRNA and TGF-beta(1) mRNA at day 7 and CTGF mRNA at day 21 were also elevated. CONCLUSION: The present study demonstrated for the first time that one-time injection of BLM-PLA microspheres can induce dermal fibrosis in C3H mice. BLM-PLA microspheres thus offer a labour-saving, simple and powerful tool to establish an animal model of BLM-induced dermal fibrosis.

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice.

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.

Distribution of MEL-14+ cells in various lymphoid tissues.

The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.

Sequential analysis of the thymocyte differentiation in fully allogeneic bone marrow chimera in mice. II. Further characterization of the CD4+ or CD8+ single positive thymocytes.

Differentiation of CD4+8- and CD4-8+ single-positive (SP) thymocytes in fully allogeneic bone marrow chimeras were investigated using multicolor cytometric analysis. The proportion of CD3+ cells in CD4+ SP population derived from donor mice considerably increased between day 12 and 14 after bone marrow transplantation (BMT), and gradually increased thereafter. The proportion of V beta 8+ cells in the CD3+CD4+ population remained constant (around 20%) at each period, suggesting that alpha and beta chains were used as TCR. The proportion of J11d+ cells in the CD4+ SP thymocytes transiently increased from day 12 to 14 and decreased thereafter, even though almost half of CD4+ SP cells were still dull J11d+ at day 35 after BMT. When CD8+ SP populations were analyzed, the proportion of CD3+ cells was very small until day 18. Thereafter, the proportion considerably increased and reached a maximum (83.2%) at day 21. The proportion of V beta 8+ cells in the CD3+ CD8+ SP population fell within range between 20 and 30%. However, before day 18, most of the V beta 8+ cells were dull positive, while after day 21 the majority were bright V beta 8+. Further, CD8+ SP cells at day 12, 14 and 18 were largely bright J11d+. After day 21, however, the proportion of bright J11d+ cells rapidly decreased. Similar results were obtained when the sequence of appearance of CD4+ and CD8+ SP cells was compared among bright CD3+, bright V beta 8+ or J11d- mature populations. The CD4+ SP cells regularly appeared earlier than CD8+ SP cells in the mature populations. These findings indicate that a considerable heterogeneity exists within both CD4+ and CD8+ SP populations and that the differentiation process for CD4+ SP cells precedes that for CD8+ SP cells.

Donor and recipient specific tolerance in cells from semi-allogeneic, H-2 subregion compatible or fully allogeneic bone marrow chimeras attributable to clonal deletion.

Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsen's splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.

Delayed clearance of zymosan-induced granuloma and depressed phagocytosis of macrophages with concomitant up-regulated kinase activities of Src-family in a human monocyte chemoattractant protein-1 transgenic mouse.

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

[Positive selection of a T cell repertoire during intrathymic differentiation].

In SWR mice most of thymocytes bearing a high density V beta chain of T cell antigen receptor (TCR), V beta 17a brightly positive cells, were biased to CD4+8- subpopulation, whereas such thymocytes from SJL mice resided in both CD4+8- and CD4-8+ subpopulations. To elucidate the mechanism underlying the apparent difference in the thymocyte population, CD4/CD8 phenotype of thymocytes bearing high V beta 17a was analyzed in bone marrow (BM) chimeras in which SWR or SJL mice were used as BM donors and various strains of mice as recipients. When SWR precursor cells developed in SJL or B10 thymus, the proportion of V beta 17a+CD4-8+ thymocytes was expanded to the level of that seen in normal SJL mice. By contrast, the proportion of SJL derived V beta 17a+ CD4-8+ thymocytes which had developed in SWR or DBA/1 thymus was reduced to the level of that in normal SWR mice. An intermediate proportion was observed when SWR precursor cells had developed in the thymus of B10.A(4R). It was revealed that H-2K molecules expressed on thymic epithelial cells is one of the determinants which influence the proportion of V beta 17a+CD4-8+ thymocytes. In addition, a hierarchy of selective pressure among the different H-2K haplotypes was present, such as Ks,b greater than Ks/q,k greater than Kq. Fine analysis of the H-2K molecules in which several kinds of H-2Kb mutant (bm) mice were used as recipients to prepare bone marrow chimeras demonstrated that substitutions of amino acids at a region on the beta-pleated floor of antigen recognition site reduced significantly the proportion of V beta 17a+CD4-8+ cells. According to the three-dimensional class I structure, TCR are unable to access directly to this region. Thus, the present finding suggests that the substitutions of amino acids at this site alter the shape and charge of peptide binding site of H-2K molecules and eventually influence the positive selection of the V beta 17a+ T cell repertoire during differentiation.

Compound heterozygosity in sibling patients with recessive dystrophic epidermolysis bullosa associated with a mild phenotype.

We describe two cases of a 3-year-old Japanese boy and his 1-year-old sister presenting recessive dystrophic epidermolysis bullosa; a relatively mild phenotype. Blistering and scarring were limited to the acral region, and some fingernails and toenails were lost. PCR-RFLP and DNA sequencing analyses revealed compound heterozygotes for a splice-site mutation (6573 +1GtoC) and a nonsense mutation (E2857X) in the type VII collagen gene (COL7A1). Both mutations caused a premature termination codon (PTC). The mutation E2857X was located behind the candidate cleavage site within the NC-2 domain required for the assembly of anchoring fibrils. This PTC position may explain their mild phenotype.

Sex difference in the development of fatty liver by orotic acid.

Effects of orotic acid on liver lipid accumulation and incorporation of methionine [methyl-14C] into liver phosphatidylcholine and protein, and into serum beta-lipoprotein were studied. Male and female rats of Wistar strain were fed a semisynthetic diet supplemented with 1 per cent orotic acid for 7 days. Feeding of orotic acid induced a marked fatty liver in female rats, but not in males. In female rats, radioactivity in liver phosphatidylcholine was significantly decreased by orotic acid, and that in liver protein was slightly decreased. In male rats, incorporation of methionine [methyl-14C] into liver phosphatidylcholine and protein was unchanged between the control and the rats fed orotic acid. Radioactivity in serum beta-lipoprotein was decreased to a greater extent in female rats than in males. These results suggest that sex difference in the development of fatty liver may be due to the difference in the effect of orotic acid on liver phosphatidylcholine biosynthesis.

Possible contribution of microchimerism to the pathogenesis of Sjögren's syndrome.

OBJECTIVES: Microchimerism of foetal cells occurs during most pregnancies. Two autoimmune diseases, systemic sclerosis (SSc) and Sjögren's syndrome (SS), have many clinical and pathological similarities to chronic graft-vs-host disease (GVHD). These findings suggest that anti-maternal graft-vs-host reaction by foetal cells may be involved in the pathogenesis of the diseases. To explore this hypothesis, we examined foetal DNA in peripheral blood of 59 women and in salivary glands from 28 women. METHODS: DNA extracted from peripheral blood and the affected minor salivary glands was analysed for the Y-chromosome-specific gene using a nested polymerase chain reaction (PCR) test. In the minor salivary gland specimens, the Y-chromosome-positive foetal cells were identified by in situ hybridization with a Y-chromosome-specific DNA probe. RESULTS: In peripheral blood, there was no significant difference between controls and patients with SSc or SS. In salivary glands, foetal DNA was detected in 11 of 20 women with SS but in only one of eight normal controls using PCR test. Additionally, foetal cells were clearly detected in three out of eight women with SS by the use of in situ hybridization. CONCLUSIONS: The identification of foetal cells in salivary glands suggests that anti-maternal GVHD may be involved in the development of SS.

H-2K molecules positively select V beta 17a+ CD4(-)8+ T cells in bone marrow and thymic chimeras.

Population size of V beta 17a brightly positive cells among CD4(-)8+ thymocytes was analyzed in thymic chimeras as well as bone marrow (BM) chimeras in which SWR/J mice were used as BM donors and various strains of mice including H-2Kb mutant (bm) mice as recipients. It was shown that the proportion of V beta 17a+ CD4(-)8+ thymocytes was determined by H-2K molecules expressed on thymic epithelial cells. The highest proportion was observed in Ks and Kb thymuses, the intermediate proportion in Ks/q and Kk, and the lowest in Kq thymuses. Fine analysis of the H-2Kbm molecules involved in the positive selection revealed that the region important to the selection was located on the beta-pleated floor of antigen recognition site. According to the three-dimensional class I structure, this site appears not to be directly accessible to the T cell antigen receptor. Thus, the present finding suggests that the substitutions of amino acids at this site alter the shape and charge of the peptide binding site and eventually influence the positive selection of the V beta 17a+ T cell repertoire during differentiation.

Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.

Mice carrying ablated coding regions of the bcl-2 alpha and bcl-2 beta transcripts have been made. bcl-2-/- mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+ T cells disappeared most quickly followed by CD4+ T cells, whereas B cells were least affected. bcl-2-/- lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice.

Naive CD28-deficient T cells can initiate but not sustain an in vitro antigen-specific immune response.

Naive T cells require an Ag-specific signal, as well as a costimulatory signal to mount a primary Ag-specific response. Because of their low precursor frequency, it has been difficult to study costimulatory requirements of these Ag-specific T cells. We have generated a CD28-deficient mouse that has been bred to a TCR transgenic (Tg) mouse to better study the function of CD28 during CD4+ T cell responses to Ag. In the absence of CD28, naive TCR Tg T cells responded vigorously to peptide, but responded poorly to mitogen activation. Comparison of activation-induced cell-surface molecules, including CD25, CD44, CD69, and CD71, showed no significant differences between CD28+ and CD28- TCR Tg T cells during the first 24 to 48 h after Ag stimulation. Despite relatively normal surface phenotype and normal proliferative response to Ag, CD28- T cells produced little IL-2, had a decreased sensitivity to lower Ag concentrations, and were unable to maintain their proliferative response. These results suggest that naive T cells are able to utilize other costimulatory signals to initiate a primary Ag-specific response, but require CD28 for optimal, sustained proliferation.

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells.

CD4+ helper T lymphocytes and CD8+ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4 CD8+ thymocytes into mature 'single-positive' T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4+ CD8+ thymocytes determines their fate to become either CD4+ or CD8+ T cells, the molecular signals that direct precursor thymocytes to become CD4+ and CD8+ T cells are unclear. By using ZAP-70 mutant thymus organ cultures in which T cell development is arrested at the CD4+ CD8+ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4+ and CD8+ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4+ CD8- TCR(high) cells, consistent with previous results. On the other hand, we find that the generation of CD4- CD8+ TCR(high) cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4+ and CD8+ T cells, and that biochemical treatment can manipulate the development and choice of CD4+ and CD8+ T cells.

Defective development of NK1.1+ T-cell antigen receptor alphabeta+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+ CD3- NK-like cells in the thymus.

Development of natural killer 1.1+ (NK1.1+) CD3+ (NK1.1+ T) cells was analyzed in zeta-associated protein 70 (ZAP-70) null ((-/-)) mice. Both NK1.1+ TCRalphabeta+ and NK1.1+ TCRgammadelta+ cell populations were absent in the thymus and spleen. By contrast, the number of NK1.1+ CD3- cells was increased in these tissues. The NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice had surface phenotypes in common with NK or NK1.1+ T cells. However, some of them were discordant either with NK cells or with NK1.1+ T cells. The NK1.1+ CD3- cells produced interferon-gamma upon stimulation with NK1.1 cross-linking in the presence of interleukin-2 and exhibited a substantial cytotoxicity against YAC-1 cells. Moreover, the generation of NK1.1+ T cells with invariant Valpha14Jalpha281 chains was induced from the NK1.1+ CD3- thymocytes following stimulation with phorbol myristate acetate and ionomycin in a neonatal thymic organ culture. An introduction of TCRalpha and beta transgenes to the ZAP-70(-/-) mice resulted in generation of an NK1.1+ TCRalphabeta(dim) population, whereas no substantial CD4+ CD8- or CD4- CD8+ population that expressed the introduced TCRalphabeta was generated in the mainstream T lineage. These findings demonstrate that ZAP-70 kinase is indispensable for the development of NK1.1+ T cells and that the unique NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice contain immediate precursors of NK1.1+ T cells.