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.

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.

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.

Deficiency of phospholipase C-gamma1 impairs renal development and hematopoiesis.

Phospholipase C-gamma1 (PLC-gamma1) is involved in a variety of intracellular signaling via many growth factor receptors and T-cell receptor. To explore the role of PLC-gamma1 in vivo, we generated the PLC-gamma1-deficient (plc-gamma1(-/-)) mice, which died of growth retardation at embryonic day 8.5-9.5 in utero. Therefore, we examined plc-gamma1(-/-) chimeric mice generated with plc-gamma1(-/-) embryonic stem (ES) cells for further study. Pathologically, plc-gamma1(-/-) chimeras showed multicystic kidney due to severe renal dysplasia and renal tube dilation. Flow cytometric analysis and glucose phosphate isomerase assay revealed very few hematopoietic cells derived from the plc-gamma1(-/-) ES cells in the mutant chimeras. However, differentiation of plc-gamma1(-/-) ES cells into erythrocytes and monocytes/macrophages in vitro was observed to a lesser extent compared with control wild-type ES cells. These data suggest that PLC-gamma1 plays an essential role in the renal development and hematopoiesis in vivo.

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.

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.

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.

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.

Multiple gene duplication and expression of mouse bcl-2-related genes, A1.

Here we report the genomic cloning and characterization of the murine A1 genes, which belong to the bcl-2 gene family. Southern analysis indicated the existence of at least four A1 genes in the murine genome and four different A1 genes, designated A1-a, -b, -c and -d, were cloned from the murine genomic library. The A1-a, -b and -d genes consisted of two exons, whereas the A1-c gene contained 1 bp insertion in the coding region which may result in an aberrant and truncated protein by frame-shift. With the exception of A1-c, the coding regions among A1 genes are highly conserved at >97% at the nucleotide level and at >96% at the amino acid level. A1-a, -b and -d genes appeared to be expressed specifically in organs containing many neutrophils. In neutrophils, A1-a, -b and -d transcripts were detected at a comparable level. Our data suggest that the multiple A1 genes in mice were generated by gene duplication and each of them may function as anti-apoptotic molecules in neutrophils.

Restoration of thymocyte development and function in zap-70-/- mice by the Syk protein tyrosine kinase.

The Syk family of protein tyrosine kinases, consisting of ZAP-70 and Syk, associate with the pre- and alphabeta T cell antigen receptors (TCRs) and undergo tyrosine phosphorylation and activation following receptor engagement. Thymocyte development in zap-70-/- mice is blocked at the CD4+CD8+ TCR(lo) stage. The presence of Syk in the thymus has raised the possibility that Syk may be able to mediate TCR function. To determine if Syk can play a role in thymocyte development, we generated zap-70-/- mice expressing a human syk cDNA. Syk expression restored both thymocyte development and function. In addition, Syk function required the CD45 transmembrane protein tyrosine phosphatase. Hence, ZAP-70 and Syk can play overlapping functions and exhibit similar regulatory mechanisms in mediating alphabeta T cell development.

Interaction of pigeon cytochrome c-(43-58) peptide analogs with either T cell antigen receptor or I-Ab molecule.

We determined that a pigeon cytochrome c-derived peptide, p43-58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43-58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43-58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vbeta usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR beta chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR beta chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling.

An early stage in thymocyte development, after rearrangement of the beta chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4(-)CD8(-) double negative (DN) cells to CD4(+)CD8(+) double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo beta chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development.

Development of immature CD4+ CD8+ thymocytes into functionally mature CD4+ and CD8+ T cells is driven by selection events that require signals transduced through the T cell antigen receptor (TCR). Transduction of TCR signals in the thymus involves tyrosine phosphorylation of the protein tyrosine kinase ZAP-70 by p56(lck) and results in induction of ZAP-70 enzymatic activity. We have identified a novel, spontaneously arising point mutation within a highly conserved motif (DLAARN) in the kinase domain of murine ZAP-70 that uncouples tyrosine phosphorylation of ZAP-70 from induction of ZAP-70 kinase activity. Mice homozygous for this mutation are devoid of mature T cells because thymocyte development is arrested at the CD4+ CD8+ stage of differentiation. The developmental arrest is due to the inability of CD4+ CD8+ thymocytes to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules.

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.

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.

Functional significance of the Fas molecule in naive lymphocytes.

The Fas molecule mediates apoptotic signal in many cell types. Mouse mutations (lpr, lprcg, gld), which impair the function of Fas, cause spontaneous autoimmune disease. We generated Fas-deficient (Fas-/-) mice by homologous recombination. In embryonic stem cells Fas-/- mice developed lpr-like disease, confirming that the abnormality of Fas is causal in the lpr phenotype. We also made Fas-/- chimeric mice composed of a mixture of Fas+/+ and Fas-/- cells. The chimeric mice also showed the lpr phenotype. In Fas-/-, chimeric mice, the Fas-deficient population expanded progressively among mature T and B lymphocytes. The expansion of Fas-deficient lymphocytes occurred at the naive, pre-primed, lymphocyte stage. These results suggest that the Fas molecule functions not only after antigenic stimulation, as previously hypothesized, but also at the naive lymphocyte stage.

Essential role for ZAP-70 in both positive and negative selection of thymocytes.

During thymic development, T cells that can recognize foreign antigen in association with self major histocompatibility complex (MHC) are selected for survival (positive selection) and autoreactive T cells are eliminated (negative selection). Both of these selective events are mediated by interaction between the T-cell receptor (TCR) and the peptide-MHC complex. But the signalling pathways that lead to cell survival or to cell death are still unclear. ZAP-70 is a protein tyrosine kinase (PTK) that is associated with the TCR signalling subunits (CD3 and zeta) and is expressed in T cells and natural killer cells. It has been shown that ZAP-70 plays a crucial role in T-cell activation and development. Here we show that mice lacking ZAP-70 had neither CD4 nor CD8 single-positive T cells, but human ZAP-70 reconstituted both CD4 and CD8 single-positive populations. Moreover, ZAP-70-/- thymocytes were not deleted by peptide antigens. Natural killer cell function was intact in the absence of ZAP-70. These data suggest that ZAP-70 is a central signalling molecule during thymic selection for CD4 and CD8 lineage.

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.