Marrow allograft success inW/W v anemic mice: Relation to skin graft survival times

Genetically anemicW/W ^v mice were cured by marrow allografts from donors of 13 out of 18 tested strains that differed at non-H-2 histocompatibility alleles defined by skin or tumor grafting. They were also cured by donors from all four tested congenic lines whose antigenic differences had been defined by induction of serum antibodies. They were not cured acrossH-2 differences. Tail skin graft survival times on uncuredW/W ^v recipients were determined for all congenic lines used as marrow donors. The longest and shortest skin graft survival times predicted correctly marrow graft success or failure. NoW/W ^v mice were cured by marrow grafts from donors of the three congenic lines whose skin grafts were rejected in fewer than three weeks. Almost everyW/W ^v mouse grafted was cured by marrow grafts from donors of the 13 congenic lines whose skin grafts survived longest, from 11 to more than 25 weeks. Intermediate skin graft survival times failed to predict whether marrow grafts would succeed.W/W ^v mice were cured by marrow from four congenic lines with mean skin graft survival times of 4.2, 4.4, 8, and 9 weeks, while marrow grafts failed from other congenic lines with mean skin graft survival times of 3.3, 3.4, 4.8, and 8.7 weeks. The simplest explanation for these results is that the antigens specified by theH-2, H-3, H-4, H-25, andH-28 loci are strongly immunogenic on both marrow precursor cells and skin,H-17 andH-24 are strongly immunogenic on skin but not on marrow, andH-12 is strongly immunogenic on marrow precursor cells but less strongly on skin.     

Haematopoiesis In Mice Heterozygous For the W Trait: Defective Formation of Transient Endogenous Spleen Colonies

It has been determined that W/+ and W^v/+ heterozygous mice, as compared with normal +/+ homozygous littermates, form significantly lower numbers of transient 5-day endogenous spleen colonies in response to X-irradiation. This defect was evident for doses of irradiation between 2–6 Gy (200–600 rad) and was associated with a slightly increased radiosensitivity of the assayed precursor cells (TE-CFU) in W heterozygotic mice. Moreover, the defect was transplantable, i.e., intrinsic to the marrow cells and not to the microenvironment, and was not associated with a similar decrease in cells which form erythropoietic bursts in vitro (BFUe). This study provides a cellular basis for increased radiosensitivity of W/+ and W^v/+ mice and suggests that the ‘W’ mutation is semi-dominant, both with respect to the white spotting and TE-CFU formation.     

Effects of Alleles at the W Locus on Testicular Luteinizing Hormone Receptors in Adult Mice

Abstract

The autoregulation of testicular LH receptors was studied in W^x/W^v mice with germ cell aplasia and in normal (±/±) mice. To assess the effects of each individual allele, W^x/± and W^v/± mice were also examined. Basal testicular LH receptor concentration was higher in W^x/W^v mice than in all other genotypes, and higher in W^x/± than in ±/± mice. Twenty-four h after injection of 0.3 IU hCG/g bw, LH receptor concentration was decreased in ±/± and W^v/± mice, but not in W^x/W^v or W^x/± animals. Administration of hCG caused a significant increase in plasma testosterone levels in all genotypes. However, injection of the highest dose of hCG used (0.9 IU/g bw) caused a significantly greater elevation in plasma testosterone in W^x/W^v than in ±/± mice. Plasma gonadotropin levels were significantly higher in W^x/W^v mice than in all other genotypes. The present results indicate that the W^x allele is responsible for the changes in testicular function observed in W^x/W^v mice, and suggest that this allele may be involved in the genetic regulation of testicular LH receptors in the mouse.     

Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro

The proliferation and differentiation of hemopoietic cells from genetically anemic W^v/W^x,W/W^v, and W^v/W^v mice, and from nonanemic carrier W/+, W^b/+, and W^v/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method. Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice. Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis. The severity of the hemopoietic impairment was gene-dose dependent. The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis. The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in vitro were neither decreased in number nor impaired in proliferation and differentiation. Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells. The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in vitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line. Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series. W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation.     

Concentration of mast-cell progenitors in bone marrow,spleen, and blood of mice determined by limiting dilution analysis

When hematopoietic cells of congenic +/+ mice were injected into the skin of genetically mast-cell-depleted (WB × C57BL/6)F₁-W/W^v mice, mast cells appeared at the injection site. The donor origin of developing mast cells was confirmed by using granules of C57BL/6-bgl/bgl mice as a marker. When the number of injected cells was decreased, the proportion of injection sites at which mast cells did not appear increased according to the expected frequency of null response in a Poisson distribution. Therefore, such proportions were used to calculate the concentration of mast-cell precursors in the bone marrow, spleen, and peripheral blood. The relative concentration of mast-cell precursors in these tissues was similar to that of spleen-colony-forming cells. The present method seems useful as a semiquantitative in vivo assay for a population of progenitor cells which are committed to differentiate into mast cells.     

The cardiac repair benefits of inflammation do not persist: evidence from mast cell implantation

Multiple mechanisms contribute to progressive cardiac dysfunction after myocardial infarction (MI) and inflammation is an important mediator. Mast cells (MCs) trigger inflammation after MI by releasing bio‐active factors that contribute to healing. c‐Kit‐deficient (Kit^W/W‐v) mice have dysfunctional MCs and develop severe ventricular dilatation post‐MI. We explored the role of MCs in post‐MI repair. Mouse wild‐type (WT) and Kit^W/W‐v MCs were obtained from bone marrow (BM). MC effects on fibroblasts were examined in vitro by proliferation and gel contraction assays. MCs were implanted into infarcted mouse hearts and their effects were evaluated using molecular, cellular and cardiac functional analyses. In contrast to WT, Kit^W/W‐v MC transplantation into Kit^W/W‐v mice did not improve cardiac function or scar size post‐MI. Kit^W/W‐v MCs induced significantly reduced fibroblast proliferation and contraction compared to WT MCs. MC influence on fibroblast proliferation was Basic fibroblast growth factor (bFGF)‐dependent and MC‐induced fibroblast contractility functioned through transforming growth factor (TGF)‐β. WT MCs transiently rescue cardiac function early post‐MI, but the benefits of BM cell implantation lasted longer. MCs induced increased inflammation compared to the BM‐injected mice, with increased neutrophil infiltration and infarct tumour necrosis factor‐α (TNF‐α) concentration. This augmented inflammation was followed by increased angiogenesis and myofibroblast formation and reduced scar size at early time‐points. Similar to the functional data, these beneficial effects were transient, largely vanishing by day 28. Dysfunctional Kit^W/W‐v MCs were unable to rescue cardiac function post‐MI. WT MC implantation transiently enhanced angiogenesis and cardiac function. These data suggest that increased inflammation is beneficial to cardiac repair, but these effects are not persistent.     

A kinetic study of the genetic control of hemopoietic progenitor cells assayed in culture and in vivo

The kinetics of growth of bone marrow cells from normal or genetically anemic mice (Sl/Sl^d and W/W^v) were studied in irradiated normal and genetically anemic hosts. The parameters followed included total cellularity, the number of peroxidase positive cells, and the number of cells capable of forming colonies in vivo (CFU-S) or in culture (CFU-C). The results of these experiments demonstrate that W and Sl defects alter the growth of CFU-C and peroxidase-positive cells to a modest degree; that the defects are more obvious when studied in spleen rather than in bone marrow; and that there is no additivity of W and Sl defects. Nineteen irradiated recipients of marrow from W/W^v mice were studied after three to six months. Of these, 18 showed host-type erythrocytes, while in one mouse the erythrocytes had the size distribution of W/W^v cells. This finding indicated that occasionally genetically defective stem cells may repopulate irradiated hosts.     

Murine adult hematopoietic cells produce adult erythrocytes in fetal recipients

Bone marrow cells from normal adult mice were introduced by microinjection via the placenta into W/W^v genetically anemic fetuses of 11 days' gestation. After birth, erythrocytes were fractionated by fluorescence-activated cell sorting on the basis of antibody binding to a fetal-specific antigen (Ft). Lysates of Ft-positive, i.e., fetal, erythrocytes did not detectably contain hemoglobin of the donor type, as judged from electrophoresis of strain-specific hemoglobin variants. Thus, adult hematopoietic bone marrow cells did not resume fetal differentiation despite their post-transplant maturation in a fetal environment.     

Absence of heparin or heparine-like compounds in mast-cell-free tissues and animals

Three models were used for the analysis of heparin concentration and the presence of mast cells, namely different fetal and adult bovine tissues, mast-cell-deficient mice and athymic mice. It was observed that heparin and mast cells are present mainly in spleen and liver during fetal development and in skin, lung and ileum in adults. A good correlation between the concentration of heparin and the number of mast cells was observed in all tissues examined. No heparin was detected in animals that did not have mast cells, such as the WBB₆Fl W/W^v mice, again suggesting a correlation between mast cells and heparin. No differences in the other sulfated glycosaminoglycans were observed between the mast cell-deficient mice and the normal littermates and breeders. Studies in ‘nude’ mice have shown that the heparin concentration in different tissues is similar to normal strains.     

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE.We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1^R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1^R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1^R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1^R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1^R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1^R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.     

Analysis of Pleiotropism at the Dominant White-Spotting (W) Locus of the House Mouse: A Description of Ten New W Alleles

Characterization of the pleiotropic effects of ten new putative W locus mutations, nine co-isogenic and one highly congenic with the C57BL/6J strain, reveals a wide variety of influences upon pigmentation, blood formation and gametogenesis. None of the putative alleles, each of which is closely linked to Ph, a gene 0.1 cM from W, gave evidence of complementation with W³⁹, a new allele previously shown to be allelic to W^v. All W*/W³⁹ genotypes resulted in black-eyed-white anemics with reduced gametogenic activity.¹ Homozygotes for seven of these mutations are lethal during perinatal life; anemic embryos have been identified in litters produced by intercross matings involving each of these alleles.—Phenotypes of mice of several mutant genotypes provide exceptions to the frequent observation that a double dose of dominant W alleles (e.g., W/W^v or W/W) results in defects of corresponding severity in each of the three affected tissues. One viable homozygote has little or no defect in blood formation, and another appears to have normal fertility. The phenotypes of these homozygotes support the conclusion that the three tissue defects are not dependent on each other for their appearance and probably do not result from a single physiological disturbance during the development of the embryo.—Although homozygosity for members of this series results in a wide range of phenotypes, the absence of complementation of any allele with W³⁹, the close proximity of each mutant to Ph, and the fact that all alleles produce detectable (though sometimes marginal) defects in the same tissues affected by W and W^v, support the hypothesis that each new mutant gene is a W allele.     

Roles of mast cells in the pathogenesis of inflammatory myopathy

Introduction

In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases.

Methods

The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-Kit^W/Kit^Wv mice (W/W^v mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/W^v mice to CIM was also evaluated.

Results

The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/W^v mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8⁺ T cells and macrophages in the skeletal muscles of CIM decreased in W/W^v mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/W^v mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/W^v mice upon CIM induction.

Conclusion

Mast cells are involved in the pathogenesis of inflammatory myopathy.     

Histamine from Brain Resident MAST Cells Promotes Wakefulness and Modulates Behavioral States

Mast cell activation and degranulation can result in the release of various chemical mediators, such as histamine and cytokines, which significantly affect sleep. Mast cells also exist in the central nervous system (CNS). Since up to 50% of histamine contents in the brain are from brain mast cells, mediators from brain mast cells may significantly influence sleep and other behaviors. In this study, we examined potential involvement of brain mast cells in sleep/wake regulations, focusing especially on the histaminergic system, using mast cell deficient (W/W^v) mice. No significant difference was found in the basal amount of sleep/wake between W/W^v mice and their wild-type littermates (WT), although W/W^v mice showed increased EEG delta power and attenuated rebound response after sleep deprivation. Intracerebroventricular injection of compound 48/80, a histamine releaser from mast cells, significantly increased histamine levels in the ventricular region and enhanced wakefulness in WT mice, while it had no effect in W/W^v mice. Injection of H1 antagonists (triprolidine and mepyramine) significantly increased the amounts of slow-wave sleep in WT mice, but not in W/W^v mice. Most strikingly, the food-seeking behavior observed in WT mice during food deprivation was completely abolished in W/W^v mice. W/W^v mice also exhibited higher anxiety and depression levels compared to WT mice. Our findings suggest that histamine released from brain mast cells is wake-promoting, and emphasizes the physiological and pharmacological importance of brain mast cells in the regulation of sleep and fundamental neurobehavior.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

Survival of marrow allografts inW/W v anemic mice: Effect of disparity at theEa-2 locus

Erythrocyte producing tissues of genetically anemicW/W ^v mice were completely populated and the anemic mice were permanently cured by marrow cell grafts from donors that differed at theEa-2 locus. Circulating erythrocytes contained ≧ 80% donor Ea-2 antigen and ≧ 90% donor hemoglobin. Population occurred without immunosuppressive treatment. No sign of graft-versus-host reaction was observed, although donors were congenic with one parent strain of the F₁ hybrid recipients. When partially congenic donors carrying the T6 chromosome marker were used, the immune systems of curedW-anemic mice were populated to an appreciable extent by donor cells. The dependence of cure upon route of injection and the persistence of functioning donor type cells across the strong immunological barrier of the Ea-2 system are discussed.     

The ret oncogene can induce melanogenesis and melanocyte development in W/W mice

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in W^v/W^v mice crossed to one of the transgenic mouse lines. The pigmented hair of W^v/W^v mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neural tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in W^v mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.     

Precursor of mast cells fixed in the skin of mice

The presence and origin of mast-cell precursors fixed in the skin tissue of mice were investigated. Giant granules of beige (C57BL/6-bg^j/bg^j, Chediak-Higashi syndrome) mice were used to distinguish different populations of mast cells. Pieces of the skin were grafted from the intact WBB6F¹ (WB × C57BL/6)F¹?+/+ mice onto the back of the WBB6F¹?+/+ mice which had been irradiated and injected with bone marrow cells of C57BL/6-bg^j/bj^j mice (bg^j/bg^j ?+/+ chimeras). Although the number of mast cells in the skin grafts decreased after the transplantation, the mast-cell precursors circulating in the bloodstream of bg^j/bg^j ?+/+ chimeras (bg^j/bg^j type) did not seem to enter into the skin grafts, because most of mast cells were of +/+ type after the recovery of mast-cell number to pregrafting levels. As a considerable proportion of +/+-type mast cells was labeled with ³H-thymidine, the recovery of mast-cell number in the grafts was attributed to the proliferation and differentiation of +/+-type precursor cells fixed in the skin tissue of the donor. On the other hand, the skin of WBB6F¹-W/W^v mice seemed to be depleted of fixed precursors, because most of mast cells were of bg^j/bg^j type in skin grafted from WBB6F¹-W/W^v mice to bg^j/bg^j?+/+ chimeras. Since the fixed precursor cells which proliferate and differentiate into mast cells after skin grafting may be transferred to WBB6F¹-W/W^v mice by bone marrow transplantation, such precursor cells seem to have been derived from the bone marrow.     

Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis

W/W^v mice are deficient intissue mast cells, and mast cells cultured from these mice do notproliferate in response to the c-kit ligand, stem cell factor (SCF). Inthis paper, we report that mouse bone marrow cultured mast cellsderived from W/W^v mice do adhereto fibronectin in the presence of SCF and exhibit chemotaxis to SCF,and we explore this model for the understanding of c-kit-mediatedsignaling pathways. Both in vitro and in vivo (in intact cells)phosphorylation experiments demonstrated a low residual level ofW/W^v c-kit proteinphosphorylation. SCF-induced responses inW/W^v mast cells were abolished bythe tyrosine kinase inhibitor herbimycin A and by thephospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin butwere not affected by protein kinase C inhibitors. These observationsare consistent with the conclusions thatW^v c-kit initiates a signalingprocess that is PI 3-kinase dependent and that mutatedW^v c-kit retains the ability toinitiate mast cell adhesion and migration.

     

Skin graft rejection in mice repopulated with marrow of the skin donor type: A SKn gene in a congenic line

Genetically anemic W/W ^c mice and lethally irradiated wild-type mice were cured and populated by grafted marrow cells from donor mice of three congenic lines that differed at non-H-2 histocompatibility loci. Tail skin from mice of the same congenic lines was grafted 3–4 weeks later. In two cases, the recipients behaved as expected, no longer rejecting skin syngeneic with the marrow graft that had repopulated them. However, B6-H-24 ^c skin was rejected by WBB6F₁-W/W mice that were cured with B6-H-24 ^c marrow showing a mean survival time of 9.9 weeks. It was rejected somewhat faster, with a mean survival time of 5.9 weeks, by W/W mice cured with marrow from other types of donors. Results were more variable in lethally irradiated WBB6F₁-+/+ recipients of B6-H-24 ^c marrow, but they also rejected B6-H-24 ^c skin. Both types of recipients remained chimeras after the skin was rejected, showing more than 90% of the B6-H-24 ^c hemoglobin type. This is the first report of a Skn gene in a congenic line.     

Histamine Synthesis by Bone Marrow-derived Macrophages

Histamine may be involved in the regulation of hematopoiesis. However, it remained obscure what kind of cells are responsible for its synthesis in bone marrow (BM). In this study, a pure population of macrophages were raised from BM of W/W^v mice, which are genetically deficient in mast cells. The addition of Escherichia coli lipopolysaccharide (LPS) or murine recombinant granulocyte/macrophage colony-stimulating factor (mrGM-CSF) alone had essentially no effect on histamine synthesis. However, the latter rendered the cells responsive to LPS, leading to a marked increase in HDC-dependent histamine synthesis.