Akio Matsuzawa

Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes

To study the contribution of putative perforin-independent mechanism in the antigen-specific target destruction by cytotoxic T lymphocytes CD8+ CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-/-) embryonic stem cells into blastocysts of RAG-2(-/-) mice. When tested on normal concanavalin A blasts, these perforin-deficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas-mediated pathway in antigen-specific cytolysis.

Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle

Fas, also called APO‐1, belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits an apoptotic signal within the cell by binding to the Fas ligand. Fas has been implicated in the activation‐induced suicide of T cells and cytotoxic T cell activity in the immune system. Non‐immune cells such as those in liver, lung and ovary also express Fas, but its role in these cells remains unclear. Ovariectomy has been used to study homeostasis of female reproductive organs, which is regulated by sex hormones. Here we analyzed Fas function in the ovariectomy‐induced regression of mouse vaginal epithelial cells. Fas expression was detected in vagina and was elevated after ovariectomy. Fas‐deficient lpr and lpr(cg) mice did not exhibit ovariectomy‐induced regression of vaginal epithelia, whereas uterine regression induced by ovariectomy was not affected in these mice. The vaginas of lpr and lpr(cg) mice were in a persistent estrous stage with cornification of vaginal epithelia, as judged from the cell types in the vaginal fluid. Thus, Fas appears to be involved directly in the regression of vaginal epithelia induced by ovariectomy and during the estrous cycle, suggesting that the physiological role of this receptor extends beyond that exerted on immune cells. This is the first evidence of a role for Fas inducing physiological apoptosis in non‐immune cells.

CD95/Fas Signaling in T Lymphocytes Induces the Cell Cycle Control Protein p21cip-1/WAF-1, Which Promotes Apoptosis

Ligation of CD95 on T lymphocytes resulted in the up-regulation of a cell cycle control protein, p21cip-1/WAF-1, an inhibitor of cyclin-dependent kinases. This up-regulation was completely blocked by the cysteine protease inhibitor Z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), whereas DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde), a caspase 3 inhibitor, had no effect. In Faslpr-cg mice, a point mutation in the death domain of CD95 results in failure to recruit FADD (Fas-associated death domain), and in the present study this mutation prevented both CD95-mediated apoptosis and p21cip-1/WAF-1 induction. During apoptotic cell death due to irradiation, p21cip-1/WAF-1 is up-regulated by a p53-dependent pathway that responds to DNA damage. However, CD95-induced up-regulation of p21cip-1/WAF-1 in T cells was p53-independent. T cells deficient in p21cip-1/WAF-1 were less susceptible to CD95-induced apoptosis. We conclude that in T cells, ligation of CD95 and activation of caspases cause the induction of p21cip-1/WAF-1, which acts to promote cell death.

Immunohistochemical localization of myoepithelial cells and basement membrane in normal, benign and malignant human breast lesions

Distributions of actin and type IV collagen were investigated immunohistochemically as markers for myoepithelial cells and basement membranes. Carnoys and Methacarn-fixed, paraffin-embedded tissues from 103 human breast lesions from 103 patients were examined; 65 with carcinomas, 27 with mastopathies, 9 with fibroadenomas and 2 with phyllodes tumours. Fifty-five samples of the normal mammary gland tissue adjacent to tumours were also included for comparison. In normal breast and benign breast diseases, type IV collagen was identified around the mammary glandular cells and actin-positive cells were demonstrated to attach to basement membranes. In noninvasive carcinomas, type IV collagen was found as a continuous lining around a cell nest, while actin-positive cells were usually absent in ductal but quite numerous in lobular carcinomas. In invasive carcinomas, type IV collagen was fragmented or absent and actin-positive cells were very uncommon around the fragmentary basement membranes. These results suggest that the different distributions of myoepithelial cells and basement membrane material is useful in the differential diagnosis of surgical pathology of the breast.

Autoimmunity in Mice Bearing lprcs: A Novel Mutant Gene

A novel mutation at the lpr (lymphoproliferation)(Fas) locus, lprcg, that can complement gld (generalized lymphoproliferative disease) in induction of lymphadenopathy was discovered in CBA/K1Jms mice. The lpr and lprcg mutations are a defective allele of the Fas locus that encodes an apoptosis-mediating receptor. The former does not express the receptor and the latter expresses the point-mutated nonfunctional receptor. The gld locus is hypothesized to encode a ligand for the receptor and the gld mutation to have a defect that leads to incompetent expression of the ligand. The absence and non-functioning of the receptor in lpr/lpr and lprcg/lprcg mice, respectively, and the lack of the ligand in gld/gld mice may arrest apoptosis of lymphoid cells in the thymus, resulting in the same type of lymphadenopathy characterized by expansion of unusual CD4-CD8- (DN) T cells. Less severe lymphadenopathy induced by complementarity between lprcg and gld may be explained by less efficient apoptosis resulting from competition for the ligand between the functional and nonfunctional receptors. Phenotypically, lpr and lprcg are different from gld in the function at bone marrow (BM) and lymph node (LN) levels: lpr/lpr and lprcg/lprcg BM cause atrophy but gld/gld BM hyperplasia of wild-type (+/+) LNs, and lpr/lpr and lprcg/lprcg LNs but not gld/gld LNs allow the homing of lpr- and lprcg-induced DN T cells. Lymphadenopathy is equally prominent in CBA-lprcg/lprcg and MRL-lprcg/lprcg mice. Hypergammaglobulinemia, autoantibodies and circulating immune complexes are detectable at significant levels in both lprcg/lprcg mice but at higher levels on the MRL background. Pathological signs like glomerulonephritis and vasculitis are clinically unimportant in CBA-lprcg/lprcg but strikingly severe in MRL-lprcg/lprcg mice. Noticeably, clinically significant glomerulonephritis and vasculitis also develop with slight but significant serological aberrations in MRL-lprcg/+ heterozygotes. Graft-vs.-host disease-like syndrome in the lprcg/lprcg BM-->+/+ chimera is minimal on the CBA but as severe as life-threatening on the MRL background as in the MRL-lpr/lpr BM-->MRL(-)+/+ chimera. Thus, autoimmune diseases induced by the lpr, lprcg and gld genes are actually indistinguishable in the clinical, serological and pathological aspects on the same strain background and the disease caused by the interaction between lprcg and gld is less severe in all the aspects, consistent with the receptor-ligand theory. The lprcg/lprcg mice with different strain backgrounds together with lpr/lpr and gld/gld mice will serve as a powerful tool for elucidation of the mechanism of development of single-gene autoimmune diseases at a molecular biological level.

Eosinophilia, IgE production, and cytokine production by lung T cells in surface CD4-deficient mutant mice infected with Toxocara canis.

Mutant mice deficient in CD4+ T cells and their normal and heterozygous littermates were infected with Toxocara canis, and compared for eosinophilia, total and Toxocara‐specific immunoglobulin E (IgE) production, and in vitro cytokine production by lung cells. The numbers of eosinophils in the peripheral blood of normal and heterozygous mice peaked on days 10 and 21, although mutant mice showed eosinophilia with a peak on day 10. This indicates that the first peak on day 10 is CD4 independent and the second peak is CD4 dependent. Before infection, the levels of total IgE had no significant difference among the three groups of mice. Total and Toxocara‐specific IgE in all genotypes of mice increased after infection, and was the highest in normal mice and the lowest in mutant mice. In vitro production of interleukin (IL)‐5 and IL‐4 by total lung cells was the highest in normal mice and the lowest in mutant mice. CD4+ and CD4− CD8− T lymphocytes, but not CD8+ T lymphocytes produced IL‐5 and IL‐4 when incubated with anti‐CD3 monoclonal antibody (mAb) and lung‐adherent cells. These results indicated that IL‐5 and IL‐4 were produced mainly by CD4+ cells and partly by CD4− CD8− cells, but not by CD8+ cells. In addition, cytokine production by CD4+ cells was affected by the number of CD4 molecules on their surface.

Engraftment of human non-Hodgkin lymphomas in mice with severe combined immunodeficiency

Background. Malignant non‐Hodgkin lymphoma (NHL) is one of the most difficult neoplasms to transplant into nude mice. Mice with severe combined immunodeficiency (SCID) accept various human cancers much more efficiently than do nude mice. The authors investigated whether SCID mice could be used as convenient hosts in which to grow human NHL in vivo.

Hormone dependence and independence of mammary tumors in mice

Publisher Summary This chapter reveals that the hormone dependence of mammary tumors is considered a rare phenomenon in mice. Endocrine milieus with higher hormone levels—such as pregnancy, ectopic pituitary isografts, and continuous treatment with estrogen and progesterone—are prerequisites for the development of these tumors. A variant of mouse mammary tumor virus transmitted by these strains has an appreciable role in the induction of the tumors. Chemical carcinogens can also induce hormone-dependent mammary tumors. Therefore, certain combinations of carcinogenic agents and mouse strains may be favorable to the development of hormone-dependent mammary tumors, although it is impossible at present to predict what combination is best. The chapter describes the characteristics of prototypic hormone-dependent mouse mammary tumors. Hormone-dependent tumor cells survive long in a quiescent state in the endocrine milieu where the hormonal stimulation is insufficient to cause tumorous growth: they produce an interaction with the normal mammary epithelium and form the structures mimicking the mammary gland in the gland-free fat pad. Hormone-dependent mouse mammary tumors progress toward autonomy via less dependent stages with time.

Serological and histological characterization of the new mutant strain of lpr mice, CBA/KUms-lprcg/lprcg

CBA/KIJms‐lprcg/lprcg mice with a novel mutation producing systemic lymphoproliferation wereinvestigated for their serological and histological characteristics. The mutant mice showed elevatedlevels of serum immunoglobulin, C1q‐binding immune complexes and antibodies to nuclear antigenssuch as dsDNA and ssDNA and poly(ADP‐ribose). In contrast, histopathological lesions, e.g.glomerulonephritis, vasculitis or interstitial pneumonitis, were not revealed by histological andimmunofluorescent examinations, except for lymphocytic infiltration in various organs. These resultssuggest that this mutant mouse strain may provide a new animal model For autoimmunity. However, further investigations are required to clarify whether this strain is unique as compared with otherwell‐known lupus‐prone strains of mice with respect to serological and histological abnormalities andbecome to be a new model of systemic autoimmune disease.

Studies on the mechanism of dimethylnitrosamine-induced acute liver injury in mice

Male and female adult C3H- +/+, C3H-gld/gld.lpr/lpr (gld.lpr) and CBA-lprcg/lprcg (lprcg) mice were given a single i.p. dose of 30 mg/kg dimethylnitrosamine (DMN). Liver tissues were collected from mice killed 6, 12, 24 and 36 hrs post treatment, and the progression of the lesions was characterized morphologically and by the TUNEL method. DMN induced centrilobular hepatic injury accompanied with acute hemorrhage, and all mice died 36 to 48 hrs after the dosing. At 12 hrs after DMN administration, centrilobular hepatocytes revealed nuclear chromatin clumping. At 24 hrs, hepatocyte nuclei became fragmented to form apoptotic cells. Ultrastructurally, chromatin was condensed into a compact granular mass or crescent granular cap at the nuclear periphery. At 36 hrs, the number of apoptotic cells increased and they protruded into the sinusoid or were engulfed by the neighboring hepatocytes. A TUNEL-positive signal preceded the morphological changes and a few normal appearing centrilobular hepatocytes were positive 6 hrs post dosing. Endothelial damage was seen immunohistochemically at 24 hrs by disruption of type IV collagen and factor VIII-related antigen, resulting in massive hemorrhage in the centrilobular to mid zone. No inflammatory reactions were observed throughout the degeneration. The findings indicate that a single i.p. administration of DMN induced severe and fatal toxicity in liver tissues in mice which resembled human fulminant hepatitis. However, as gld-lpr and lprcg mice defective in apoptosis through the Fas system also showed similar severe liver damage, the Fas/Fas ligand system is not involved in DMN-induced liver apoptosis. No other organs or tissues were damaged, and the control mouse liver was intact.