Shinji Sunaga

Defective B-1 Cell Development and Impaired Immunity against Angiostrongylus cantonensis in IL-5Rα-Deficient Mice

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Murine interleukin 4 transgenic heart allograft survival prolonged with down-regulation of the TH1 cytokine mRNA in grafts

BACKGROUND Data supporting the differential activation of T helper (Th) 2 cells in transplantation acceptance/tolerance in rodents have been presented by several investigators. However, the differential activation of Th2 cells may be simply the result of allograft acceptance/tolerance induction instead of a contribution to the maintenance of grafts. METHODS Therefore, we established interleukin (IL)-4 transgenic mice under the control of a cardiac alpha-myosin heavy chain promotor and transplanted IL-4-expressing heart allografts into unmodified recipients to determine the actual contribution of the Th2 bias to allograft acceptance. RESULTS Among 16 newborn C57BL/6J (B6) mice, three were positive for the IL-4 transgene. Serum IL-4 levels of transgenic versus control B6xC3H F1 mice were not statistically different. Reverse-transcriptase polymerase chain reaction showed that the transgenic B6xC3H F1 mice expressed IL-4 mRNA in the heart and in the lung, whereas control mice did not express IL-4 in any organ. IL-4 mRNA expression in the transgenic but not in the control heart was also confirmed by RNAse protection assay and fluorescence in situ hybridization. The survival of IL-4 transgenic B6xC3H heart grafts heterotopically placed in C3H recipients was prolonged compared with that of nontransgenic grafts (19.0+/-9.1 vs. 6.8+/-2.2 days, P=0.003). Interferon-gamma mRNA expression in IL-4 transgenic heart grafts on the fifth posttransplant day as assessed by Northern blotting was suppressed compared with that in control grafts. Reverse-transcriptase polymerase chain reaction analysis showed that IL-2 mRNA was suppressed in the IL-4 transgenic grafts compared with that in control grafts, while IL-4 mRNA was observed only in IL-4 transgenic grafts. IL-10 mRNA was detected at similar levels in both transgenic and control grafts. CONCLUSIONS A Th2 bias may contribute to allograft acceptance in part by inducing the down-regulation of Th1-cytokine mRNAs, but it may not be sufficient to induce indefinite graft survival.

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.

The bacteriophage P1 Cre/loxP site‐specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP‐flanked DNA sequences in a gene‐targeted locus in fertilized eggs. We replaced a part of the T‐cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP‐flanked neogene in mouse embryonic stem (ES) cells by gene‐targeting technique. The resulting ES cell clones containing the mutant allele (VγLNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild‐type female mice. One hundred and seventy‐six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS‐Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre‐mediated recombination, resulting in a deletion of the loxP‐flanked sequences (VγΔ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the VγΔ locus to their offspring. The other 8 pups carried only wild‐type alleles. There were no pups carrying the unrecombined VγLNL locus. Thus, the frequency of Cre‐mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS‐Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the VγLNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997.

Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor β

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, we generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR β in myeloid cells. In these Tg mice the transgene is expected to suppress the function of heterodimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5‐fluorouracil (5‐FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5‐FU or granulocyte‐colony stimulating factor (G‐CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G‐CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.

Case Report: Cytokine-Induced Hypoalbuminemia in a Patient with Hemophagocytic Syndrome (Direct in Vitro Evidence for the Role of Tumor Necrosis Factor-α)

Hemophagocytic syndrome (HPS) is a rare, potentially lethal hematological disorde r, characterized by in® ltration of hemophagocytic histiocyte s into various tissues and organs. It can take a varie ty of clinical forms such as familial erythrophagocytic lymphohistiocytosis (FEL), virus-assoc iated hemophagocytic syndrome (VAHS), histiocytic medullary reticulosis, malignant histiocytosis, or malignancy-assoc iated hemophagocyti c syndrome (1± 6). Common clinical manife stations include high fever, weight loss, pancytopenia, hepatosplenomegaly, jaundice , and disseminated intravascular coagulopathy (1± 6). Hypoalbuminemia has also been observed frequently in HPS, although its pathogenesis has not been fully elucidated (5). Cytokine s are multifunctional local peptide mediators released from reticuloendothe lial cells including the macrophage /monocyte /histiocyte system (7). In the live r, in ̄ ammatory cytokine s, ie , tumor necrosis factora (TNFa ), interleukins (IL-1 and IL-6), together with interferong (IFNg ), have been shown in both in vitro and in vivo studie s to enhance the synthesis of acute -phase proteins, but to suppress that of albumin (8 ± 10) . Clinically, elevated plasma concentrations of these cytokine s have been detected in patients with septic shock, cirrhosis, or alcoholic live r disease, disorders that are frequently accompanie d by hypoalbum inemia (11± 15) . It has therefore been postulated that these cytokine s may be involve d in the pathoge nesis of hypoalbumine mia unde r such conditions (11± 15) . In HPS, an increase in the serum leve ls of these cytokine s has also been reported, and this is known to be corre lated positive ly with disease activity and negative ly with prognosis (2, 3). It is therefore reasonable to speculate that these cytokine s play a role in the pathoge nesis of hypoalbum inemia in patients with HPS, although the relationships between overproduction of cytokine s and hypoalbum inemia in HPS have not been reported. In this communication, we report a case of HPS characterized by marked hypoalbumine mia and ele vated plasma levels of TNFa and IFNg and evidence obtained from an in vitro experiment supporting the crucial pathogenetic role of TNFa in severe hypoalbum inemia in this case .

Establishment and characterization of a new human mesothelioma cell line (T‐85) from malignant peritoneal mesothelioma with remarkable thrombocytosis

A mesothelioma cell line, termed T‐85, was established from a patient with malignant peritoneal mesothelioma and remarkable thrombocytosis (1.4 × 106/mm3). Electron microscopically, two types of mesothelioma cells have been characterized; the major type of cells with dense‐cored granules in the cytoplasm and the minor one with evenly dense granules. Immunologically, the cells showed staining for inter‐leukin‐6 (IL‐6), cytokeratin, collagen type IV, vimentin, laminin, fibronectin and Factor VIII‐related antigen. Quanti‐tation by ELISA revealed a high concentration of IL‐6 in T‐85 cell culture supernatants. RT‐polymerase chain reaction of T‐85 cells showed two positive bands of cDNA at 628 and 251 base pairs indicating the constitutive expression of IL‐6 and IL‐6 receptor mRNA. Moreover, prominent pro‐platelet process formation activity in T‐85 cell culture supernatants indicated the presence of a thrombopoietic activity due mainly to IL‐6 but not the c‐MpI ligand or erythropoietin. However, the fact that 15% of PPF activity remained in the supernatants treated with anti‐IL‐6 antibody indicated the presence of another thrombopoietic substance. T‐85 is so far the first mesothelioma cell line derived from a case with remarkable thrombocytosis.

Protein kinase C plays a key role in the cross-talk between intracellular signalings via prostanoid receptors in a megakaryoblastic cell line, MEG-01s

In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.

CD8+ aggressive lymphoma in a human T‐cell leukaemia virus type‐1 carrier

Dear Editor, Primary cutaneous CD8 aggressive epidermotropic cytotoxic T-cell lymphoma is a rare variant of cutaneous T-cell lymphoma (CTCL) characterized by eruptive papules, plaques, and nodules partly with central ulceration. Here we report a case of this type of lymphoma in a human T-cell leukaemia virus type-1 (HTLV-1) carrier. A 69-year-old Japanese man was referred to our hospital with a 4-month history of multiple indurated erythematous plaques and nodules on the whole body including the head and face (Fig. 1a). Some nodular lesions showed central ulcerations (Fig. 1b). He had small nodules in the oral cavity (Fig. 1c). Superficial lymph nodes on bilateral axilla and groin were swollen. The peripheral white cell count was normal without any atypical lymphocytes. Laboratory tests showed increased levels of serum soluble interleukin-2 receptor (2391 U/mL, normal: 167–497 U/mL) and thymus and activation-regulated chemokine (4766 pg/mL, normal: 0–449 pg/mL). He was HTLV-1seropositive. A skin biopsy specimen showed diffuse infiltration of atypical lymphocytes with remarkable epidermotropism (Fig. 1d). Atypical lymphocytes were positive for CD3 (Fig. 1e), CD5, CD7, CD 8 (Fig. 1g), and CD25 (Fig. 1h). They were negative for CD4 (Fig. 1f), CD20, and cytotoxic molecules. Part of tumour cells expressed CD45RA and CXCR3, but not CCR4 (Fig. 1i). Southern blotting analysis showed a clonal T-cell receptor gene rearrangement but no clonal integration of HTLV-1 proviral DNA using the skin specimen. A biopsy of inguinal lymph nodes revealed invasion of the same CD8 T cell clone. Systemic examination revealed no other visceral or bone marrow involvement. He was diagnosed with primary cutaneous CD8 aggressive epidermotropic cytotoxic T-cell lymphoma. Chemotherapy consisting of two cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and one cycle of ESHAP (etoposide, methylprednisolone, highdose cytarabine, and cisplatin) was not effective and the patient died 4 months after the initial visit to our hospital. In primary cutaneous CD8 aggressive epidermotropic cytotoxic T-cell lymphoma, tumor cells are usually positive for CD3, CD7, CD8, CD45RA, CXCR3, and negative for CD4, CD5, CD25, CD45RO, and CCR4. They also express cytotoxic molecules such as TIA-1, Granzyme B and perforin. So far, specific genetic abnormalities and association with specific viruses have not been described. Interestingly, we recently experienced another case of this type of lymphoma in an HTLV-1 carrier. HTLV-1 infection is associated with an increased risk of immunosuppression and malignancies other than adult T-cell