Masaki Hikida

Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells.

Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.

Molecular Cloning of a Novel Murine Cell-surface Glycoprotein Homologous to Killer Cell Inhibitory Receptors

We have isolated a cDNA clone encoding a novel murine cell-surface glycoprotein. This polypeptide is predicted to be composed of a signal peptide of 23 amino acids, an extracellular region of 620 amino acids that contains six immunoglobulin-like domains with five potential N-glycosylation sites, a transmembrane sequence of 20 amino acids, and a cytoplasmic tail of 178 amino acids with four sets of sequences similar to the immunoreceptor tyrosine-based inhibition motif. The relative molecular mass of the mature polypeptide is calculated to be 90,520 Da. The polypeptide, designated as p91, shows striking homologies to human killer cell inhibitory receptors, a murine gp49B1 protein, a bovine Fcγ2 receptor, and a human Fcα receptor. The mRNA of p91 was especially abundant in murine macrophages. Western blot analysis using p91-specific anti-peptide sera detected a 130-kDa polypeptide in macrophages. Surface biotinylation and immunoprecipitation analysis verified the surface expression of the translation products on COS-1 cells transfected with the p91 cDNA, but the cells failed to show any Fc binding activity.

Expression of human and mouse genes encoding polκ: testis-specific developmental regulation and AhR-dependent inducible transcription

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers

Background. Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT). Methods. Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3–TWNT-1 co-culture system. Results. TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. Conclusions. TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.

Contribution of light chain rearrangement in peripheral B cells to the generation of high-affinity antibodies

Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high‐affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)‐knockin mouse whose B cell diversity is limited due to the expression of the site‐directed transgene. Immunization of the mouse with p‐nitrophenylacetyl (pNP)‐conjugated chicken γ‐globulin preferentially led to the production of anti‐pNP IgG Ab comprised of non‐VHT‐encoded H chains and λ chains. λ+ IgG constituted a majority of high‐affinity Ab to this hapten. RAG‐2 mRNA and the recombination signal sequence break of the λ1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating λ gene rearrangement and the production of λ+ high‐affinity anti‐pNP IgG. These observations were reproduced in RAG‐1‐deficient mice that were reconstituted with the spleen cells ofthe knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.

B Cell Selection and Affinity Maturation During an Antibody Response in the Mouse with Limited B Cell Diversity

The quasi-monoclonal mouse has limited B cell diversity, whose major (∼80%) B cell Ag receptors are comprised of the knockin VH 17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/λ1 and VHT/λ2 IgM were equally produced, VHT/λ2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded γ-chains could form λ1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/λ1 anti-pNP IgG may not be due to the incompatibility between the γ-chains and the λ1-chain, but may be explained by the fact that VHT/λ1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/λ2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Induction of antigen-specific IgE response in murine lymphocytes by IL-10

When murine spleen cells that had been primed with trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) were stimulated in vitro with the same antigen, anti-TNP IgE, as well as anti-TNP IgM and IgG1, was secreted into the culture medium. On the other hand, anti-TNP IgM and IgG1 were produced, but anti-TNP IgE secretion was negligible when the carrier (KLH)-primed spleen cells were cultured with the hapten-carrier antigen (TNP-KLH) under the same conditions. Anti-TNP Ig responses in the latter cultures are thought to reflect the interaction between normal TNP-specific B cells and KLH-primed helper T cells. By using this culture system, we investigated the requirements of exogenous cytokines for inducing anti-TNP IgE response. The addition of interleukin-4 (IL-4), that is known to induce IgE response in LPS-stimulated murine B cells, failed to elicit anti-TNP IgE response. The combination of IL-4 with IL-2 and/or IL-5 was also ineffective. Interestingly, a significant level of anti-TNP IgE was induced when IL-10, another cytokine from type 2 helper T cells, was added to the culture. Although IL-10 enhanced the production of anti-TNP IgM and IgG1, as well as that of anti-TNP IgE, the rate of enhancement was at least 3-fold higher in the IgE response than in the IgM and IgG1 responses. Simultaneous addition of IL-4, IL-5 or IL-13 with IL-10 did not augment but rather reduced the enhancing effects of IL-10. IL-10 did not further stimulate the spontaneous secretion of IgE from antigen-primed B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of immunocyte functions by thymosin α1 in cyclophosphamide-induced immunodeficient mice

Thymosin α1 (Tα1) is an oligopeptide hormone originally isolated from the thymus gland, and has been reported to have stimulating effects on the differentiation of T cells and NK cells. These immunostimulating properties have been considered to be useful for improving immune disorders associated with various diseases including cancer, AIDS and hepatitis. Here, we characterized immunostimulating properties of Tα1 in experimental immunodeficiency of mice that was induced by the administration of cyclophosphamide (CY). Repeated injection of 30–300 μg/kg/day of Tα1 after CY-treatment significantly accelerated the restoration of the reduced number of CD4+CD8+ T cells in the thymus. Tα1 administration was effective in restoring the suppressed activities of helper T cells and cytotoxic T cells in CY-treated mice. Tα1 also had stimulating effects on reduced activity of lymphokine-activated killer cells in CY-treated mice. These results indicate that Tα1 is stimulatory for both humoral and cellular immune responses, thus providing the immunological basis for the clinical benefit of this compound.

Prevention of collagen-induced arthritis in DBA/1 mice by oral administration of AZ-9, a bacterial polysaccharide from Klebsiella oxytoca

Collagen-induced arthritis (CIA) is an excellent model of rheumatoid arthritis (RA) in humans that is induced in DBA/1 mice immunized with bovine type II collagen (CII). Here, we report that the induction of CIA was effectively suppressed by oral administration of AZ-9, a purified polysaccharide with the average molecular weight of approximately 200 kDa that was produced by a soil bacterium, Klebsiella oxytoca. When AZ-9 was administered at 125-250 mg/kg/day orally for 9 consecutive days after immunization with CII followed by its administration every 3 days, resulted in a marked reduction of the incidence and the severity of CIA. The serum level of anti-CII IgG2a and the production of IFN-gamma and IL-12 in the draining lymph node (LN) cells were significantly lower in AZ-9-administered mice than the untreated control. These findings suggest that orally administered AZ-9 suppressed CIA through attenuating a Th1-type response to CII. AZ-9 could be fragmented into smaller molecules (3-4 kDa) without losing its suppressive activity.

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab)2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).