DNA polymerase beta is not essential for the formation of palindromic (P) region of T cell receptor gene.

Formation of palindromic (P) region at the variable (V)-diversity (D)-joining (J) junction in DNA polymerase beta (pol-beta) deficient mice were investigated by sequencing of reverse transcriptase-polymerase chain reaction (RT-PCR) products of mRNAs encoding the beta chain of T cell receptor (TCR). Total 42 and 43 cDNA clones encoding V(beta8)-D(beta)-J(beta)-C(beta) from E18.5 embryonic thymocytes of pol-beta gene knocked-out and wild type control mouse, respectively, were sequenced. Among them five and six clones from pol-beta knocked-out and wild type, respectively, have P insertions of two nucleotides. This result unequivocally indicates that pol-beta, which is one of the repair-type DNA polymerases most abundantly expressed in thymus and spleen, is not essential for the formation of P region.

Histone acetylation determines the developmentally regulated accessibility for T cell receptor gamma gene recombination.

Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-gamma gene, wherein predominant rearrangement is switched from Vgamma3 to Vgamma2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vgamma3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow-derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vgamma3 gene. These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

Targeting of Krüppel-associated box-containing zinc finger proteins to centromeric heterochromatin. Implication for the gene silencing mechanisms.

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) repress transcription via functional interaction with the corepressor KRAB-associated protein-1 (KAP-1). KAP-1 directly interacts with heterochromatin protein 1 (HP1), a dose-dependent regulator of heterochromatin-mediated silencing. Here we show that two KRAB-ZFPs that we previously identified, KRAZ1 and KRAZ2, are targeted to foci of centromeric heterochromatin containing HP1alpha through the interaction with KAP-1. Centromeric targeting potential of KRAZ1 and KAP-1 is strictly correlated with their silencing activities; a KRAB mutant of KRAZ1 that is unable to bind KAP-1 and KAP-1 deletions unable to bind HP1 cannot localize to centromeric foci nor repress transcription. We provide evidence that this correlation is likely to be functionally relevant. First, overexpression of the VP16 transactivation domain fused with the KAP-1 deletion that binds to KRAB but not to HP1 leads to dramatic redistribution of KRAZ1 from centromeric foci and simultaneously converts KRAZ1-mediated silencing into strong transcriptional activation. Second, a specific inhibitor of histone deacetylases, trichostatin A, effectively redistributes KRAZ1 and KAP-1 from centromeric foci and partially relieves their silencing activities. These data strongly suggest that KRAB-ZFPs/KAP-1 silence transcription by dynamic recruitment of the target locus to the specific gene silencing compartment, centromeric heterochromatin, in a histone deacetylase-dependent manner.

Induction of follicular gastritis following postthymectomy autoimmune gastritis in Helicobacter pylori-infected BALB/c mice.

Helicobacter pylori is the major causative agent of chronic antral gastritis and is thought to be involved in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (MALToma) developing in the human stomach. The aim of this study was to clarify whether corporal autoimmune gastritis (AIG), which is known to decrease acidity due to destruction of parietal cells, predisposes mice to H. pylori infection, thereby leading to MALToma-like pathology. BALB/c mice in which AIG had been induced by thymectomy 3 days after birth (AIG mice) were used. The AIG mice were orally administered mouse-adapted H. pylori at the age of 6 weeks and were examined histologically and serologically after 2 to 12 months. The results were compared with those obtained from uninfected AIG mice and infected normal mice. Germinal centers were induced in the corpus in 57% of the H. pylori-infected AIG mice, which elicited anti-H. pylori antibody responses in association with upregulation of interleukin-4 (IL-4) mRNA. In these mice, parietal cells remained in the corpus mucosa. These findings were in contrast to those with the uninfected AIG mice: fundic gland atrophy due to disappearance of parietal cells associated with upregulation of gamma interferon, but not IL-4, mRNA and no germinal center formation in the corpus. These observations suggest that AIG alters the infectivity of H. pylori, leading to MALToma-like follicular gastritis, at an early stage after H. pylori infection.

Molecular cloning and splicing isoforms of mouse p144, a homologue of CA150.

We previously characterized p144 bearing N-acetylglucosamine residues in a rat liver nuclear matrix fraction. Based on partial amino acid sequences of rat p144, mouse p144 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence revealed that p144 is a rat homologue of CA150, which is a transcription factor involved in Tat-activated human immunodeficiency virus type 1 transcription. The reported human CA150 consists of 1098 amino acids and has a leucine zipper-like motif in its carboxyl-region. However, a clone of mouse p144 cDNA encoded a CA150 consisting of 1,034 amino acids. The mouse CA150 was shorter by 64 amino acids than hitherto known human CA150 and lacked the leucine zipper-like motif. We designated the longer and shorter CA150 species as CA150a and CA150b, respectively. The partial nucleotide sequences of other mouse p144 cDNA clones were examined and it was found that some clones encode CA150a having a leucine zipper-like motif. It was suggested that CA150a and CA150b are splicing isoforms. All rat and mouse tissues examined contained transcripts for both CA150a and CA150b. Both transcripts were detected in human blood and Jurkat cells as well as mouse CD4(+) T-cells, which are the HIV-1-sensitive counterpart in humans.

Differential localization of Th1 and Th2 cells in autoimmune gastritis.

The vast majority of CD4+ T cells infiltrating into gastric mucosa (GM) and in the draining (gastric) lymph node (GLN) shows an activated/memory phenotype, CD45RB(low) L-selectin(low) CD44(high), in neonataly thymectomized BALB/c mice bearing autoimmune gastritis (AIG), indicating that these cells are actively involved in this disease. CD4+ T cells sort-purified from GLN expressed mRNAs encoding for both IFN-gamma and IL-4. However, those infiltrating into GM expressed very low levels of IL-4 mRNA, even though they strongly expressed IFN-gamma mRNA. Among CD4+ T cells separated from AIG mice expressing detectable levels of either IFN-gamma or IL-4 by intracellular staining, less than one-seventh expressed IL-4 and thus most of them expressed IFN-gamma in GM, whereas roughly half and one-third expressed IL-4 in GLN and spleen respectively. These findings indicate that the Th1 cells predominantly infiltrate into autoimmune lesions and Th2 cells are mainly resident in the regional LN. We further set up an in vitro model system of transendothelial migration using a murine endothelial cell line, F-2, and found that Th1 cells in CD4+ T cells separated from lymphoid tissues of AIG mice preferentially passed through the monolayer of endothelial cells while only a small portion of Th2 cells did so. This differing ability of transendothelial migration and localization might explain the dominance of Th1 cells destroying the tissue in focal lesions without inhibition by the Th2 cells, in spite of both subsets being simultaneously activated in AIG mice, and the functions of each T cell subset seems to be mutually exclusive.

Destruction of hematopoietic microenvironment by cytotoxic T cells.

Coculture of cytotoxic T cells (STIL-3 C5) derived from L8313 leukemic mice with hematopoietic supportive stromal cells (MS-5) resulted in the detachment of MS-5 cells from the culture dish, whereas helper T cells (STIL-3 DF) did not induce this detachment. The response of bone marrow (BM) adherent cells to the same treatment was similar to that of MS-5 cells. The detached cells were unable to proliferate further, and genomic DNA of these cells showed fragmentation, suggesting that hematopoietic stromal cells died of apoptosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that STIL-3 C5 cells, but not STIL-3 DF cells expressed perforin, granzyme A & B, and Fas ligand. Fas was expressed in MS-5, BM adherent cells, MS-K and NIH/3T3 cells, which do not support hematopoiesis. These data suggest that the aforementioned factors mediate induction of apoptosis in MS-5 cells induced by direct cell-to-cell interaction with STIL-3 C5. This may explain the mechanism responsible for the destruction of the hematopoietic microenvironment by cytotoxic T cells in L8313 leukemia, from which STIL-3 cells are derived; it also suggests that destruction of hematopoietic tissue may be caused by leukemic cytotoxic T cells in some cases of leukemia.

Structure of the TCR expressed on a gastritogenic T cell clone, II-6, and frequent appearance of similar clonotypes in mice bearing autoimmune gastritis.

A parietal cell-specific Th1 clone, II-6, which was established from a BALB/c mouse bearing post-thymectomy autoimmune gastritis (AIG), recognizes a peptide of the alpha subunit (alpha891-905) of H+/K+-ATPase and induces gastritis in nu/nu BALB/c mice by adoptive cell transfer. In the present study, the primary structure of the TCR of II-6 was determined as Valpha10-Jalpha c5a-Calpha and Vbeta14-Jbeta2.3-Cbeta2 by cDNA cloning. Using PCR with specific primers, we defined the use of this II-6 TCR in nu/nu mice with transferred II-6 cells and in mice that spontaneously developed AIG by thymectomy on day 3 after birth (d3-Tx). II-6 TCR mRNAs were detected in the gastric mucosa of all of the nu/nu mice, suggesting that II-6 cells indeed home to the gastric mucosa and thereby were directly involved in the destruction of target parietal cells. TCR beta chain mRNAs encoding CDR3 region sequences almost identical with that of II-6 were also found in the gastric mucosa in 43% (six of 14 mice tested) of the d3-Tx AIG mice at 4-12 weeks old by nested RT-PCR. Such a frequent appearance of similar clonotypes in independent individuals suggests that T cells bearing II-6-like TCR including the II-6 itself might be directly involved in, although not essential for, the pathogenesis of AIG in 3d-Tx mice.

A possible involvement of Fas-Fas ligand signaling in the pathogenesis of murine autoimmune gastritis.

BACKGROUND & AIMS: A Th1 clone, II-6, established from an autoimmune gastritis BALB/c mouse that underwent thymectomy 3 days after birth, recognized a 15 mer peptide constructing the alpha subunit of H+, K(+)-adenosine triphosphatase as antigen and induced gastritis in nu/nu mice by adoptive transfer. The aim of this study was to examine the molecular mechanism of target (parietal cells) destruction in either thymectomized or II-6 cell-transferred nu/nu mice. METHODS: Expression of Fas, major histocompatibility complex class II, and intercellular adhesion molecule 1 molecules on the gastric mucosa of these mice were immunohistochemically examined. In situ DNA fragmentation in these thymectomized or nu/nu mice was tested by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end label (TUNEL) method. Moreover, activity of II-6 cells to induce apoptosis was tested by using the 15 mer peptide-pulsed B lymphoma cells, A20.2J, as the target. RESULTS: A portion of parietal cells in gastritis-bearing thymectomized or nu/nu mice at an early stage expressed Fas, major histocompatibility complex class II, and intercellular adhesion molecule 1 molecules and was TUNEL positive. Fas-ligand message was induced on activated II-6 cells and caused DNA fragmentation of the antigen-pulsed A20.2J cells. CONCLUSIONS: Cognate interaction between Fas antigen on the target and Fas ligand on the effector seems to be one possible mechanism for the target cell destruction in organ-specific autoimmune gastritis.

Breakdown of self-tolerance by intrathymic injection of a T-cell line inducing autoimmune gastritis in mice.

Autoimmune gastritis (AIG) develops spontaneously in BALB/c mice thymectomized 3 days after birth (3d-Tx). We first confirmed our previous observations that CD4+ splenic T cells in AIG mice induced AIG in nu/nu mice, while those in normal mice suppressed the development of the disease. In addition, we found that a quantitative balance between these effector (Te) and suppressor (Ts) T cells determined either onset or prevention of the disease. Peripheralization of Ts seemed to begin around 3 days after birth, since the incidence of AIG in mice that underwent Tx 6 days after birth (6d-Tx) decreased markedly, compared with that of 3d-Tx mice; 12% in the former, while 79% in the latter. Notably, Ts existed in the 6d-Tx mice that escaped AIG. We next examined the target specificity of such Ts using syngeneic parietal cells known as autoantigens and two kinds of T-cell lines established from an AIG mouse; one is gastritis inducible in vivo, termed A-II, while another is not, named AC-II. Intrathymic injection of parietal cells into mice 3 days after birth followed by 6d-Tx completely prevented the development of AIG. In contrast, injection of irradiated A-II, but not AC-II cells resulted in AIG in 67% of the mice. No autoimmune oophoritis (AIO) was induced in female mice, implying that the breakdown of tolerance is organ specific. Taken together, peripheral tolerance for organ-specific autoantigens seems to be maintained by CD4+ Ts responding to Te, which induces the disease.