Yuji Tsuruta

Pirfenidone suppresses tumor necrosis factor-α, enhances interleukin-10 and protects mice from endotoxic shock

A new experimental drug, pirfenidone (5-methyl-1-phenyl-1H-pyridine-one; S-7701), has been reported to have beneficial effects for the treatment of certain fibrotic diseases. We investigated the anti-inflammatory properties in murine endotoxic shock to determine the pharmacological characteristics. The present study describes the prophylactic effect, cytokine regulatory profiles and therapeutic effect of pirfenidone in murine endotoxic shock, which was induced in mice using an intraperitoneal (i.p.) injection of lipopolysaccharide and D-galactosamine. First, we examined the prophylactic effect and cytokine regulatory profiles. A single oral administration of pirfenidone prior to lipopolysaccharide/D-galactosamine challenge inhibited the production of circulating tumor necrosis factor-alpha (TNF-alpha), interleukin-12 and interferon-gamma, markedly enhanced that of interleukin-10, and offered protection from subsequent lethal symptoms in a dose-dependent manner. Second, we examined the therapeutic effect. A single oral administration of pirfenidone 1, 2, 3, 4 and 5 h post lipopolysaccharide/D-galactosamine challenge provided protection against lethal shock in a time-dependent manner. At the histopathological level, apoptotic positive cells were found to be suppressed in the liver. The transforming growth factor (TGF)-beta1 level was markedly elevated in the liver of lipopolysaccharide/D-galactosamine-challenged mice, suppressed in pirfenidone-treated mice. These findings may offer an alternative for both protective and therapeutical treatment of several human acute or chronic inflammatory diseases by pirfenidone.

A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-α at the translational level

A new experimental drug pirfenidone (5-methyl-1-phenyl-2-1H-pyridine-2-one) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. Here, we studied the anti-inflammatory activities of pirfenidone by investigating the mechanism of its inhibitory effect on cytokine production. In RAW264.7 cells, a murine macrophage-like cell line, pirfenidone suppressed the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) by a translational mechanism, which was independent of activation of the mitogen-activated protain kinase (MAPK) 2, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). In the murine endotoxin shock model, pirfenidone potently inhibited the production of the proinflammatory cytokines, TNF-alpha, interferon-gamma, and interleukin-6, but enhanced the production of the anti-inflammatory cytokine, interleukin-10. The in vivo model also showed that pirfenidone suppressed the cytokine production by a translational mechanism, though interleukin-10 transcription was activated by pirfenidone. These findings show that pirfenidone inhibits the production of the proinflammatory cytokine selectively at the translational level. Therefore, cytokine inhibitory activities play an important role in the anti-inflammatory activities of pirfenidone. Coupled with the fact that this inhibitory effect is selective, translational, and not for total protein synthesis, this drug may have a clinical effect on inflammation and fibrosis with very low toxicity.

Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung.

The content of mRNA for a pancreatic-type phospholipase A2 present in rat gastric mucosa was much greater than that in pancreas. In lung the mRNA for this pancreatic-type phospholipase A2 was also detected, but less than in pancreas. Nucleotide sequence analysis showed that these pancreatic-type phospholipase A2 cDNAs derived from rat gastric mucosa and lung were completely identical to that from rat pancreas (Ohara et al. (1986) J. Biochem. 99, 733-739). This demonstrates that the pancreatic-type phospholipase A2 present in gastric mucosa and lung does not originate from pancreas.

Constitutive expression of interleukin-7- mRNA and production of IL-7 by a cloned murine thymic stromal cell line

Mouse interleukin‐7 (IL‐7) cDNA was cloned from mouse thymic stromal cell clone MRL104.8a using a polymerase chain reaction (PCR) technique and expressed in COS‐7 cells. The resulting recombinant interleukin‐7 (rlL‐7) supported the proliferation of mouse antigen‐specific helper T cell (Th) clone 9–16 in the absence of IL‐2 and antigen as well as mouse pre‐B cell line DW34. It was also found that high levels of the mRNA for IL‐7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL‐7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL‐7 mRNA and for production of IL‐7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL‐7 and the previously described cytokines produced by thymic stromal cells.

Characterization of dermatitis arising spontaneously in DS-Nh mice maintained under conventional conditions: another possible model for atopic dermatitis

DS-Nh (DS Nh/+) mice spontaneously develop dermatitis when they are housed in a conventional environment. In this study, we analyzed the clinical and histopathological features of dermatitis in DS-Nh mice, which is characterized by erythema, edema, and erosion on the face, neck, chest and flexor surfaces of their forelegs with marked scratching behavior. Histopathological examination, including immunohistochemistry, revealed that inflammatory cells consisting of mast cells, eosinophils, CD4-positive T cell-dominant lymphocytes and CD11b-positive macrophages infiltrated the skin lesions. The cytokine production pattern of inflammatory cells in a lesional skin tissue was shifted to the Th2-type (IL-4) rather than the Th1 type (IFN-gamma). Serum IgE levels were elevated and correlated with the severity of the clinical skin conditions. These skin symptoms were observed in association with a colonization of Staphylococcus aureus. Similar clinical and histopathological symptoms were inducible with repeated percutaneous immunization of heat-killed S. aureus on the back of SPF DS-Nh mice. These results suggest that the spontaneous dermatitis that occurs in conventionally raised DS-Nh mice is comparable to a certain type of human atopic dermatitis (AD), which is associated with S. aureus, a recognized environmental factor. Thus, we consider that DS-Nh mice offer a useful model for investigating the pathogenesis of AD and for developing new therapeutic approaches or drugs for treating AD.

Restricted usage of T-cell receptor α-chain variable region (TCRAV) and T-cell receptor β-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation

We analysed T‐cell receptor α‐chain variable region (TCRAV) and T‐cell receptor β‐chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo‐BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6–7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1‐4 (26%, 11 of 41 recipients), VA3‐1 (32%) and VB24‐1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity‐determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0·05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T‐cell regeneration via a thymic‐dependent pathway in children.

Determination of T-cell receptors of clonal CD8-positive T-cells in myelodysplastic syndrome with erythroid hypoplasia

We determined T-cell receptor alpha-chain variable (TCRAV) and T-cell receptor beta-chain variable (TCRBV) region repertoires in peripheral bloods from patients with myelodysplastic syndrome (MDS) with erythroid hypoplasia. T-cells bearing TCR ADV14S1/BV5S2, AV21S1/BV21S4, and AV2S2/BV7S2 segments were markedly increased in three of four MDS patients, respectively. In addition, there was a positive relationship between the increase in the number of CD8-positive T-cells and the expression levels of these TCR transcripts. These findings suggest that CD8-positive T-cells monoclonally or oligoclonally expanded in the peripheral blood. We also determined the nucleotide and amino acid sequences of the complementarity-determining region 3 (CDR3) of TCR alpha- and beta-chains of the expanded T-cells. Unique sequences were detected in a high percentage of the respective CDR3 clones. The gene segment of the variable and joining regions, however, varied among the patients. The deduced amino acid sequences of CDR3 were heterogeneous among the patients, and there was no common motif. These results indicate there is monoclonal or oligoclonal proliferation of CD8-positive T-cells in MDS patients with erythroid hypoplasia, and suggest that these proliferating T-cells are responsible for the pathogenesis of the MDS entity.

Detection of human T cell receptor cDNAs (α, β, γ and δ) by ligation of a universal adaptor to variable region

Abstract The study of T cell receptor (TCR) genes has been hampered by their large repertoires and elusive methods for gene amplification. We have developed a new method for amplification of all human TCR genes (α, β, γ, and δ) with the ligation of a universal adaptor to the leader sequence of variable (V) regions, which permitted effective and reproducible amplification of all four types of TCR genes. cDNA sequencing of TCR-γ, -δ, -α, -β was carried out in respectively 15, 13, 28, and 26 T cell clones from human peripheral blood T cells using a newly developed universal adaptor and these methods. TCR-γ V-II (Vγ 9) was a major population, and V-I (Vγ 2 and 3) and V-III (Vγ 10) were next major populations among TCR-γ subfamilies, and confirmed the previous observations determined using mAbs specific to TCR-γ. All five clones of TCR-γ V-II and three of five clones of TCR-γ V-I subfamilies had in-frame V-N-J junctions. In contrast, sequences from both TCR-γ V-III (4/4 clones) and V-IV (1/1 clones) subfamilies had intron-like regions that caused out-of-frame cDNA, suggesting that most of TCR-γ V-III and V-IV in PBL are not functional. Vδ 2 was a major population and Vδ 1 was a next predominant population among TCR-δ subfamilies, also confirming the previous observations determined using mAbs to TCR-δ. With regards to TCR-α and -β, this new method randomly amplified TCR cDNAs. In addition, the sequences of 5′ portions of three TCR-V-α and one TCR-Vβ were extended. Two new TCR-α subfamilies and one new TCR-β family were also identified. In summary, this new method will provide a scientific tool for understanding structures of the human TCR genes involved in specific immune responses.

Quantitative analysis of the usage of human T cell receptor α and β chain variable regions by reverse dot blot hybridization

Abstract We previously developed an adaptor ligation-mediated PCR method to amplify the T cell receptor (TCR) cDNA pools. In the present study we applied reverse dot blot hybridization to PCR-amplified specimens for quantitative analysis of the usage of TCR α and β chain variable (V) region. 44 VA sequence-specific oligonucleotide probes (SSOPs) and 38 VB SSOPs were synthesized corresponding to unique sequences of VA and VB subfamilies. Peripheral blood lymphocytes of ten healthy donors and five T cell clones established from bone marrow cells were examined for VA and VB usage using this method. The results were consistent with those obtained by a colony hybridization method and those by immunofluorescence staining using monoclonal antibodies to VA and VB. Thus, reverse dot blot hybridization for TCR Vα and Vβ is a new, easy and dependable technique useful for analysis of VA and VB usage by human T cells.

Arthritis and pneumonitis produced by the same T cell clones from mice with spontaneous autoimmune arthritis

SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.