Kenji Sekikawa

Lipopolysaccharide induction of indoleamine 2,3‐dioxygenase is mediated dominantly by an IFN‐γ‐independent mechanism

Indoleamine 2,3‐dioxygenase (IDO) is a rate‐limiting enzyme in the L‐tryptophan‐kynurenine pathway, which converts an essential amino acid, L‐tryptophan, to N‐formylkynurenine. It has been speculated that IFN‐γ is a dominant IDO inducer in vivo. The present study used IFN‐γ or TNF‐α gene‐disrupted mice and IFN‐γ antibody‐treated mice to demonstrate that lipopolysaccharide (LPS)‐induced systemic IDO is largely dependent on TNF‐α rather than IFN‐γ. IFN‐γ‐independent IDO induction was also demonstrated in vitro with LPS‐stimulated monocytic THP‐1 cells. These findings clearly indicate that there is an IFN‐γ‐independent mechanism of IDO induction in addition to the IFN‐γ‐dependent mechanism.

Tumor Necrosis Factor-α (TNF-α) Plays a Protective Role in Acute Viral Myocarditis in Mice A Study Using Mice Lacking TNF-α

BACKGROUND It has been reported that tumor necrosis factor-alpha (TNF-alpha) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-alpha on viral myocarditis, however, have not been fully elucidated. METHODS AND RESULTS To investigate the role of TNF-alpha in the progression of viral myocarditis, we used TNF-alpha gene-deficient mice (TNF-alpha(-/-)) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-alpha(-/-) mice after EMCV infection was significantly lower than that of TNF-alpha(+/+) mice (0% versus 67% on day 14). Injection of recombinant human TNF-alpha (0.2 to 4.0 microg/mouse IV) improved the survival of TNF-alpha(-/-) mice in a dose-dependent manner, indicating that TNF-alpha is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-alpha(-/-) mice compared with TNF-alpha(+/+) mice. CONCLUSIONS These observations suggested that TNF-alpha is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-alpha plays a protective role in the acute stage of viral myocarditis.Background—It has been reported that tumor necrosis factor-α (TNF-α) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-α on viral myocarditis, however, have not been fully elucidated. Methods and Results—To investigate the role of TNF-α in the progression of viral myocarditis, we used TNF-α gene–deficient mice (TNF-α−/−) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-α−/− mice after EMCV infection was significantly lower than that of TNF-α+/+ mice (0% versus 67% on day 14). Injection of recombinant human TNF-α (0.2 to 4.0 μg/mouse IV) improved the survival of TNF-α−/− mice in a dose-dependent manner, indicating that TNF-α is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-α−/− than in TNF-α+/+ mice. Histopathological examination showed that the inflam...

Endovascular Injury Induces Rapid Phenotypic Changes in Perivascular Adipose Tissue

Objective—Accumulating evidence suggests that adipose tissue not only stores energy but also secretes various bioactive substances called adipocytokines. Periadventitial fat is distributed ubiquitously around arteries throughout the body. It was reported that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular diseases accelerated by obesity. We investigated the effect of endovascular injury on the phenotype of perivascular fat. Methods and Results—Endovascular injury significantly upregulated proinflammatory adipocytokines and downregulated adiponectin within periadventitial fat tissue in models of mouse femoral artery wire injury and rat iliac artery balloon injury. Genetic disruption of tumor necrosis factor (TNF)-&agr; attenuated upregulation of proinflammatory adipocytokine expression, with reduced neointimal hyperplasia after vascular injury. Local delivery of TNF-&agr; to the periadventitial area enhanced inflammatory adipocytokine expression, which was associated with augmented neointimal hyperplasia in TNF-&agr;-deficient mice. Conditioned medium from a coculture of 3T3-L1 and RAW264 cells stimulated vascular smooth muscle cell proliferation. An anti-TNF-&agr; neutralizing antibody in the coculture abrogated the stimulating effect of the conditioned medium. Conclusion—Our findings indicate that endovascular injury induces rapid and marked changes in perivascular adipose tissue, mainly mediated by TNF-&agr;. It is suggested that the phenotypic changes in perivascular adipose tissue may have a role in the pathogenesis of neointimal hyperplasia after angioplasty.

Role of Tumor Necrosis Factor-Alpha and Interferon-Gamma in Helicobacter pylori Infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor‐α (TNF‐α) and interferon‐7 (IFN‐γ) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF‐α gene‐knockout (TNF‐α−/−) mice and IFN‐γ gene‐knockout (IFN‐γ−/−) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild‐type and knockout mice. The number of H. pylori colonized in the stomach of IFN‐γ−/− and TNF‐α−/− mice was higher than that of wild‐type mice. These findings suggest that TNF‐α and IFN‐γ may play a protective role in H pylori infection. Furthermore, we examined the contribution of TNF‐α and IFN‐γ to gastric inflammation. The CPY2052‐infected TNF‐α−/− mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild‐type mice, whereas the CPY2052‐infected IFN‐γ−/− mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN‐γ may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF‐α may not participate in the development of inflammatory response.

Role of IFN-γ and Tumor Necrosis Factor-α in Herpes Simplex Virus Type 1 Infection

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.

Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.

Overexpression of the Wiskott-Aldrich Syndrome Protein N-Terminal Domain in Transgenic Mice Inhibits T Cell Proliferative Responses Via TCR Signaling Without Affecting Cytoskeletal Rearrangements

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with small platelets, severe eczema, and recurrent infections due to defects in the immune system. The disease arises from mutations in the gene encoding the WAS protein (WASP), which plays a role as an adaptor molecule in signal transduction accompanied by cytoskeletal rearrangement in T cells. To investigate the functional domain of WASP, we developed transgenic mice overexpressing the WASP N-terminal region (exon 1–5) including the Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain, in which the majority of mutations in WAS patients have been observed. WASP transgenic mice develop and grow normally under the specific pathogen-free environment, and showed normal lymphocyte development. However, proliferative responses and cytokine production induced by TCR stimulation were strongly inhibited in transgenic mice, whereas Ag receptor capping and actin polymerization were normal. These findings suggest that overexpressed Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain of WASP inhibits the signaling from TCR without coupling of cytoskeletal rearrangement. WASP transgenic mice shown here could be valuable tools for further understanding the WASP-mediated processes.

Intrabodies against the EVH1 domain of Wiskott–Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single‐chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (VH) leader signal sequence at the N‐terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott–Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)‐2 synthesis in the T‐cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N‐terminal region of WASP may participate in IL‐2 synthesis. In transgenic mice expressing anti‐EVH1 scFvs derived from hybridoma cells producing WASP‐EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti‐EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL‐2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T‐cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL‐2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.

Impaired Contact Hypersensitivity Reaction and Reduced Production of Vascular Endothelial Growth Factor in Tumor Necrosis Factor-α Gene-Deficient Mice

Tumor necrosis factor (TNF)‐α is an important proinflammatory cytokine in contact hypersensitivity (CHS) reactions. Previous efforts to assay CHS in TNF‐α gene‐deficient (‐/‐) mice have demonstrated a significant reduction in ear skin weight at 24 h following challenge by oxazolone, although the mechanisms of this suppression have not been examined. To further characterize the impaired CHS during evolution of the elicitation phase in TNF‐α ‐/‐ mice and to clarify its mechanisms, focusing on the roles of TNF‐α and vascular endothelial growth factor (VEGF), we used an established method of CHS assay‐sensitization and challenge by trinitrochlorobenzene (TNCB)‐ in TNF‐α ‐/‐ and wild‐type mice. We compared the histopathology of the sequential evolution of CHS between the two groups of mice and assessed both the extent of inflammatory cell infiltration and the degree of dilatation in dermal vessels labeled with CD31. We quantified the production of VEGF in the epidermis at specific time points by using a murine VEGF ELISA kit. The CHS reaction was markedly suppressed in TNF‐α ‐/‐ mice at all time points of the elicitation phase. Histologically, in TNF‐α ‐/‐ mice we observed diminished vascular permeability, reduced numbers of infiltrating inflammatory cells, neutrophils at 12 h, mononuclear cells and eosinophils at 24 h, and a decreased area of dilatation of vessels labeled with CD31. The level of epidermal VEGF in wild type mice increased rapidly after challenge and peaked at 24 h, paralleling the peak of ear swelling. In contrast, the peak level of epidermal VEGF in TNF‐α ‐/‐ mice was significantly reduced. These results suggest that TNF‐α plays an enhancing role in the elicitation phase of the CHS reaction. Diminished degrees of vascular permeability, dilatation of CD31+ vessels, and inflammatory cell infiltration in TNF‐α ‐/‐ mice are likely to be the result of a lack of TNF‐α and reduced production of epidermal VEGF.

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.