Toshiaki Mizuochi

Both cathepsin B and cathepsin D are necessary for processing of ovalbumin as well as for degradation of class II MHC invariant chain.

The effect of highly selective inhibitors of cathepsins on the processing of ovalbumin (OVA) and the presentation of an OVA-derived antigenic peptide (OVA323-339) by antigen presenting cells (APC) was investigated. Both CA-074 (a specific inhibitor of cathepsin B) and pepstatin A (a specific inhibitor of cathepsin D) showed an inhibitory effect on the IL-2 production from an OVA-specific, I-Ad-restricted helper T (Th) cell clone upon stimulation with OVA presented by the I-Ad-positive APC. In contrast, the presentation of the antigenic epitope, OVA323-339, to the same Th clone was not inhibited by either CA-074 or pepstatin A alone, nor even by the mixture of both inhibitors. When APC were treated with cathepsin inhibitor for 24 h, and then antigen and Th were added to the culture, the presentation of not only OVA but also an OVA-derived antigenic peptide was inhibited by either cathepsin inhibitor alone. In addition, the expression of invariant chain on APC was significantly augmented by the pretreatment of APC with either cathepsin inhibitor. Two main conclusions are drawn from these results. First, not only aspartyl protease, such as cathepsin D, but also thiol protease, such as cathepsin B, is involved in antigen processing by APC. Second, both cathepsin B and cathepsin D are necessary for degradation of the invariant chain (Ii) from the MHC class II alpha beta heterodimer in endosomes in order to express functional MHC class II molecules for binding antigenic peptides.

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: Implications for resolution of the inflammatory response

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-α, and addition of IFN-γ inhibited SLPI but augmented TNF-α production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-α production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.

Recognition Requirements for the Activation, Differentiation and Function of T‐Helper Cells Specific for Class I MHC Alloantigens

The present review has focused on the specificity of the T-helper cell populations initiating MHC class I alloreactions. In contrast to conventional immune responses against soluble antigens, responses against membrane-bound class I alloantigens are initiated by two distinct antigen-specific T-helper cell populations that can be distinguished by their Lyt phenotype, MHC restriction specificity, antigen specificity, and requirement for thymically determined self-recognition. Alloresponses were shown to be a composite consisting of two distinct components: one mediated by L3T4+ Th cells and very similar to conventional self + X responses; and one mediated by Lyt2+ Th cells and unique to alloresponses against MHC class I antigens. As would befit an unusual Th cell population, the recognition/response spectrum of Lyt2+ Th cells was highly unusual and was found to be the basis for much of the uniqueness we attribute to immune alloreactions, including rapid rejection of tissue allografts in vivo.

Autophagic Compartments Gain Access to the MHC Class II Compartments in Thymic Epithelium

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell’s components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

Enhanced expression of lymphomagenesis-related genes in peripheral blood B cells of chronic hepatitis C patients.

Epidemiological data indicate a close relationship between chronic hepatitis C virus (HCV) infection and B-cell non-Hodgkins lymphoma (B-NHL), suggesting that chronic HCV infection is, at least in part, associated with B-lymphomagenesis. However, experimental data concerning these conditions remains elusive. In this study, we confirmed that peripheral blood B cells of chronic hepatitis C (CHC) patients were infected with HCV. Expression levels of activation-induced cytidine deaminase (AID) which are thought to be associated with occurrence of B-NHL were analyzed in these CHC B cells. It was demonstrated that AID mRNA/protein levels in CHC B cells were dramatically increased compared with those of healthy subjects. Furthermore, expression levels of several previously reported prognostic B-NHL marker genes in the B cell subset of CHC patients were increased. These results suggest a possible relationship between chronic HCV infection and B-lymphomagenesis.

Peripheral B Cells May Serve as a Reservoir for Persistent Hepatitis C Virus Infection

A recent study by our group indicated that peripheral B cells in chronic hepatitis C (CHC) patients are infected with hepatitis C virus (HCV). This raised the logical question of how HCV circumvents the antiviral immune responses of B cells. Because type I interferon (IFN) plays a critical role in the innate antiviral immune response, IFNβ expression levels in peripheral B cells from CHC patients were analyzed, and these levels were found to be comparable to those in normal B cells, which suggested that HCV infection failed to trigger antiviral immune responses in B cells. Sensing mechanisms for invading viruses in host immune cells involve Toll-like receptor-mediated and retinoic acid-inducible gene-I (RIG-I)-mediated pathways. Both pathways culminate in IFN regulatory factor-3 (IRF-3) translocation into the nucleus for IFNβ gene transcription. Although the expression levels of RIG-I and its adaptor molecule, IFN promoter-stimulator-1, were substantially enhanced in CHC B cells, dimerization and subsequent nuclear translocation of IRF-3 were not detectable. TANK-binding kinase-1 (TBK1) and IĸB kinase Ε (IKKΕ) are essential for IRF-3 phosphorylation. Constitutive expression of both kinases was markedly enhanced in CHC B cells. However, reduced expression of heat shock protein of 90 kDa, a TBK1 stabilizer, and enhanced expression of SIKE, an IKKΕ suppressor, were observed in CHC B cells, which might suppress the kinase activity of TBK1/IKKΕ for IRF-3 phosphorylation. In addition, the expression of vesicle-associated membrane protein-associated protein-C, a putative inhibitor of HCV replication, was negligible in B cells. These results strongly suggest that HCV utilizes B cells as a reservoir for persistent infection.

The antigen presentation pathway in medullary thymic epithelial cells, but not that in cortical thymic epithelial cells, conforms to the endocytic pathway.

Murine medullary thymic epithelial cells (mTEC), but not cortical thymic epithelial cells (cTEC), are able to present a soluble antigen, ovalbumin, to helper T cells (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601 – 1605). This functional difference between the mTEC and the cTEC is particularly important when we consider the thymic selection of the T cell repertoire. In the previous report, we proposed that mTEC and cTEC utilize two distinct antigen processing/presenting pathways (Kasai, M. et al., Eur. J. Immunol. 1996. 26: 2101 – 2107). In this report, we further confirmed this difference by analyzing (a) localization of MHC class II, H2‐DM, and invariant chain (Ii) molecules, (b) the biochemical nature of MHC class II molecules, (c) the sensitivity of MHC class II α β heterodimer formation to concanamycin A, a vacuolar H+‐ATPase inhibitor, and (d) the subcellular distribution of MHC class II, H2‐DM, and Ii molecules, in both TEC. Our results demonstrated that, in the mTEC, MHC class II, H2‐DM and Ii molecules gain access to the endocytic pathway, where the luminal condition is acidic and thus Ii molecules are efficiently degraded and H2‐DM molecules function well. In the cTEC, however, such molecules seemed to gain access to an alternative transport pathway, e.g. a secretory pathway, where the luminal condition is not fully acidic. These two distinct antigen processing pathways may account for the functional difference between mTEC and cTEC.

Role of Self Antigens in the Selection of the Developing T Cell Repertoire

The thymus plays a central role in determining the receptor repertoire that mature T cells express. The cellular elements involved in thymic selection of the T cell repertoire remain controversial, as do the rules by which it occurs. There is general agreement that mature L3T4+ T helper cells utilize as self-Ia determinants those Ia determinants their precursors encountered on thymic elements during their differentiation into functional competence. Thus, the thymus selects for further differentiation and expansion those T cell precursors that appear to express low affinity receptors for thymic Ia determinants. The present study explores the possibility that thymic selection of the L3T4+ T cell repertoire is based on more than the specificity of their receptors for thymic Ia determinants. Indeed, the results of the present study indicate that thymic selection of the L3T4+ Th cell repertoire is based on T cell recognition of self-complexes composed of nominal self-antigens plus thymic Ia. Thus, nominal antigens as well as Ia antigens present in the thymus during T cell ontogeny can play a significant role in shaping the mature T cell repertoire. Moreover, the demonstration that thymic selection occurs by recognition of self-complexes suggests that thymic selection and thymic tolerization may be alternative outcomes of a single selection event.

Possible Recruitment of Peripheral Blood CXCR3+ CD27+ CD19+ B Cells to the Liver of Chronic Hepatitis C Patients

It has been suggested that hepatitis C virus (HCV) infects not only hepatocytes but also immune cells, including B cells. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkins lymphoma. To clarify the effects of chronic HCV infection on B-cell dynamics, peripheral B cells from chronic hepatitis C patients (CHC) were characterized. We found that the frequency of CD27(+) B cells, that is memory phenotype, was significantly reduced in the peripheral blood of CHC. At the same time, the amount of IFN-gamma-inducible protein-10 (IP-10), a CXCR3 ligand, was markedly elevated in the plasma of CHC. Furthermore, the CD27(+) B-cell population was found to highly express CXCR3 in CHC, thus suggesting that the CD27(+) B-cell population was recruited from peripheral blood to the inflammatory site of the liver of CHC, where IP-10 is produced. Immunohistochemical analyses of intrahepatic lymphocytes indicated that CXCR3(+) B cells were infiltrated in the liver of CHC. Our results thus offer new insight into the role of memory B cells in the HCV pathogenesis.

HCV Infection and B-Cell Lymphomagenesis

Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkins lymphoma (NHL). Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.