Tomoko Inubushi

Novel physiological functions of cathepsins B and L on antigen processing and osteoclastic bone resorption

Lysosomal cathepsin B plays an essential role in the processing of ovalbumin as an exogenous antigen to produce the complex between antigenic-peptide and major histocompatibility-complex class II. Administration of cathepsin B inhibitors, E-64, CA-074 and vitamin B6, caused the strong suppression of the Th-2 type immune responses. We found that pyridoxal phosphate (PAP), a coenzyme form of vitamin B6, inhibits the activities of cathepsin B and L in vitro and vitamin B6 administration induces the inhibition of the lysosomal cathepsin activities in vivo. The production of an antigenic epitope (I323-R339) of ovalbumin by antigen presenting cells was suppressed by cathepsin B specific inhibitors. The ovalbumin dependent production of immunoglobulins (IgE and IgG1) and of the corresponding interleukin (IL-4) was suppressed by cathepsin B inhibitors, while the production of IgG2a and interferon (INF-gamma) was increased. The switch of helper T lymphocyte functions from the type-2 to the type-1 may be induced by the cathepsin B inhibition. The experimental bone pit formation, i.e., osteoclastic bone collagen degradation test, induced by parathyroid hormone was markedly suppressed by the administration of pyridoxal, because of the inhibition of cathepsin L type cysteine proteases in bone.

Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2+

Abstract Biphenyl is found both in natural and anthropogenic sources and is used as a fungistat in the packaging of citrus fruits. Acute exposure to high levels of biphenyl has been observed to cause skin irritation and toxic effects on the liver and kidneys. However, the mechanisms of cytotoxicity induced by biphenyl are not yet well understood. In the present study, the cytotoxicity of biphenyl was studied by flow cytometry with fluorescent probes. Biphenyl at 100 μM significantly increased cell lethality after 3 h in rat thymocytes. In addition, biphenyl at 100 μM or more elevated intracellular Zn2+ levels. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an intracellular and extracellular Zn2+ chelator, but not diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), a membrane-impermeable Zn2+ chelator, attenuated the biphenyl-induced increase in intracellular Zn2+ levels and cell death. These results suggested that biphenyl-induced cytotoxicity caused an increase in intracellular Zn2+ levels, which was dependent on internal Zn2+. Moreover, biphenyl led to an increase in sensitivity to oxidative stress, while TPEN inhibited this biphenyl-induced increase. Our findings revealed that biphenyl caused an increase in the intracellular free Zn2+ concentration, inducing cytotoxicity, cell death, and an increase in sensitivity to oxidative stress.