Kyoko Yamashiro

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Chymase Participates in Chronic Dermatitis by Inducing Eosinophil Infiltration

An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to a mouse ear caused a transient skin swelling, and the repetition of the challenge enlarged the contact dermatitis. The repeated challenge with DNFB also induced eosinophil infiltration on the application site. Administration of a chymase inhibitor significantly inhibited the ear swelling as well as eosinophil accumulation. An intradermal injection of human chymase to the mouse ear also elicited transient skin swelling and eosinophil infiltration, both of which were augmented in proportion to the number of injections. Human serum albumin and heat-inactivated chymase failed to induce such skin reactions, suggesting the participation of proteolytic activity of the enzyme. In addition, chymase stimulated eosinophil migration in vitro in a concentration-dependent manner. Taken together, these observations suggest that mast cell chymase may contribute to development of the DNFB-induced dermatitis, probably by promoting eosinophil infiltration. It is therefore possible that chymase plays a role in pathogenesis of chronic dermatitis such as atopic dermatitis.

Role of mast cell chymase in allergen-induced biphasic skin reaction

Intradermal injection of human chymase (EC 3.4.21.39) into the mouse ear elicited an edematous skin reaction in a biphasic manner, with a transient reaction peaking at 1 hr, followed by a delayed response persisting for at least 24hr. The kinetics of this reaction was analogous to the biphasic skin reaction induced by ascaris extract in actively sensitized mice. A similarity between the two dermatitis models was also shown by histological analysis, i.e. accumulation of inflammatory cells was observed exclusively in the later phases of the skin reaction. A chymase inhibitor, SUN-C8077 [3-(3-aminophenylsulfonyl)-7-chloroquinazorine 2,4(1H, 3H)-dione], significantly inhibited both the early- and late-phase responses of the skin reaction induced by ascaris extract. These findings suggest that chymase may play an important role in the allergen-induced biphasic skin reaction. A histamine receptor antagonist, homochlorcyclizine, inhibited the early-phase but not the late-phase of the chymase-induced skin reaction. In addition, human chymase showed chemotactic activity to human polymorphonuclear leukocytes in vitro. Mast cell chymase may participate in the two phases of allergic skin inflammation by two distinct mechanisms, i.e. histamine- and leukocyte-dependent mechanisms, respectively.

Mouse mast cell protease-1 cleaves angiotensin I to form angiotensin II.

The ability to convert angiotensin (Ang) I to Ang II was compared between human alpha-chymase and two mouse beta-chymases, mouse mast cell protease (mMCP)-1 and mMCP-4. Human chymase hydrolyzed Ang I to produce Ang II without further degradation. mMCP-1 similarly generated Ang II from Ang I in a time-dependent manner and the formation of the fragment other than Ang II was marginal. In contrast, mMCP-4 hydrolyzed Ang I at two sites, Tyr(4)-Ile(5) and Phe(8)-His(9), with Ang II formation being tentative. Consistently, mMCP-4 but not human chymase hydrolyzed Ang II and mMCP-1 showed little hydrolytic activity against Ang II. These data suggest that not only human chymase but also mMCP-1 might possess a physiological role in Ang II formation. Our findings also imply that the Ang-converting activity of chymase may not be related to the categorization of chymase into alpha- or beta-type based on their primary structure.

Purification, cDNA Cloning, and Characterization of a New Serpin with Megakaryocyte Maturation Activity

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturationin vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

Eosinophils Negatively Regulate Eosinophilopoiesis by Decreasing IL-5 Levels

The effect of mature eosinophils on eosinophilopoiesis was studied. Mature eosinophils decreased the interleukin-5 (IL-5) level after incubation with IL-5 and suppressed IL-5 release from non-adherent mononuclear cells. These findings suggest that eosinophils negatively regulate eosinophilopoiesis by decreasing the IL-5 level.