Eiji Ichimaru

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: Identification by immunological and enzymological methods

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous, cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in, GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.

Immunohistological Study of Plasma Cells Bearing Immunoglobulins Reactive to Human IgG in Human Periodontitis.

辺縁性歯周炎罹患歯肉における抗ヒトIgG抗体保有細胞の存在を検索する目的で, まずヒトIgGで感作したラット脾臓から凍結切片を作製し, ペルオキシダーゼ標識ヒトIgGを用いた標識抗原法を行い, 抗ヒトIgG抗体保有細胞を観察するための手法を確立した。そして実際にヒト歯肉において同法を行った結果, 41人から採取した63部位中, 9人14部位に抗ヒトIgG抗体保有細胞が観察された。抗ヒトIgG抗体保有細胞は今回分類した成人性歯周炎, 早期発症型歯周炎, 増殖性歯肉炎のすべてのグループにおいて観察され, その割合は早期発症型歯周炎が最も高く, 以下成人性歯周炎, 増殖性歯肉炎の順であった。抗ヒトIgG抗体保有細胞はおもに口腔上皮近くのリンパ球や形質細胞が密集して浸潤した部位に存在していた。今回の検索では, 年齢, 種々の臨床パラメーターで抗ヒトIgG抗体保有細胞の存在の有無を分けることはできなかった。しかし, 早期発症型歯周炎の一部においてはその歯肉中に多数の抗ヒトIgG抗体保有細胞が確認され, IgGに対する特異的な抗体産生が起こっている可能性が示唆された。

A role of human neutrophil lysosomal proteinases in periodontal tissue breakdown.

To clarify a role of neutrophil lysosomal proteinases in periodontal tissue breakdown, their nature in neutrophils and their levels in gingival crevicular fluid (GCF) of periodontitis patients were investigated. When the distribution of these proteinases in the neutrophil granular subfractions was analyzed, cathepsins B and H were made soluble by hypotonic shock. Cathepsins L and G were made soluble after washing the membrane with 1M NaCI. On the other hand, about 30% of hemoglobin (Hb) -hydrolase (s) was tightly associated with the membrane after both treatments. Immunochemical analyses revealed that about 35% of the total Hb-hydrolase activity was attributed to cathepsin D, which was found in the content fraction, and the rest were due to pepstatin-insensitive serine proteinase (s) . Differing from rat neutrophils, human neutrophils do not contain cathepsin E. The ratio of cathepsin D to the serine proteinase (s) in neutrophils was not changed in GCF from periodontitis patients and these enzymes were associated with development of the disease.The quantity of cathepsin B in GCF of periodontitis patients determined by enzyme immunoassay was closely correlated with that by activity measurement, indicating that cathepsin B increases with the increase of severity of the disease and that its level in GCF can be inferred by activity measurement.

Studies on the roles of lysosomal proteinases in periodontal breakdown. Purification and properties of cathepsins D and E from human polymorphonuclear leucocytes.

本研究の目的は, 歯周疾患の発症ならびに進行過程におけるリソゾーム性アスパルチックプロテアーゼの役割を解明することである。歯肉満滲出液中の酵素量を酵素免疫測定法で定量するために, ヒト白血球を材料としてカテプシンDおよびEの精製を行った。両酵素は, Pepstatin-Sepharose4B, DEAE-Sephace1およびSephadex G―100の各種クロマトグラフィーによって同時精製された。最終酵素標品において, カテプシンDは比活性23, 000, 回収率21%, 精製度7, 000倍であり, カテプシンEは比活性75, 000, 回収率11%, 精製度23, 000倍であった。ゲル濾過法で求めた分子量は, カテプシンDが45, 000, カテプシンEは90, 000であった。ヘモグロビンを基質とした至適pHは, ともに3.0～3.5であった。両酵素はPepstatin, DAN, EPNPなどのアスパルチックプロテアーゼインヒビターによって特異的に阻害を受け, Cu2+, Pb2+, Fe3+およびPCMBによっても明らかに阻害を受けた。