JP4787445B2 - Antigen-specific IgE antibody production inhibitor - Google Patents
Antigen-specific IgE antibody production inhibitor Download PDFInfo
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- JP4787445B2 JP4787445B2 JP2001551502A JP2001551502A JP4787445B2 JP 4787445 B2 JP4787445 B2 JP 4787445B2 JP 2001551502 A JP2001551502 A JP 2001551502A JP 2001551502 A JP2001551502 A JP 2001551502A JP 4787445 B2 JP4787445 B2 JP 4787445B2
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Description
技術分野
本発明は、アトピー性皮膚炎等のアレルギー性疾患の予防ないしは改善に有効なＩｇＥ抗体産生抑制剤に関する。また、本発明は生体内における免疫系を刺激して免疫を賦活化する組成物に関する。
背景技術
ラクトフェリン（ＬＦ）は、乳汁だけでなく、唾液、涙、胆汁、膵液などのような外分泌液に含まれる鉄結合性糖タンパク質であり、また、炎症部位において活性化した好中球により放出される。
ＬＦは広範なスペクトラムの生物活性を有する。例えば、抗菌作用、抗ウイルス作用、抗腫瘍作用、癌転移巣形成抑制作用等である。
また、ＬＦはサイトカイン分泌に対して影響を及ぼす。例えば、ヒト好中球からのインターロイキン−８（ＩＬ−８）の分泌促進、ＩＬ−１やＩＬ−６、ＴＮＦαの分泌制御、ＧＭ−ＣＳＦの遺伝子レベルでの発現の抑制等である。
サイトカインの選択的な産生は、様々な病態において、保護的役割あるいは病因的役割を果たしている。例えば、大部分のアレルギーは、ＩｇＥ抗体の関与するＩ型アレルギーであるが、このＩｇＥ産生は、ＩＬ−４を中心とするシステムにより誘導される一方、インターフェロン−γ（ＩＦＮ−γ）を中心とするシステムにより産生制御されている。ＩＦＮ−γはその上位のＩＬ−１２やＩＬ−１８により産生誘導される。
ＩＦＮ−γは、また、様々な免疫修飾機能による抗ウイルス活性、抗微生物活性、及び抗腫瘍活性を有している（Ｊ．Ｉｍｍｕｎｏｌ．，１３０：２０１１−２０１３，１９８３；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ８５：４８７４−４８７８，１９８８）。
Ｎａｋａｊｉｍａらは、ウシＬＦ（ｂＬＦ）経口投与後のマウスの脾細胞をマイトジェン（Ｃｏｎ Ａ）の存在下で培養すると、ＩＦＮ−γの産生は増強されたが、一方、ＩＬ−４の産生は増強されなかった、と報告している（Ｂｉｏｍｅｄｉｃａｌ Ｒｅｓｅａｒｃｈ，２９（１）：２７−３３，１９９９）。
上述したように、ＬＦは、多彩な生物活性やサイトカイン産生制御に関与している。そこで、本発明は、ＬＦの新たな生物活性を見出し、該活性に基づき、ＬＦを有効成分とする医薬品や機能性食品を提供することを課題とする。また、ＬＦと組み合わせることにより、ＬＦの生物活性を増強する作用を有する経口摂取可能な物質を見出すことを課題とする。
発明の開示
本発明者らは、ＬＦ、加熱処理したＬＦ、あるいはＬＦ加水分解物を経口摂取したマウスに、主要食品アレルゲンである卵白アルブミン（ＯＶＡ）を免疫すると、ＯＶＡ特異的ＩｇＥ抗体の産生が抑制されること、及び脾細胞培養系における抗原特異的ＩＬ−４の産生が抑制されることを見出した。また、ＬＦの存在下でマクロファージを培養すると、マクロファージのＩＬ−１２の産生が増強されることを見出した。さらに、ＬＦとセラミド類を組み合わせて用いると、脾細胞培養系におけるＩＦＮ−γの産生が著明に増強することを見出した。ＬＦの経口摂取による抗原特異的ＩｇＥ抗体の産生抑制は、ＩＬ−４産生の抑制によりＩｇＥ抗体産生Ｂリンパ球の活性化の抑制やＩＬ−１２産生増強によるＴｈ１／Ｔｈ２バランスのＴｈ１優位へのシフトによるものと考えられる。すなわち、ＬＦを経口摂取することにより、Ｔｈ２免疫の方向へのシフトによる細胞性免疫の低下を防ぎ、アレルギー疾患や抗体を介した腎炎などを予防あるいは改善することができる。さらに、ＬＦとセラミド類との組み合わせによるＩＦＮ−γの著明な産生増強は、ＬＦとセラミド類とを組み合わせが、ウイルス性疾患、細菌感染症、あるいは腫瘍に対して有効であることがわかる。
すなわち、本発明はＬＦまたはその加水分解物を有効成分として含有するヒトを含む哺乳動物のＩｇＥ抗体産生抑制剤を提供するものである。
また、本発明はＬＦまたはその加水分解物を有効成分として含有するヒトを含む哺乳動物のＩｇＥ抗体産生を抑制して免疫を調節するための組成物を提供するものである。
また、本発明はＬＦまたはその加水分解物及びセラミド類を含有するヒトを含む哺乳動物のＩＦＮ−γ産生増強剤組成物、抗ウイルス剤組成物、抗細菌感染症剤組成物及び抗腫瘍剤組成物を提供するものである。
また、本発明はＬＦまたはその加水分解物及びセラミド類を含有するヒトを含む哺乳動物の免疫賦活剤組成物を提供するものである。
また、本発明はＬＦまたはその加水分解物のヒトを含む哺乳動物のＩｇＥ抗体産生抑制剤製造のための使用を提供するものである。
また、本発明はＬＦまたはその加水分解物の、ヒトを含む哺乳動物のＩｇＥ抗体産生を抑制して免疫を調節するための組成物製造のための使用を提供するものである。
また、本発明はＬＦまたはその加水分解物及びセラミド類の、ヒトを含む哺乳動物のＩＦＮ−γ産生増強剤組成物、抗ウイルス剤組成物、抗細菌感染症剤組成物及び抗腫瘍剤組成物製造のための使用を提供するものである。
また、本発明はＬＦまたはその加水分解物及びセラミド類の、ヒトを含む哺乳動物の免疫賦活剤組成物製造のための使用を提供するものである。
また、本発明はＬＦまたはその加水分解物の有効量を投与することを特徴とするヒトを含む哺乳動物のＩｇＥ抗体の産生抑制方法を提供するものである。
また、本発明はＬＦまたはその加水分解物の有効量を投与することを特徴とするヒトを含む哺乳動物のＩｇＥ抗体産生を抑制して免疫を調節する方法を提供するものである。
また、本発明はＬＦまたはその加水分解物及びセラミド類の有効量を投与することを特徴とするヒトを含む哺乳動物のＩＦＮ−γ産生増強方法、又はウイルス性疾患、細菌感染症もしくは腫瘍の処置方法を提供するものである。
さらに本発明はＬＦまたはその加水分解物及びセラミド類の有効量を投与することを特徴とするヒトを含む哺乳動物の免疫賦活方法を提供するものである。
発明を実施するための最良の形態
多くのアレルギー性疾患においては、アレルゲン特異的ＩｇＥがその発症に関与することが知られており、実際、アレルギー患者においては、血清中のアレルゲン特異的ＩｇＥがしばしば認められる（Ａｌｌｅｒｇｙ Ｃｌｉｎ．Ｉｍｍｕｎｏｌ．，１６：１６１，１９９６）。したがって、ＩｇＥ産生を抑制することがアレルギーの予防、治療のために効果的方法の一つとされている。
ＩｇＥの産生には、ＣＤ４＋ヘルパーＴ細胞（Ｔｈ細胞）の産生するサイトカインが重要な役割を果たしている。Ｔｈ細胞は、産生するサイトカインおよびその機能の違いにより、細胞性免疫に関わるＴｈ１細胞と液性免疫に関与するＴｈ２細胞の２つの亜集団に大別されるが、そのうちＩｇＥの産生に関与するのはＴｈ２細胞である（Ｊ．Ｉｍｍｕｎｏｌ．，１３６：２３４８，１９８６）。
Ｔｈ１細胞、Ｔｈ２細胞はそれぞれ産生するサイトカインによって互いにその分化・機能発現を抑制しあう関係にある。すなわち、Ｔｈ２細胞はＩＬ−４、ＩＬ−５、およびＩＬ−６を産生してＩｇＥ産生を正に調節している。逆にＴｈ１細胞の産生するＩＦＮ−γは、ＩｇＥクラススイッチやＴｈ２細胞応答を抑制することでＩｇＥ産生を抑制する。このため、ＩｇＥ産生やアレルギー性疾患はＴｈ２細胞応答の亢進を特徴とする（Ｎａｔｕｒｅ，３８３：７８７，１９９６）。したがって、Ｔｈ１細胞応答を誘導してＴｈ１／Ｔｈ２バランスを正常に戻すことでその予防や改善ができる。そこで食品成分中から、Ｔｈ１応答を誘導しやすい因子を検索する試みがなされており、これまである種の乳酸菌（Ｉｎｔ．Ａｒｃｈ．Ａｌｌｅｒｇｙ Ｉｍｍｕｎｏｌ．，１１５：２７８，１９９８）やヌクレオチド（Ｉｎｔ．Ａｒｃｈ．Ａｌｌｅｒｇｙ Ｉｍｍｕｎｏｌ．，１２２：３３，２０００）などが、その効果を示すことが明らかにされている。
近年、ＬＦの経口投与による生体防御効果がさまざまな動物モデルで報告されている。そこで本発明者らは、先ず、マウスを用い、ＬＦを自由に経口摂取させた場合の、血清中の全ＩｇＥおよび抗原特異的ＩｇＥの産生について調べた。その結果、ＬＦを自由に経口摂取すると、全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生を抑制することが明らかとなった。
ＬＦの生理活性は熱処理によって低下することが知られている（Ｊ．Ｐｅｄｉａｔｒ．，９０：２９，１９７７）。したがって、ＬＦを加熱処理することによって、ＬＦの全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生抑制能が低下する可能性が考えられる。そこで、加熱処理したＬＦの、全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生に与える影響を検討した。さらに、ＯＶＡ存在下で脾細胞を培養し、培養上清中のサイトカイン（ＩＦＮ−γ、ＩＬ−４）レベルを調べた。その結果、ＬＦは加熱することにより、抗原特異的ＩｇＥ抗体産生抑制作用およびＩＬ−４産生抑制作用が失われることはないことが明らかとなった。
ＬＦおよび加熱処理したＬＦを経口摂取すると、全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生が対照群に対し有意に抑制されることが明らかとなった。また、抗原で感作された脾細胞を該抗原存在下で培養するとＩＬ−４産生が対照群に対し有意に低下することが明らかとなった。
そこでさらに加水分解したＬＦを自由に経口摂取させた場合における全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生に与える影響を検討した。また、抗原感作された脾細胞を該抗原存在下で培養し、上清中のサイトカイン（ＩＦＮ−γ、ＩＬ−４）レベルを調べた。その結果、全ＩｇＥ抗体産生および抗原特異的ＩｇＥ産生は、ＬＦ加水分解物摂取群において低下傾向が見られた。一方、ＩＦＮ−γの産生は、各濃度のＯＶＡ存在下でＬＦ加水分解物摂取群で増加傾向が示されたが、ＩＬ−４については、ＬＦ加水分解物摂取群と対照群との間に差はみられなかった。
ＩＬ−１２は、単球、マクロファージ、樹状細胞などの抗原提示細胞が産生するサイトカインで、ＩＦＮ−γの産生やヘルパーＴ細胞のＴｈ１への分化の誘導に重要な役割を果たす（Ｂｌｏｏｄ，８４：４００８，１９９４；Ｊ．Ｌｅｕｋｏｃ．Ｂｉｏｌ．，５５：２８０，１９９４）。Ｇａｚｚｉｎｅｌｌｉら（Ｊ．Ｉｍｍｕｎｏｌ．，１５３：２５３３，１９９４）は、Ｔｏｘｏｐｌａｓｍａ ｇｏｎｄｉｉをマウスに感染させると、脾臓や腹腔の細胞のＩＬ−１２産生が上昇し、このマウスにさらに抗ＩＬ−１２抗体を投与すると、脾臓細胞のＩＦＮ−γ産生が低下し、ＩＬ−４やＩＬ−１０産生が上昇することを報告した。また、Ｎａｓｔａｌａら（Ｊ．Ｉｍｍｕｎｏｌ．，１５３：１６９７，１９９４）は、ガンを移植したマウスにＩＬ−１２を投与すると、ガンの増殖を抑制し、血中のＩＦＮ−γ濃度が上昇することを見出している。
そこで、チオグリコレート培地で誘導したマウス腹腔マクロファージをＬＦと共に培養しＩＬ−１２産生を調べた。その結果、ＬＦはマクロファージのＩＬ−１２産生を誘導することが明らかとなった。
ところで、以前は軽い皮膚炎と考えられていたアトピー性皮膚炎は、現在では乳児から成人にまで見られる難病の一つに数えられ、社会的にも大きな問題となっている。その原因の一つとして、表皮におけるスフィンゴ脂質の代謝異常が疑われている。実際アトピー患者の多くは表皮のセラミド量が減少している。セラミド類は、美容食品としても利用され始めている。そこで、本発明者らはＬＦとセラミド類との組み合わせの経口摂取は、アレルギー性疾患の予防・改善において、ＬＦ単独よりもさらにその効果を増強するのではないかと考えた。
そこで、ＬＦとセラミド類との組み合わせが、ＩＦＮ−γ産生に及ぼす効果について調べた。その結果、ＬＦとセラミド類との組み合わせは、脾細胞におけるＩＦＮ−γ産生を著明に増強することが明らかとなった。スフィンゴ脂質では、ケフィア（発酵乳）中のリゾスフィンゴ脂質（スフィンゴシン、スフィンゴミエリン、ロゾフォスファチジルコリンなど）がＩＦＮ−β産生を促進することが知られている（Ｂｉｏｔｈｅｒａｐｙ：１１５−１２３，１９９４）。また、スフィンゴシン投与によって、感染マウスの血中ＩＦＮ−β及びＩＦＮ−γが上昇したことか報告されている（Ｏｓａｄａ，Ｋ．ｅｔ ａｌ．：Ａｎｉｍａｌ Ｃｅｌｌ ｔｅｃｈｎｏｌｏｇｙ：Ｄｅｖｅｌｏｐｍｅｎｔ ｔｏｗａｒｄｓ ｔｈｅ ２１ｓｔ Ｃｅｎｔｕｒｙ １９９５，１０６７−１０７１）。
これらの結果から、ＬＦの経口摂取による抗原特異的ＩｇＥ抗体の産生抑制は、ＩＬ−４産生の抑制によりＩｇＥ抗体産生Ｂリンパ球の活性化の抑制やＩＬ−１２産生増強によるＴｈ１／Ｔｈ２バランスのＴｈ１優位へのシフトによるものと考えられる。したがって、ＬＦの経口摂取は、アレルギー炎症性疾患の予防ならびに改善に有効である。そこで、食品への栄養補強物として、その有効量を添加すれば、日常の食生活のなかでアレルギー性疾患の予防ならびに改善ができ、また、該疾患に対する医薬品としても利用できる。アレルギー炎症性疾患としては、例えば、慢性気管支喘息、アトピー性皮膚炎、花粉症（アレルギー性鼻炎）、アレルギー性血管炎、アレルギー性結膜炎、アレルギー胃腸炎、アレルギー性肝障害、アレルギー性膀胱炎、及びアレルギー性紫斑病等が挙げられる。あるいは抗体を介した腎炎などを予防あるいは改善することが期待できる。
また、ＬＦとセラミド類を組み合わせて用いると、ＩＦＮ−γの産生が著明に増強することから、ＬＦとセラミド類とを組み合わせて用いることにより、Ｔｈ１／Ｔｈ２バランスのＴｈ１優位へのシフトによるＩｇＥ抗体の産生抑制がわかる。ＩＦＮ−γは抗ウイルス作用、抗腫瘍作用、細胞障害性Ｔリンパ球の誘導作用、ナチュラルキラー細胞（ＮＫ細胞）活性の誘導作用、好中球の活性化作用、マクロファージの活性化作用、ＭＨＣクラスＩＩの発現促進作用、ＩＬ−２レセプターの発現促進作用、Ｆｃレセプターの発現促進作用などが知られている。また、ＩＦＮは、もともと生体の異物に対する防御機構の一端を担う生体物質であり、極めて選択毒性が高く、生体にとって安全性の高いものである。そこで、ＬＦとセラミド類を組み合わせた組成物を含む食品などを経口摂取すると、生体細胞を刺激して、ＩＦＮ−γの分泌を促進し、ＩＦＮ−γの上記生理活性を引き出すことができる。したがって、ＬＦとセラミド類を組み合わせた組成物は、抗ウイルス剤、抗腫瘍剤、抗微生物感染症剤となり得る。ＬＦの安全性については、ラットを用いたウシＬＦの急性毒性試験及び亜急性毒性試験では、最大用量である２，０００ｍｇ／ｋｇ／体重／ｄａｙのウシＬＦの投与によっても、何ら異常は認められてない（Ｍｉｌｋ Ｓｃｉｅｎｃｅ Ｖｏｌ．４８，Ｎｏ．３，ｐ２２７−２３２，１９９９）。また、セラミドの安全性については、マウスに対するオリザセラミドのＬＤ５０は５，０００ｍｇ／ｋｇ以上である（ＦＯＯＤ Ｓｔｙｌｅ Ｖｏｌ．４，Ｎｏ．１０：９９−１０５，２０００）。
本発明で用いることのできるＬＦは、例えば、市販のＬＦ、哺乳動物（例えば、ヒト、ウシ、ヒツジ、ヤギ、ウマ等）の初乳、移行乳、常乳、末期乳等、またはこれらの乳由来の脱脂乳、ホエー等から常法（例えば、イオン交換クロマトグラフィー等）により分離したＬＦ、酸又は酵素による加水分解物、それらを塩酸、クエン酸等により脱鉄したアポＬＦ、アポＬＦを鉄、銅、亜鉛、マンガン等の金属でキレートさせた金属飽和又は部分飽和ＬＦ等を含む。
本発明のＬＦ加水分解物の調製には公知の加水分解手段を用いることができる。加水分解に酵素を用いる場合、酵素として、本発明ではトリプシンを用いたが、その他の酵素、例えばペプシン、パパインも用いられるが、加水分解物が本発明の活性を有する限りこれらの酵素に限定されない。加水分解物は加熱により酵素を失活させた後、限外濾過などの常法により分画し濃縮操作を施してもよい。得られた液状の加水分解物はそのまま用いてもよいし、凍結乾燥して用いてもよい。
組換えＬＦは、Ｏｒｌａ Ｍ．Ｃｏｎｎｅｅｌｙら（米国特許５，７６６，９３９）により記載されたのと実質上同じアミノ酸配列を有するポリペプチドを含む。また、天然（ｎａｔｉｖｅ）に生じる対立遺伝子変異体、及び天然のＬＦに比べて、１以上のアミノ酸の挿入、置換、又は欠失により修飾されたＬＦを含む。組換えＬＦは、また、トランスジェニック動物、例えば、ウシにおいて発現された組換えＬＦを包含し、ここでグリコシル化パターンは、ヒトミルクから得られた天然のＬＦのグリコシル化パターンと異なりうる。
本発明に用いるセラミド類はセラミド関連物質を含み、例えば、スフィンゴ脂質、とりわけスフィンゴ糖脂質が好ましい。スフィンゴ糖脂質としては、例えば、最も単純なスフィンゴ糖脂質は乳、脳および腎などに見出されるセレブロシド、さらにそれに硫酸基のついたスルファチド、中性糖が数分子ついたセラミドオリゴヘキソシド、アミノ糖のついたグロボシド、シアル酸のついたガングリオシド類などが挙げられる。スフィンゴ糖脂質糖鎖の違いにより構造が判明しているもので１００種類を超えるが、本発明の作用を有するものは全て含まれる。好ましくはラクトシルセラミド、ガラクトシルセラミド、グルコシルセラミドなどが挙げられる。また、リン脂質の一種でもあるスフィンゴミェリン（牛乳中に含まれる）なども好ましい。このほか、不斉合成技術などにより、天然のセラミドの化学合成が可能となり、光学活性なセラミドの開発も行われている。セラミド２、コスモファームのセラミド３がそれである。本発明においてはこれらのセラミドも本発明の作用を有する限り用いることができる。さらに植物由来のセラミドが注目され、利用されはじめている。例えば、米由来のスフィンゴ脂質は、動物性のスフィンゴ脂質と同様に、長鎖塩基スフィンゴシンに脂肪酸が酸アミド結合した疎水性セラミドが基本骨格となっている。また、米由来のスフィンゴ脂質は、長鎖塩基スフィンゴシンおよび脂肪酸の炭素数の違い、水酸基や二重結合の有無によって、分種に多様性がある。帯広畜産大学の藤野教授らの報告によると、少なくとも２０種類を越えるスフィンゴ脂質分子種が存在する。本発明はこれらのセラミドも含む。
本発明において、ＬＦ（全てのＬＦおよびそれらと同等の生理活性をもつＬＦの誘導体並びにそれらの加水分解物を含む）とセラミド類の含量は、セラミド類が、該組成物の総重量に基づき、好ましくは０．００００１〜０．１重量％、より好ましくは０．００００５〜０．０１重量％、特に好ましくは０．０００１〜０．０５重量％、ＬＦが、該組成物の総重量に基づき、好ましくは０．００５〜１０重量％、特に好ましくは０．０１〜５重量％、最も好ましくは０．０１〜３重量％と推定されるが、当業者であれば、対照とする組成物に応じて、例えば後述する実験により最適な量を容易に決定できると考えられる。ちなみに母乳中のセラミド含量は公知であり、セラミド類配合量において一応の目安となると考えられる。
本発明により、ＬＦの経口摂取が抗原特異的ＩｇＥ抗体産生を抑制することが明らかにされた。したがって、当業者はこの発明に基づき、上述した各種ＬＦのなかから、本発明に適用できる好ましい形態のＬＦ及び有効量を、例えば、ＩｇＥ抗体産生を指標にして、選択し、臨床データ、或いは臨床症状等と、患者における血清ＩｇＥとの関連を勘案し、食品、栄養補助食品、病者用食品、乳児用調製粉乳、健康食品、健康強調表示食品、機能性食品、特定保健用食品、或いは医薬品等に用いる場合の、該ＬＦの形状及び有効投与量を決定することが可能である。したがって、このようにして決定されたＬＦの種類及びその有効投与量は、本発明に包含される。ＬＦを食品等に添加・加工する技術は周知慣用である。医薬品としてＬＦの有効量を用いる場合、当業者公知の種々の製剤形態に加工して用いることができる。
実施例
以下に本発明を試験例および実施例を挙げて説明するが、本発明はこれらの試験例および実施例により限定されるものではない。
以下の試験例において、試験例１〜４及び６は３週齢の雄性幼若ＢＡＬＢ／ｃマウス（日本ＳＬＣ；Ｓｈｉｚｕｏｋａ，Ｊａｐａｎ）を用い、試験例５は８週齢の雌性ＢＡＬＢ／ｃマウス（日本ＳＬＣ；Ｓｈｉｚｕｏｋａ，Ｊａｐａｎ）を用いた。対照群と実験群との有意差検定はＳｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔにより行った。
［試験例１］ ウシＬＦ（ｂＬＦ）のＩｇＥ抗体産生抑制作用
マウスには実験期間中、ＬＦ投与群（ｎ＝７）にはカゼインをタンパク質原とした食餌（ＡＩＮ７６準拠）および１％ウシＬＦ（ｂＬＦ、ＤＭＶジャパン社製）を含む水を自由摂取させた。対照群（ｎ＝８）には上記食餌とｂＬＦを含まない水を自由摂取させた。
実験開始後、５日目および１９日目に、マウス１匹当たり、卵白アルブミン（ＯＶＡ、生化学工業社製）１０μｇを水酸化アルミニウム４ｍｇとともに腹腔免疫した。２６日目に、眼窩から採血して、血清中の総ＩｇＥ抗体レベル及びＯＶＡ特異ＩｇＥ抗体レベルをＥＬＩＳＡ法で測定した。血清中の総ＩｇＥ抗体レベル（図１）及びＯＶＡ特異ＩｇＥ抗体レベル（図２）ともにｂＬＦ群のマウスの方が対照群に比べ有意（＊ｐ＜０．０５）に低下した。
［試験例２］ ｂＬＦのＩＬ−４産生抑制作用
試験例１において、２６日目に脾臓を摘出し脾細胞浮遊液を調製した。溶血処理をした後、脾細胞を種々の濃度（５、１０、５０、および１００μｇ／ｍｌ）のＯＶＡ存在下で７２時間培養した。培養後に培養上清中のＩＬ−４レベルをＥＬＩＳＡ法で測定した。ｂＬＦ投与群（ｎ＝７）では、各濃度のＯＶＡにおけるＩＬ−４レベルは、対照群（ｎ＝８）におけるそれらに比べすべて低下した（図３）。
［試験例３］ 加熱処理ｂＬＦのＩｇＥ抗体およびサイトカイン産生抑制作用
試験例１の食餌にｂＬＦを２．２％添加した食餌［ｂＬＦ（＋）］を７０℃で１時間加熱した。対照として、ｂＬＦ無添加の食餌［ｂＬＦ（−）］も同様に７０℃で１時間加熱した。
マウスに、実験期間中、ｂＬＦ（＋）（実験群）あるいはｂＬＦ（−）（対照群）を自由摂取させた。実験開始後５日目、１４日目、２３日目、および３３日目に、１０μｇの卵白アルブミン（ＯＶＡ、生化学工業社製）を４ｍｇの水酸化アルミニウムとともにマウスの腹腔に免疫した。３回目の腹腔免疫後８日目（３０日目）（７週齢）に尾部から採血し、血清中の抗体をＥＬＩＳＡ法で測定した。また、３回目の腹腔免疫後１１日目（３３日目）および４回目の腹腔免疫後８日目（４０日目）に脾細胞を個体別に採取した。脾細胞は各種濃度のＯＶＡ（０、５０、１００、２００および４００μｇ／ｍｌ）とともに７２時間、１０％ＦＣＳ、１００Ｕ／ｍｌペニシリンＧ、１００μｇ／ｍｌストレプトマイシンおよび５×１０−５Ｍの２−メルカプトエタノールを含むＲＰＭＩ １６４０培地で培養した。培養後、培養上清中のＩＦＮ−γ、およびＩＬ−４レベルをＥＬＩＳＡ法で測定した。
免疫を３回行った場合、実験群の血清中の全ＩｇＥ抗体レベルおよびおよびＯＶＡ特異的ＩｇＥ抗体レベルは、対照群のそれらに比べて有意に低下した（図４及び５）。また、脾細胞の、各濃度のＯＶＡ（０、５０、１００、および２００μｇ／ｍｌ）におけるＩＦＮ−γ産生は、実験群と対照群との間でほとんど差が見られなかった（図６）。一方、脾細胞の、上記各濃度のＯＶＡにおけるＩＬ−４産生は、実験群の方が対照群に比べて全般的に低く、２００μｇ／ｍｌのＯＶＡの刺激下では、実験群の方が対照群に比べて有意に低下した（図７）。
免疫を４回行った場合、脾細胞の、各濃度のＯＶＡ（０、５０、１００、２００、および４００μｇ／ｍｌ）におけるＩＦＮ−γ産生は、免疫３回の場合と同様に、両群間でほとんど差が見られなかった（図８）。一方、上記各濃度のＯＶＡにおけるＩＬ−４産生は、実験群の方が対照群に比べて、全般的に有意に低下した（図９）。
以上の結果から、ｂＬＦは加熱することにより、抗原特異的ＩｇＥ抗体産生抑制作用およびＩＬ−４産生抑制作用が失われることはないことが判明した。
［試験例４］ 加水分解ｂＬＦのＩｇＥ抗体およびサイトカイン産生抑制作用
（１）加水分解ｂＬＦの作製
トリプシン（和光純薬（株）、２０７−０９８９１、生化学用、ブタ膵臓製、４５００ ＵＳＰ ｔｒｙｐｓｉｎ ｕｎｉｔｓ／ｍｇ）を滅菌ＰＢＳに溶解し、トリプシン原液（×５００、５０ｍｇ／ｍｌ）とした。１０ｇのｂＬＦ（和光純薬（株）、１２７−０４１２２、ｌｏｔ．ＫＳＧ７７２４）を２５ｍＭ ＣａＣｌ２−０．１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．２）中に１％となるように溶解した。この１ＬのｂＬＦ溶液を３７℃に加温した後、トリプシン原液２ｍｌを添加し、３７℃で４時間反応させた。反応後、８０℃で３０分間加熱し酵素を失活させた。１７００×ｇにて２０分間遠心分離した後、上清を回収し、０．４５μｍのフィルターにより濾過滅菌した。サンプルをマイクロアシライザーＳ１（旭化成工業製）により処理し、サンプル中の塩を除いた。０．４５μｍのフィルターにより再度濾過滅菌した後、試験に供するまでの間−２０℃で保存した。ｂＬＦ加水分解物の収率はほぼ１００％であった。したがって、ｂＬＦ加水分解物濃度は１％となった。
マウスに、試験例１の食餌およびｂＬＦ加水分解物を１％含む溶液を自由摂取させた。対照群には、同様の固形飼料とｂＬＦを含まない水を自由摂取させた。実験開始後５日目と１９日目に、１０μｇの卵白アルブミン（ＯＶＡ、生化学工業社製）を４ｍｇの水酸化アルミニウムとともにマウス腹腔に免疫した。２回目の免疫後８日目（２６日目）に採血し、血清中の抗体をＥＬＩＳＡ法で測定した。また、このとき脾細胞を個体別に採取した。脾細胞はＯＶＡ（０〜４００μｇ／ｍｌ）とともに７２時間培養し、培養上清中のＩＦＮ−γ、およびＩＬ−４レベルをＥＬＩＳＡ法で測定した。
実験群における血清中の全ＩｇＥ抗体レベルおよびＯＶＡ特異的なＩｇＥレベルは、対照群のそれらに比べて低下する傾向が見られた（図１０及び１１）。
脾細胞の、各種ＯＶＡ濃度におけるＩＦＮ−γ産生は、実験群の方が対照群に比べて全般的に高くなる傾向が認められた（図１２）。一方、ＩＬ−４産生は、実験群と対照群との間でほとんど差が見られなかった（図１３）。
［試験例５］ ＬＦのＩＬ−１２産生に及ぼす影響
ＩＬ−１２は比較的新しく見出されたサイトカインであるが、Ｔ細胞やＮＫ細胞に作用してＩＦＮ−γの産生を誘導することが知られている（Ｊ．Ｅｘｐ．Ｍｅｄ．，１７３：８６９−８７９，１９９１；Ｊ．Ｅｘｐ．Ｍｅｄ．，１７７：１１９９−１２０４，１９９３）。ＩＬ−１２の産生誘導にはＬＰＳなどの菌体成分の刺激が有効であり、その産生細胞としては、マクロファージ、Ｂ細胞、好中球など多岐にわたる細胞が含まれる。
本発明者らは、ｂＬＦ（和光社製）を添加した培地中で腹腔マクロファージを培養し、その培養上清中のＩＬ−１２のレベルをＥＬＩＳＡ法で測定した。
ＭＦ食（オリエンタル酵母社製）を自由摂取させたマウスに、２．５ｍｌのチオグリコレート培地（ＤＩＦＣＯ社製）を腹腔内注射し、腹腔マクロファージを活性化した。４日後に腹腔に１％ＦＣＳを含むＤｕｌｂｅｃｃｏのリン酸緩衝液（Ｄｕｌｂｅｃｃｏｓ’ＰＢＳ、ニッスイ）を注入し、腹腔細胞を回収した。細胞を２回洗浄後、再度ＤｕｌｂｅｃｃｏのＰＢＳに懸濁し、９６ウェルプレートに注入（２×１０５細胞／０．２ｍｌ ｗｅｌｌ）した後、プレートをＣＯ２インキュベーターで２時間インキュベートした。吸着していない細胞を除いた後、各ウェルに、１０％ＦＣＳ、１００Ｕ／ｍｌペニシリンＧ、１００μｇ／ｍｌストレプトマイシンおよび５×１０−５Ｍの２−メルカプトエタノール、及び１００μｇ／ｍｌのｂＬＦ（和光社製）を含むＲＰＭＩ １６４０培地を２００μｌ注入した。対照として、ｂＬＦを含まない上記培地を用いた。プレートはＣＯ２インキュベーター中１８時間培養した。培養後、上清中のＩＬ−１２レベルをＥＬＩＳＡ法で測定した。結果をを図１４に示す。ｂＬＦを培地に添加すると、マクロファージのＩＬ−１２産生が著明に増強した。
以上の結果から、ラクトフェリンは、マクロファージのＩＬ−１２産生を誘導することが示された。
［試験例６］ セラミドのＩＦＮ−γ産生に与える影響
日本ＳＬＣ社のＢＡＬＢ／ｃマウス（３週齢、雄）を実験に供した。マウスは体重の平均値およびばらつきがほぼ等しくなるように１群３匹で、ラクトシルセラミド投与群（実験群）および、ラクトシルセラミド非投与群対照群）に群分けした。
セラミドは、ラクトシルセラミド（ウシバターミルク由来、和光純薬工業（株）、生化学用試薬１２６−０４４９１、ｌｏｔ．ＥＬＫ６１９１）を用いた。ラクトシルセラミドは０．５％ Ｔｗｅｅｎ２０を含む０．９％ ＮａＣｌ溶液に２００μｇ／ｍｌの濃度で分散させ、使用時まで−２０℃で保存した。使用時に解凍し、等量の滅菌ＰＢＳを加えよく混合した後、マウス一匹当たりラクトシルセラミド溶液２００μｌを１４日間毎日（ただし、土曜と日曜日を除く）腹腔内投与した（２０μｇ／マウス）。対照群には溶媒のみ（０．５％ Ｔｗｅｅｎ２０を含む０．９％ ＮａＣｌ溶液に等量の滅菌ＰＢＳを加えたもの）を投与した。
投与後１４日目にマウスから得た脾細胞を９６穴マイクロプレート（Ｆａｌｃｏｎ社製、Ｎｏ．３０７２）に１ウェル当たり４×１０５個播種した。培養には１０％ウシ胎児血清（日本バイオテスト社製、ｌｏｔ．１００８６−１）、ペニシリン１００Ｕ／ｍｌ、ストレプトマイシン１００μｇ／ｍｌ、および２−メルカプトエタノール（５×１０−５Ｍ）を含むＲＰＭＩ１６４０培地（Ｇｉｂｃｏ社製、Ｎｏ．１１８７５−０９３）を用いた。培養は４連で行い、ｂＬＦ（Ｍｅｇｇｌｅ社製、０または１００μｇ／ｍｌ）の存在下で２０時間培養した。培養後、培養上清中のＩＦＮ−γをＥＬＩＳＡキット（ＥＮＤＯＧＥＮ社製）により測定した。データはラクトシルセラミドの投与、およびｂＬＦによる刺激を要因とする２元配置分散分析（２×２、危険率５％）で分析した。
結果を図１５に示した。セラミドを投与しなかった場合、ｂＬＦの存在の有無に関係なくＩＦＮ−γの産生は見られなかった。セラミドを予め投与した場合、ｂＬＦの非存在下および存在下共にＩＦＮ−γ産生が有意に増加することが示された。さらに、これらセラミドおよびｂＬＦの二つの要因には相互作用が確認され（ｐ＝０．００１６）たことから、ラクトシルセラミドの投与はウシＬＦ刺激によるＩＦＮ−γ産生を増強することが判明した。
実施例１
乳清タンパク質を原料としない下記処方の牛乳アレルギー治療ミルクエピトレス（明治乳業（株）製）にウシＬＦを０．０１％配合した。
乳清タンパク質を原料として含む加水分解乳、明治のびやか（明治乳業（株）製）にウシＬＦを０．０１％配合した。
本発明により、ＬＦの経口摂取により、生体内のＩｇＥ抗体産生を抑制することが明らかにされた。
【図面の簡単な説明】
図１は、マウスにおける、１％ウシＬＦ経口摂取群（■：ｎ＝７）および対照群（□：ｎ＝８）に対するＯＶＡ抗原感作後の血清中の総ＩｇＥ濃度（平均値±ＳＤ；＊ｐ＜０．０５）を示す図である。
図２は、同上における血清中のＯＶＡ特異ＩｇＥ抗体価（平均値±ＳＤ；＊ｐ＜０．０５）を示す図である。
図３は、同上における脾細胞のＩＬ−４産生能を示す図である（細胞は各群でプールした）。●：１％ウシＬＦ経口摂取群、■：対照群。
図４は、マウスにおける、２．２％加熱処理ウシＬＦ経口摂取群（ｎ＝１９）および対照群（ｎ＝１７）に対するＯＶＡ抗原感作（３回）後の血清中の全ＩｇＥ抗体濃度を示す図である。（＊ｐ＜０．０５）
図５は、同上における血清中のＯＶＡ特異的なＩｇＥ抗体価を示す図である。（＊ｐ＜０．０５）
図６は、同上における、脾細胞のＯＶＡ特異的なＩＦＮ−γ産生を示す図である。（ウシＬＦ経口摂取群：ｎ＝１１、対照群：ｎ＝１０）
図７は、同上における、脾細胞のＯＶＡ特異的なＩＬ−４産生を示す図である。（＊ｐ＜０．０５）
図８は、マウスにおける、２．２％加熱処理ウシＬＦ経口摂取群（ｎ＝８）および対照群（ｎ＝７）に対するＯＶＡ抗原感作（４回）後の脾臓細胞のＯＶＡ特異的なＩＦＮ−γ産生を示す図である。
図９は、同上における、脾細胞のＯＶＡ特異的なＩＬ−４産生を示す図である。（Ｃｏｎｔｒｏｌ群；７匹、２．２％ｂＬＦ；８匹）
図１０は、マウスにおける、１％ウシＬＦ加水分解物の経口摂取群（ｎ＝７）および対照群（ｎ＝８）に対するＯＶＡ抗原感作後の血清中の全ＩｇＥ抗体濃度を示す図である。（＊ｐ＜０．０５）
図１１は、同上における、血清中のＯＶＡ特異的なＩｇＥ抗体価に与える影響を示す図である。
図１２は、同上における、脾細胞のＯＶＡ特異的なＩＦＮ−γ産生を示す図である。
図１３は、同上における、脾細胞のＯＶＡ特異的なＩＬ−４産生を示す図である。
図１４は、マウスの腹腔マクロファージを、１００μｇ／ｍｌのウシＬＦの存在下で培養 後の培養上清中のＩＬ−１２産生を示す図である。（＊＊ｐ＜０．０１）
図１５は、ラクトシルセラミドを腹腔投与後のマウスの脾細胞あるいはラクトシルセラミド非投与後の脾細胞を、１００μｇ／ｍｌのウシＬＦの存在下あるいは非存在下で培養した場合の、培養上清中のＩＦＮ−γ産生を示す図である。 Technical field
The present invention relates to an IgE antibody production inhibitor effective for the prevention or improvement of allergic diseases such as atopic dermatitis. The present invention also relates to a composition that stimulates the immune system in vivo to activate immunity.
Background art
Lactoferrin (LF) is an iron-binding glycoprotein contained in exocrine fluids such as saliva, tears, bile, pancreatic juice as well as milk, and is also released by neutrophils activated at the site of inflammation .
LF has a broad spectrum of biological activities. For example, antibacterial action, antiviral action, antitumor action, cancer metastasis formation inhibitory action and the like.
LF also affects cytokine secretion. For example, promotion of secretion of interleukin-8 (IL-8) from human neutrophils, regulation of secretion of IL-1, IL-6, and TNFα, suppression of gene expression of GM-CSF, and the like.
Selective production of cytokines plays a protective or pathogenic role in various pathologies. For example, most allergies are type I allergies involving IgE antibodies, but this IgE production is induced by a system centered on IL-4, while interferon-γ (IFN-γ) is the center. Production is controlled by the system. Production of IFN-γ is induced by IL-12 and IL-18, which are higher in the rank.
IFN-γ also has antiviral activity, antimicrobial activity, and antitumor activity due to various immunomodulating functions (J. Immunol., 130: 2011-2013, 1983; Proc. Natl. Acad. Sci. USA 85: 4874-4878, 1988).
Nakajima et al. Showed that when mouse splenocytes after oral administration of bovine LF (bLF) were cultured in the presence of mitogen (Con A), IFN-γ production was enhanced, whereas IL-4 production was enhanced. Have been reported (Biomedical Research, 29 (1): 27-33, 1999).
As described above, LF is involved in various biological activities and cytokine production control. Then, this invention makes it a subject to discover the new biological activity of LF, and to provide the pharmaceutical and functional food which use LF as an active ingredient based on this activity. Another object is to find an orally ingestible substance having an action of enhancing the biological activity of LF by combining with LF.
Disclosure of the invention
When the present inventors immunize mice orally ingested with LF, heat-treated LF, or LF hydrolyzate with ovalbumin (OVA), a major food allergen, production of OVA-specific IgE antibodies is suppressed. And the production of antigen-specific IL-4 in the spleen cell culture system was found to be suppressed. It was also found that when macrophages are cultured in the presence of LF, IL-12 production of macrophages is enhanced. Furthermore, it has been found that when LF and ceramide are used in combination, the production of IFN-γ in the spleen cell culture system is markedly enhanced. Inhibition of antigen-specific IgE antibody production by oral ingestion of LF is due to suppression of activation of IgE antibody-producing B lymphocytes by suppression of IL-4 production and shift of Th1 / Th2 balance to Th1 dominance by enhancement of IL-12 production It is thought to be due to. That is, by ingesting LF, it is possible to prevent a decrease in cellular immunity due to a shift toward Th2 immunity, and to prevent or improve allergic diseases, antibody-mediated nephritis, and the like. Furthermore, it can be seen that the significant production enhancement of IFN-γ by the combination of LF and ceramides is effective against viral diseases, bacterial infections, or tumors when LF and ceramides are combined.
That is, the present invention provides an IgE antibody production inhibitor for mammals including humans containing LF or a hydrolyzate thereof as an active ingredient.
The present invention also provides a composition for regulating immunity by suppressing IgE antibody production in mammals including humans containing LF or a hydrolyzate thereof as an active ingredient.
The present invention also relates to an IFN-γ production enhancer composition, an antiviral agent composition, an antibacterial infection agent composition and an antitumor agent composition for mammals including humans containing LF or a hydrolyzate thereof and ceramides. It provides things.
The present invention also provides an immunostimulant composition for mammals including humans containing LF or a hydrolyzate thereof and ceramides.
The present invention also provides use of LF or a hydrolyzate thereof for producing an IgE antibody production inhibitor for mammals including humans.
The present invention also provides use of LF or a hydrolyzate thereof for producing a composition for regulating immunity by suppressing IgE antibody production in mammals including humans.
The present invention also relates to an IFN-γ production enhancer composition, an antiviral agent composition, an antibacterial infection agent composition and an antitumor agent composition of mammals including humans, such as LF or a hydrolyzate thereof and ceramides. The use for manufacturing is provided.
Moreover, this invention provides use of LF or its hydrolyzate, and ceramides for manufacture of the immunostimulant composition of mammals including a human.
The present invention also provides a method for suppressing the production of IgE antibodies in mammals including humans, which comprises administering an effective amount of LF or a hydrolyzate thereof.
The present invention also provides a method for regulating immunity by suppressing IgE antibody production in mammals including humans, which comprises administering an effective amount of LF or a hydrolyzate thereof.
The present invention also relates to a method for enhancing IFN-γ production in mammals including humans characterized by administering an effective amount of LF or a hydrolyzate thereof and ceramides, or treatment of viral diseases, bacterial infections or tumors A method is provided.
Furthermore, the present invention provides a method for immunizing mammals including humans, characterized by administering an effective amount of LF or a hydrolyzate thereof and ceramides.
BEST MODE FOR CARRYING OUT THE INVENTION
In many allergic diseases, it is known that allergen-specific IgE is involved in its development, and in fact, allergen-specific IgE in serum is often found in allergic patients (Allergy Clin. Immunol., 16: 161, 1996). Therefore, suppressing IgE production is considered as one of the effective methods for preventing and treating allergies.
For the production of IgE, CD4+Cytokines produced by helper T cells (Th cells) play an important role. Th cells are broadly divided into two subpopulations, Th1 cells involved in cellular immunity and Th2 cells involved in humoral immunity, depending on the cytokines produced and differences in their functions, of which they are involved in the production of IgE. Are Th2 cells (J. Immunol., 136: 2348, 1986).
Th1 cells and Th2 cells have a relationship in which differentiation and functional expression are mutually suppressed by cytokines produced. That is, Th2 cells produce IL-4, IL-5, and IL-6 to positively regulate IgE production. Conversely, IFN-γ produced by Th1 cells suppresses IgE production by suppressing IgE class switching and Th2 cell responses. For this reason, IgE production and allergic diseases are characterized by an enhanced Th2 cell response (Nature, 383: 787, 1996). Therefore, it can be prevented or improved by inducing a Th1 cell response and returning the Th1 / Th2 balance to normal. Thus, attempts have been made to search for factors that are likely to induce a Th1 response in food ingredients, and some types of lactic acid bacteria (Int. Arch. Allergy Immunol., 115: 278, 1998) and nucleotides (Int. Arch. Allergy Immunol., 122: 33, 2000) has been shown to show the effect.
In recent years, the biological defense effect by oral administration of LF has been reported in various animal models. Therefore, the present inventors first examined the production of total IgE and antigen-specific IgE in serum when mice were used and LF was freely orally ingested. As a result, it has been clarified that when LF is orally ingested freely, total IgE antibody production and antigen-specific IgE production are suppressed.
It is known that the physiological activity of LF decreases by heat treatment (J. Pediatr., 90:29, 1977). Therefore, it can be considered that heat treatment of LF reduces the ability of LF to inhibit total IgE antibody production and antigen-specific IgE production. Therefore, the effect of heat-treated LF on total IgE antibody production and antigen-specific IgE production was examined. Furthermore, splenocytes were cultured in the presence of OVA, and cytokine (IFN-γ, IL-4) levels in the culture supernatant were examined. As a result, it has been clarified that, by heating LF, the antigen-specific IgE antibody production inhibitory action and the IL-4 production inhibitory action are not lost.
It was revealed that when LF and heat-treated LF were orally ingested, total IgE antibody production and antigen-specific IgE production were significantly suppressed compared to the control group. In addition, when spleen cells sensitized with an antigen were cultured in the presence of the antigen, it was revealed that IL-4 production was significantly reduced compared to the control group.
Therefore, the influence of hydrolyzed LF on total IgE antibody production and antigen-specific IgE production when freely ingested was examined. Antigen-sensitized spleen cells were cultured in the presence of the antigen, and the levels of cytokines (IFN-γ, IL-4) in the supernatant were examined. As a result, total IgE antibody production and antigen-specific IgE production tended to decrease in the LF hydrolyzate intake group. On the other hand, the production of IFN-γ showed an increasing tendency in the LF hydrolyzate ingestion group in the presence of each concentration of OVA, but for IL-4, between the LF hydrolyzate ingestion group and the control group There was no difference.
IL-12 is a cytokine produced by antigen-presenting cells such as monocytes, macrophages, and dendritic cells, and plays an important role in the production of IFN-γ and induction of helper T cell differentiation into Th1 (Blood, 84). : 4008, 1994; J. Leukoc. Biol., 55: 280, 1994). Gazzinelli et al. (J. Immunol., 153: 2533, 1994) infecting mice with Toxoplasma gondii increased IL-12 production in cells of the spleen and abdominal cavity, and further administered anti-IL-12 antibodies to these mice. Then, IFN-γ production of spleen cells was decreased, and IL-4 and IL-10 production was reported to increase. Nastala et al. (J. Immunol., 153: 1697, 1994) have shown that when IL-12 is administered to mice transplanted with cancer, the growth of cancer is suppressed and the concentration of IFN-γ in the blood increases. Heading.
Therefore, mouse peritoneal macrophages induced with thioglycolate medium were cultured with LF and examined for IL-12 production. As a result, it was revealed that LF induces macrophage IL-12 production.
By the way, atopic dermatitis, formerly considered to be mild dermatitis, is now counted as one of the intractable diseases seen from infants to adults, and has become a serious social problem. One of the causes is suspected to be an abnormal metabolism of sphingolipids in the epidermis. In fact, many patients with atopy have reduced ceramide levels in the epidermis. Ceramides are beginning to be used as beauty foods. Therefore, the present inventors thought that oral ingestion of a combination of LF and ceramides may further enhance the effect of LF and ceramides in preventing and improving allergic diseases more than LF alone.
Therefore, the effect of the combination of LF and ceramides on IFN-γ production was examined. As a result, it became clear that the combination of LF and ceramides remarkably enhanced IFN-γ production in splenocytes. As for sphingolipids, it is known that lysosphingolipids (sphingosine, sphingomyelin, rozophosphatidylcholine, etc.) in kefir (fermented milk) promote IFN-β production (Biotherapy: 115-123, 1994). . In addition, it has been reported that the administration of sphingosine increased IFN-β and IFN-γ in blood of infected mice (Osada, K. et al .: Animal Cell technology: Development towers the 21st Century 1995, 1067-1071. ).
From these results, suppression of the production of antigen-specific IgE antibody by ingestion of LF is the suppression of the activation of IgE antibody-producing B lymphocytes by suppression of IL-4 production and the Th1 / Th2 balance by enhancement of IL-12 production. This is thought to be due to a shift to Th1 dominance. Therefore, oral intake of LF is effective in preventing and improving allergic inflammatory diseases. Therefore, if an effective amount is added as a nutritional supplement to foods, allergic diseases can be prevented and improved in daily eating habits, and can also be used as a medicine for the diseases. Allergic inflammatory diseases include, for example, chronic bronchial asthma, atopic dermatitis, hay fever (allergic rhinitis), allergic vasculitis, allergic conjunctivitis, allergic gastroenteritis, allergic liver disorder, allergic cystitis, and Examples include allergic purpura. Alternatively, it can be expected to prevent or improve antibody-mediated nephritis.
In addition, when LF and ceramides are used in combination, the production of IFN-γ is remarkably enhanced. Therefore, by using LF and ceramides in combination, IgE due to a shift of Th1 / Th2 balance to Th1 dominance is achieved. It can be seen that antibody production is suppressed. IFN-γ is antiviral, antitumor, cytotoxic T lymphocyte induction, natural killer cell (NK cell) activity induction, neutrophil activation, macrophage activation, MHC class II expression promoting action, IL-2 receptor expression promoting action, Fc receptor expression promoting action and the like are known. In addition, IFN is a biological substance that originally plays a part in a defense mechanism against foreign substances in a living body, and has extremely high selective toxicity and high safety for a living body. Therefore, when a food containing a composition containing LF and ceramide is orally ingested, the biological cells are stimulated to promote the secretion of IFN-γ and the physiological activity of IFN-γ can be derived. Therefore, a composition combining LF and ceramides can be an antiviral agent, an antitumor agent, or an antimicrobial infection agent. Regarding the safety of LF, in the acute toxicity test and subacute toxicity test of bovine LF using rats, no abnormality was observed even when the maximum dose of 2,000 mg / kg / body weight / day was administered. (Milk Science Vol. 48, No. 3, p227-232, 1999). As for the safety of ceramide, LD50Is 5,000 mg / kg or more (FOOD Style Vol. 4, No. 10: 99-105, 2000).
Examples of the LF that can be used in the present invention include commercially available LF, mammals (eg, humans, cows, sheep, goats, horses, etc.) colostrum, transitional milk, regular milk, terminal milk, etc., or these milks. LF separated from non-fat milk, whey, etc. by conventional methods (for example, ion exchange chromatography), hydrolyzate by acid or enzyme, Apo LF obtained by removing iron with hydrochloric acid, citric acid, etc., Apo LF Metal saturated or partially saturated LF chelated with metals such as copper, zinc and manganese.
A known hydrolysis means can be used for the preparation of the LF hydrolyzate of the present invention. In the case of using an enzyme for hydrolysis, trypsin is used as an enzyme in the present invention, but other enzymes such as pepsin and papain are also used, but the enzyme is not limited to these enzymes as long as the hydrolyzate has the activity of the present invention. . The hydrolyzate may be deactivated by heating, then fractionated by a conventional method such as ultrafiltration, and concentrated. The obtained liquid hydrolyzate may be used as it is, or may be used after lyophilization.
Recombinant LF is available from Orla M. et al. Polypeptides having substantially the same amino acid sequence as described by Connelly et al. (US Pat. No. 5,766,939) are included. Also included are naturally occurring allelic variants and LF modified by insertion, substitution, or deletion of one or more amino acids as compared to natural LF. Recombinant LF also encompasses recombinant LF expressed in transgenic animals, such as cattle, where the glycosylation pattern may differ from that of native LF obtained from human milk.
The ceramides used in the present invention contain a ceramide-related substance, and for example, sphingolipids, particularly sphingoglycolipids are preferred. As glycosphingolipids, for example, the simplest glycosphingolipids are cerebrosides found in milk, brain and kidney, sulfatides with sulfate groups, ceramide oligohexosides with several neutral sugars, amino Examples include globoside with sugar and gangliosides with sialic acid. Although the structure has been revealed by the difference in glycosphingolipid sugar chain and more than 100 types, all having the action of the present invention are included. Preferred are lactosylceramide, galactosylceramide, glucosylceramide and the like. Also preferred is sphingomyelin (which is contained in milk) which is also a kind of phospholipid. In addition, chemical synthesis of natural ceramides has become possible through asymmetric synthesis techniques, and optically active ceramides have also been developed. It is Ceramide 2 and
In the present invention, the content of LF (including all LF and derivatives of LF having physiological activity equivalent to them and their hydrolysates) and ceramides is determined based on the total weight of the composition by ceramides, Preferably 0.00001-0.1 wt%, more preferably 0.00005-0.01 wt%, particularly preferably 0.0001-0.05 wt%, LF is based on the total weight of the composition, Preferably it is estimated to be 0.005 to 10% by weight, particularly preferably 0.01 to 5% by weight, and most preferably 0.01 to 3% by weight. Thus, for example, it is considered that the optimum amount can be easily determined by an experiment described later. Incidentally, the content of ceramide in breast milk is known, and is considered to be a temporary standard in the amount of ceramides.
According to the present invention, it has been clarified that oral intake of LF suppresses antigen-specific IgE antibody production. Therefore, a person skilled in the art can select a preferred form of LF and an effective amount applicable to the present invention from the above-mentioned various LFs based on the present invention, for example, using IgE antibody production as an index, clinical data, or clinical data. Taking into account the relationship between symptoms and serum IgE in patients, foods, dietary supplements, foods for the sick, infant formulas, health foods, health claims, functional foods, foods for specified health use, or pharmaceuticals It is possible to determine the shape and effective dose of the LF when used in the above. Therefore, the type of LF determined in this way and the effective dose thereof are included in the present invention. The technique of adding and processing LF to foods is well known. When an effective amount of LF is used as a medicine, it can be processed into various preparation forms known to those skilled in the art.
Example
The present invention will be described below with reference to test examples and examples, but the present invention is not limited to these test examples and examples.
In the following test examples, Test Examples 1 to 4 and 6 use 3-week-old male young BALB / c mice (Japan SLC; Shizuoka, Japan), and Test Example 5 uses 8-week-old female BALB / c mice ( Japan SLC; Shizuoka, Japan) was used. The significant difference test between the control group and the experimental group was performed by Student's t test.
[Test Example 1] Inhibition of IgE antibody production by bovine LF (bLF)
During the experiment period, mice were allowed to freely ingest LF-administered group (n = 7) with a diet containing casein as a protein source (AIN76 compliant) and 1% bovine LF (bLF, manufactured by DMV Japan). The control group (n = 8) was allowed to freely consume the above diet and water not containing bLF.
On the 5th and 19th days after the start of the experiment, 10 μg of ovalbumin (OVA, manufactured by Seikagaku Corporation) was immunized intraperitoneally with 4 mg of aluminum hydroxide per mouse. On day 26, blood was collected from the orbit and the total IgE antibody level and the OVA-specific IgE antibody level in the serum were measured by ELISA. Both serum total IgE antibody levels (FIG. 1) and OVA-specific IgE antibody levels (FIG. 2) were more significant in mice in the bLF group than in the control group (*p <0.05).
[Test Example 2] IL-4 production inhibitory action of bLF
In Test Example 1, the spleen was removed on the 26th day to prepare a spleen cell suspension. After hemolysis, splenocytes were cultured for 72 hours in the presence of various concentrations (5, 10, 50, and 100 μg / ml) of OVA. After culture, the IL-4 level in the culture supernatant was measured by ELISA. In the bLF administration group (n = 7), IL-4 levels in each concentration of OVA were all reduced compared to those in the control group (n = 8) (FIG. 3).
[Test Example 3] IgE antibody and cytokine production inhibitory action of heat-treated bLF
A diet [bLF (+)] obtained by adding 2.2% of bLF to the diet of Test Example 1 was heated at 70 ° C. for 1 hour. As a control, a diet without bLF [bLF (−)] was similarly heated at 70 ° C. for 1 hour.
Mice were allowed to freely take bLF (+) (experimental group) or bLF (−) (control group) during the experimental period. On
When three immunizations were performed, total IgE antibody levels and OVA-specific IgE antibody levels in the serum of the experimental group were significantly reduced compared to those of the control group (FIGS. 4 and 5). In addition, IFN-γ production of splenocytes at various concentrations of OVA (0, 50, 100, and 200 μg / ml) showed almost no difference between the experimental group and the control group (FIG. 6). On the other hand, IL-4 production of spleen cells in the above-mentioned concentrations of OVA was generally lower in the experimental group than in the control group, and under the stimulation of 200 μg / ml OVA, the experimental group was the control group. (Fig. 7).
When immunization was performed 4 times, IFN-γ production in spleen cells at various concentrations of OVA (0, 50, 100, 200, and 400 μg / ml) was similar between both groups as in the case of 3 immunizations. Little difference was seen (Figure 8). On the other hand, IL-4 production in the OVA at each concentration was generally significantly decreased in the experimental group compared to the control group (FIG. 9).
From the above results, it was found that bLF does not lose its antigen-specific IgE antibody production inhibitory effect and IL-4 production inhibitory effect by heating.
[Test Example 4] Inhibitory action of hydrolyzed bLF on IgE antibody and cytokine production
(1) Preparation of hydrolyzed bLF
Trypsin (Wako Pure Chemical Industries, Ltd., 207-089891, for biochemistry, porcine pancreas, 4500 USP trypsin units / mg) was dissolved in sterile PBS to obtain a trypsin stock solution (× 500, 50 mg / ml). 10 g of bLF (Wako Pure Chemical Industries, Ltd., 127-04122, lot.KSG7724) was added to 25 mM CaCl2-It dissolved so that it might become 1% in 0.1M Tris-HCl (pH 8.2). This 1 L bLF solution was heated to 37 ° C., 2 ml of trypsin stock solution was added, and the mixture was reacted at 37 ° C. for 4 hours. After the reaction, the enzyme was inactivated by heating at 80 ° C. for 30 minutes. After centrifugation at 1700 × g for 20 minutes, the supernatant was collected and sterilized by filtration through a 0.45 μm filter. The sample was treated with a microacylator S1 (manufactured by Asahi Kasei Kogyo) to remove the salt in the sample. The solution was sterilized by filtration again with a 0.45 μm filter and then stored at −20 ° C. until the test. The yield of bLF hydrolyzate was almost 100%. Therefore, the bLF hydrolyzate concentration was 1%.
Mice were allowed to freely eat the diet of Test Example 1 and a solution containing 1% of bLF hydrolyzate. In the control group, the same solid feed and water containing no bLF were freely ingested. On
Serum total IgE antibody levels and OVA-specific IgE levels in the experimental group tended to decrease compared to those in the control group (FIGS. 10 and 11).
IFN-γ production of splenocytes at various OVA concentrations tended to be generally higher in the experimental group than in the control group (FIG. 12). On the other hand, there was almost no difference in IL-4 production between the experimental group and the control group (FIG. 13).
[Test Example 5] Effect of LF on IL-12 production
IL-12 is a relatively newly discovered cytokine, but is known to act on T cells and NK cells to induce production of IFN-γ (J. Exp. Med., 173: 869). -879, 1991; J. Exp. Med., 177: 1199-1204, 1993). Stimulation of bacterial cell components such as LPS is effective in inducing IL-12 production, and the production cells include a wide variety of cells such as macrophages, B cells, and neutrophils.
The present inventors cultured peritoneal macrophages in a medium supplemented with bLF (manufactured by Wako), and measured the level of IL-12 in the culture supernatant by ELISA.
Intraperitoneal macrophages were activated by intraperitoneally injecting 2.5 ml of thioglycolate medium (manufactured by DIFCO) into mice that freely ingested an MF diet (manufactured by Oriental Yeast). Four days later, Dulbecco's phosphate buffer containing 1% FCS (Dulbeccos'PBS, Nissui) was injected into the peritoneal cavity, and peritoneal cells were collected. The cells were washed twice and then resuspended in Dulbecco's PBS and injected into a 96-well plate (2 × 105Cells / 0.2 ml well)2Incubated for 2 hours in incubator. After removing non-adsorbed cells, each well contained 10% FCS, 100 U / ml penicillin G, 100 μg / ml streptomycin and 5 × 10-5200 μl of RPMI 1640 medium containing M 2-mercaptoethanol and 100 μg / ml bLF (manufactured by Wako) was injected. As a control, the above medium without bLF was used. Plate is CO2The cells were cultured for 18 hours in an incubator. After culture, the IL-12 level in the supernatant was measured by ELISA. The results are shown in FIG. When bLF was added to the medium, macrophage IL-12 production was markedly enhanced.
From the above results, it was shown that lactoferrin induces IL-12 production of macrophages.
[Test Example 6] Effect of ceramide on IFN-γ production
Japan SLC BALB / c mice (3 weeks old, male) were subjected to the experiment. The mice were divided into 3 groups per group so that the average values and variations in body weight were almost equal, and the group was divided into lactosylceramide administration group (experimental group) and lactosylceramide non-administration group control group.
As the ceramide, lactosylceramide (derived from bovine buttermilk, Wako Pure Chemical Industries, Ltd., biochemical reagent 126-04491, lot. ELK6191) was used. Lactosylceramide was dispersed in a 0.9% NaCl solution containing 0.5% Tween 20 at a concentration of 200 μg / ml and stored at −20 ° C. until use. After thawing at the time of use, after adding an equal volume of sterile PBS and mixing well, 200 μl of lactosylceramide solution per mouse was administered intraperitoneally every day for 14 days (excluding Saturday and Sunday) (20 μg / mouse). The control group was administered with solvent alone (0.9% NaCl solution containing 0.5% Tween 20 plus an equal amount of sterile PBS).
On the 14th day after administration, spleen cells obtained from mice were placed in a 96-well microplate (Falcon, No. 3072) at 4 × 10 4 per well.5Individual seeded. For the culture, 10% fetal bovine serum (manufactured by Nippon Biotest Co., Ltd., lot.10086-1), penicillin 100 U / ml,
The results are shown in FIG. When ceramide was not administered, IFN-γ production was not seen regardless of the presence or absence of bLF. When ceramide was pre-administered, IFN-γ production was shown to increase significantly both in the absence and presence of bLF. Furthermore, since interaction between these two factors, ceramide and bLF, was confirmed (p = 0.016), it was found that administration of lactosylceramide enhances IFN-γ production by bovine LF stimulation.
Example 1
0.01% of bovine LF was blended in milk allergy treatment milk epitres (manufactured by Meiji Dairies Co., Ltd.) having the following prescription without using whey protein as a raw material.
Hydrolyzed milk containing whey protein as a raw material, Meiji Nobiyaka (manufactured by Meiji Dairies Co., Ltd.) was mixed with 0.01% bovine LF.
According to the present invention, it has been clarified that oral ingestion of LF suppresses in vivo IgE antibody production.
[Brief description of the drawings]
FIG. 1 shows the total IgE concentration in serum after sensitization to OVA antigen (mean value ± SD; mean value ± SD;) in a 1% bovine LF oral intake group (■: n = 7) and a control group (□: n = 8).*p <0.05).
FIG. 2 shows the above-mentioned serum OVA-specific IgE antibody titer (mean ± SD;*p <0.05).
FIG. 3 is a diagram showing the IL-4 production ability of splenocytes in the same manner (cells were pooled in each group). ●: 1% bovine LF oral intake group, ■: control group.
FIG. 4 shows the total IgE antibody concentration in the serum after OVA antigen sensitization (3 times) in the mice with the 2.2% heat-treated bovine LF oral intake group (n = 19) and the control group (n = 17). FIG. (*p <0.05)
FIG. 5 is a graph showing the OVA-specific IgE antibody titer in serum as described above. (*p <0.05)
FIG. 6 is a diagram showing OVA-specific IFN-γ production of splenocytes in the same manner as above. (Bovine LF oral intake group: n = 11, control group: n = 10)
FIG. 7 is a diagram showing OVA-specific IL-4 production of splenocytes in the same manner as above. (*p <0.05)
FIG. 8 shows OVA-specific IFN of spleen cells in mice after sensitization of OVA antigen (4 times) to 2.2% heat-treated bovine LF oral intake group (n = 8) and control group (n = 7). FIG. 6 is a diagram showing γ production.
FIG. 9 is a diagram showing OVA-specific IL-4 production of splenocytes in the same manner as above. (Control group: 7 animals, 2.2% bLF; 8 animals)
FIG. 10 is a graph showing the total IgE antibody concentration in serum after OVA antigen sensitization in mice orally administered with 1% bovine LF hydrolyzate (n = 7) and control group (n = 8). . (*p <0.05)
FIG. 11 is a diagram showing the influence on serum OVA-specific IgE antibody titer in the same as above.
FIG. 12 is a diagram showing OVA-specific IFN-γ production of splenocytes in the same manner as above.
FIG. 13 is a diagram showing OVA-specific IL-4 production of splenocytes in the same manner as above.
FIG. 14 shows IL-12 production in the culture supernatant after culturing mouse peritoneal macrophages in the presence of 100 μg / ml bovine LF. (**p <0.01)
FIG. 15 shows culture supernatants when mouse spleen cells after intraperitoneal administration of lactosylceramide or spleen cells after non-administration of lactosylceramide were cultured in the presence or absence of 100 μg / ml bovine LF. It is a figure which shows IFN-gamma production in it.
Claims (7)
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| US20040152624A1 (en) * | 2002-12-06 | 2004-08-05 | Atul Varadhachary | Oral lactoferrin in the treatment of sepsis |
| JP2005350452A (en) * | 2004-05-14 | 2005-12-22 | Meiji Milk Prod Co Ltd | Composition having IgE production inhibitory action and method for inhibiting IgE production |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH01233226A (en) * | 1988-02-02 | 1989-09-19 | Roussel Uclaf | Utilization of milk protein in production of antiviral agent |
| JPH0559081A (en) * | 1991-08-29 | 1993-03-09 | Kirin Brewery Co Ltd | New sphingoglycolipid, its production and use |
| JPH09194388A (en) * | 1996-01-22 | 1997-07-29 | Morinaga Milk Ind Co Ltd | Anti-angiogenic agent |
| WO1997026891A1 (en) * | 1996-01-22 | 1997-07-31 | Beiersdorf Ag | Sphingolipids effective against bacteria, parasites, protozoans, fungi and viruses |
| JPH09294585A (en) * | 1996-04-24 | 1997-11-18 | Suu Chin-Shian | Recombined eukaryotic plasmid containing allergen gene and its use for preventing and/or treating allergic disease |
| JPH1045618A (en) * | 1996-07-26 | 1998-02-17 | Morinaga Milk Ind Co Ltd | Apoptosis inducer |
| WO1999041266A1 (en) * | 1998-02-12 | 1999-08-19 | Emory University | Sphingolipid derivatives and their methods of use |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5936076A (en) * | 1991-08-29 | 1999-08-10 | Kirin Beer Kabushiki Kaisha | αgalactosylceramide derivatives |
| FR2728793A1 (en) * | 1994-12-28 | 1996-07-05 | Oreal | Compsns. for treating sensitive skin |
| JP2001519815A (en) * | 1997-04-10 | 2001-10-23 | エイジェニックス・インコーポレイテッド | Use of lactoferrin in the treatment of allergen-induced disorders |
-
2001
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- 2001-01-12 WO PCT/JP2001/000161 patent/WO2001051079A1/en active IP Right Grant
- 2001-01-12 CN CNB2004100577068A patent/CN100384472C/en not_active Expired - Lifetime
- 2001-01-12 CN CNB018036775A patent/CN1205998C/en not_active Expired - Lifetime
- 2001-01-12 AU AU25515/01A patent/AU782539B2/en not_active Ceased
- 2001-01-12 JP JP2001551502A patent/JP4787445B2/en not_active Expired - Fee Related
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01233226A (en) * | 1988-02-02 | 1989-09-19 | Roussel Uclaf | Utilization of milk protein in production of antiviral agent |
| JPH0559081A (en) * | 1991-08-29 | 1993-03-09 | Kirin Brewery Co Ltd | New sphingoglycolipid, its production and use |
| JPH09194388A (en) * | 1996-01-22 | 1997-07-29 | Morinaga Milk Ind Co Ltd | Anti-angiogenic agent |
| WO1997026891A1 (en) * | 1996-01-22 | 1997-07-31 | Beiersdorf Ag | Sphingolipids effective against bacteria, parasites, protozoans, fungi and viruses |
| JPH09294585A (en) * | 1996-04-24 | 1997-11-18 | Suu Chin-Shian | Recombined eukaryotic plasmid containing allergen gene and its use for preventing and/or treating allergic disease |
| JPH1045618A (en) * | 1996-07-26 | 1998-02-17 | Morinaga Milk Ind Co Ltd | Apoptosis inducer |
| WO1999041266A1 (en) * | 1998-02-12 | 1999-08-19 | Emory University | Sphingolipid derivatives and their methods of use |
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| CN1395490A (en) | 2003-02-05 |
| WO2001051079A1 (en) | 2001-07-19 |
| CN1626240A (en) | 2005-06-15 |
| CN100384472C (en) | 2008-04-30 |
| CN1205998C (en) | 2005-06-15 |
| AU782539B2 (en) | 2005-08-04 |
| HK1076735A1 (en) | 2006-01-27 |
| AU2551501A (en) | 2001-07-24 |
| NZ519957A (en) | 2003-08-29 |
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