JP2004507262A - Methods for identifying substances that clearly affect the inflammatory state of chronic inflammatory airway disease - Google Patents

Abstract

The present invention relates to substances that regulate receptors involved in the inflammatory process, the regulated function of which affects inflammatory diseases.

Description

【００３３】
表２：ＦＰＲＬ−１レセプターの発現レベル：”ａｖｇ ｄｉｆｆ”値、チップ上のハイブリダイゼーションシグナルの強度の相対的指標、各患者について示してある；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。
【００３４】
【表２】

ＣＯＰＤ喫煙者と健康な喫煙者との間の比較に関するＰ値：０．０２
【００３５】
ＦＰＲＬ−１レセプターに関するチップデータはＴａｑＭａｎ解析（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｌｅｄ Ｂｉｏｓｙｓｔｅｍｓ）によって確認する。ＴａｑＭａｎによって得られる変化倍数は遺伝子チップからのデータとよく似ている（表３）。
【００３６】
【表３】
表３：遺伝子チップ及びＴａｑＭａｎによって決定された、ＣＯＰＤ喫煙者におけるＦＰＲＬ−１レセプターのアップレギュレーション。ＣＯＰＤ喫煙者と健康喫煙者間の６つの比較におけるチップデータによるＦＰＲＬ−１の変化倍数測定は同じサンプルのＴａｑＭａｎによって確認され、相対アップレギュレーションはＧＡＰＤＨをハウスキーピング遺伝子として計算する。

【００３７】
同定された別のディファレンシャル発現核酸配列は、Ｇ−プロテイン共役レセプターのファミリーに属するＨＭ７４レセプター（配列番号２０およ２１参照）をコードする。ＨＭ７４レセプターはヒト単球ライブラリーからクローニングされた（Ｎｏｍｕｒａら、１９９３）。これまでにそのリガンドは同定されていない。ＨＭ７４レセプターはＣＯＰＤ喫煙者において健康喫煙者に比較して首尾一貫してアップレギュレーションされていることが見いだされる（５４．８％）。これは、４２組の比較による「変化倍数」値（表５）および”ａｖｇ ｄｉｆｆ”値によって示してある（表６）。
【００３８】
【表４】
表５．ＨＭ７４レセプターの発現パターン：ＣＯＰＤおよび健康な喫煙者間で比較した４２組について計算した変化倍数。２倍より高い値および−２倍より低い値のみを脱調節されたものと考える。従って、ＨＭ７４レセプターは２３例でアップレギュレーションされ、１７例で調節されていない。

【００３９】
【表５】
表６：ＨＭ７４レセプターの発現レベル。”ａｖｇ ｄｉｆｆ”値、チップ上のハイブリダイゼーションシグナルの強度の相対的指標、各患者について示してある；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００４０】
ＨＭ７４レセプターに関するチップデータはＴａｑＭａｎ解析（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｌｅｄ Ｂｉｏｓｙｓｔｅｍｓ）によって確認する。ＴａｑＭａｎによって得られる変化倍数は遺伝子チップからのデータとよく似ている（表７）。
【００４１】
【表６】
表７．遺伝子チップ及びＴａｑＭａｎによって決定された、ＣＯＰＤ喫煙者におけるＨＭ７４レセプターのアップレギュレーション。ＣＯＰＤ喫煙者と健康喫煙者間の６つの比較におけるチップデータによるＨＭ７４の変化倍数測定は同じサンプルのＴａｑＭａｎによって確認され、相対アップレギュレーションはＧＡＰＤＨをハウスキーピング遺伝子として計算する。

【００４２】
同定された別の別のディファレンシャル発現核酸配列は、Ｎ−アセチルガラクトサミンまたはＮ−アセチルグルコサミンを認識し結合するＩＩ型膜タンパク質である、ＡＩＣＬレセプター（活性化−誘導Ｃ−型レクチン）（Ｏｄａら、１９８８）をコードする（配列番号５および６参照）。これはリンパ組織および造血細胞およびＮＫおよびＴ細胞で発現している。この発現はリンパ細胞活性化の過程、およびＰＭＡ刺激後に誘導される（Ｈａｍａｎｎら、１９９７）。ＡＩＣＬレセプターのホモログはリンパ細胞のシグナル伝達およびリンパ細胞の増殖に関与しているので、ＡＩＣＬレセプターがこれらの過程にも関与していると仮定することはもっともらしい（Ｈａｍａｎｎら、１９９３）。
ＡＩＣＬレセプターは健康喫煙者に比較してＣＯＰＤ喫煙者では首尾一貫してアップレギュレーション（６６．７％）されることが見いだされる。これは、４２組の比較による「変化倍数」値（表８）および”ａｖｇ ｄｉｆｆ”値によって示してある（表９）。
【００４３】
【表７】
表８．ＡＩＣＬレセプターの発現パターン：ＣＯＰＤおよび健康な喫煙者間で比較した４２組について計算した変化倍数。２倍より高い値および−２倍より低い値のみを脱調節されたものと考える。従って、ＡＩＣＬレセプターは２８例でアップレギュレーションされ、１４例で調節されていない。

【００４４】
【表８】
表９．ＡＩＣＬレセプターの発現レベル：”ａｖｇ ｄｉｆｆ”値、チップ上のハイブリダイゼーションシグナルの強度の相対的指標、各患者について示してある；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００４５】
同定された、別のディファレンシャル発現核酸配列はＩＬＴ１レセプター（イムノグロブリン様転写物１）をコードする（配列番号１１および１２参照）。ＩＬＴ１レセプターはＭＨＣクラスＩ分子に対するＮＫ細胞レセプターに類似した活性化レセプターのサブセットに関連するＩｇスーパーファミリーに属する。ＩＬＴ１レセプターは６９ｋＤａのグリコシル化膜貫通レセプターであり、肺及び肝臓および単球、顆粒球、マクロファージおよび樹状細胞で主として発現している（ＳａｍａｒｉｄｉｓとＣｏｌｏｎｎａ　１９９７）。抗体と架橋されるとＩＬＴ１レセプターはＦｃレセプターのγ−鎖と相互作用する（Ｆｃ_εＲＩ_γ）（Ｎａｋａｊｉｍａら、１９９９）。ＩＬＴ１レセプターは、健康喫煙者に比較してＣＯＰＤ喫煙者では首尾一貫してアップレギュレーション（５９．５％）されていることが見いだされる。これは、４２組の比較による”ａｖｇ ｄｉｆｆ”値によって示してある（表１０）。ＣＯＰＤ喫煙者と健康喫煙者との間の比較に関するｐ値は０．０１であった。
【００４６】
【表９】
表１０．ＩＬＴ１レセプターの発現レベル：各患者についての”ａｖｇ ｄｉｆｆ”値は３つの患者グループについての平均値及びメジアン値と共に示してある。；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００４７】
同定された、ディファレンシャル発現核酸配列は、マクロファージで高度に発現することが知られているＳＨＰＳ−１レセプター（ＳＩＲＰ−α１、ＭＹＤ１、ＭＦＲ）（Ｆｕｊｉｏｋａら、１９９６、Ｋｈａｒｉｔｏｎｅｎｋｏｖら、１９９７、Ｂｒｏｏｋｅら１９９８）（配列番号３および４参照）をコードする。ＳＨＰＳ−１レセプターはイムノグロブリンスーパーファミリーに属する膜貫通糖タンパク質である。これは３つの細胞外Ｉｇ様ドメイン、予測されるチロシンリン酸化部位を有する細胞質尾部およびイムノレセプターチロシンベースの阻害モチーフ（ＩＴＩＭ）を含む。レセプターチロシンキナーゼが活性化されるとＳＨＰＳ−１レセプターのリン酸化が起こり、ＳＨＰ−１との会合（マクロファージにおいて）またはＳＨＰ−２との会合（非造血細胞において）が生じる（Ｖｅｉｌｌｅｔｔｅら、１９９８）。さらに、他のタンパク質がＳＨＰＳ−１レセプターの細胞質内ドメインと会合することが見いだされており、従って、ＳＨＰＳ−１レセプターが足場タンパク質として機能すると仮定することはもっともらしい。
ＳＨＰＳ−１レセプターは健康喫煙者に比較してＣＯＰＤ喫煙者においては首尾一貫してダウンレギュレーション（７３．８％）されていることが見いだされる。このことは、４２組の比較による「変化倍数」値（表１１）および”ａｖｇ ｄｉｆｆ”値（表１２）によって示される。ＣＯＰＤ喫煙者と健康喫煙者との間の比較に関するｐ値は０．００５である。
【００４８】
【表１０】
表１１．ＳＨＰＳ−１レセプターの発現パターン：ＣＯＰＤおよび健康な喫煙者間で比較した４２組について計算した変化倍数。２倍より高い値および−２倍より低い値のみを脱調節されたものと考える。従って、ＳＨＰＳ−１レセプターは２９例でダウンレギュレーションされ、１３例で制御されていない。

【００４９】
【表１１】
表１２．ＳＨＰＳ−１レセプターの発現レベル：各患者についての”ａｖｇ ｄｉｆｆ”値は３つの患者グループについての平均値及びメジアン値と共に示してある。；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００５０】
同定された、別のディファレンシャル発現核酸配列は、エンドプラズミックレティキュラムにおけるタンパク質フォールディングおよびアッセンブルに重要な機能を有するレセプターであるＫＤＥＬレセプター１（配列番号７および８参照）をコードする。これは、アミノ酸配列Ｋ−Ｄ−Ｅ−Ｌを有する可溶性タンパク質を認識し、それらのタンパク質をエンドプラズミックレティキュラムに結合後回収する（Ｔｏｗｎｓｌｅｙら、１９９３）。ＫＤＥＬレセプター１はゴルジ複合体におけるタンパク質輸送の制御に関与しているかも知れない。リガンドが結合するとＫＤＥＬレセプターは二量体化しＡＲＦ ＧＡＰ（ＡＤＰリボシル化因子のＧＴＰａｓｅ活性化タンパク質）と相互作用する（Ａｏｅら、１９９７）。これは健康喫煙者に比較してＣＯＰＤ喫煙者においては首尾一貫してダウンレギュレーション（７１．４％）されていることが見いだされる。このことは、”ａｖｇ ｄｉｆｆ”値（表１３）によって示される。ＣＯＰＤ喫煙者と健康喫煙者との間の比較に関するｐ値は０．００３である。
【００５１】
【表１２】
表１３．ＫＤＥＬレセプターの発現レベル：各患者についての”ａｖｇ ｄｉｆｆ”値は３つの患者グループについての平均値及びメジアン値と共に示してある。；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００５２】
同定された別のディファレンシャル発現核酸配列はマクロファージコロニー刺激因子−１レセプター前駆体（ＣＳＦ−１レセプター、ｃ−ｆｍｓ）をコードする；配列番号９および１０参照。ＣＳＦ−１レセプターはレセプターチロシンキナーゼのサブファミリーに属する。ＣＳＦ−１レセプターの活性化は複数のタンパク質、例えば、ＣＳＦ−１レセプター、Ｓｈｃ、ＰＩ３Ｋ、Ｇｒｂ２、Ｃｂｌ、ＳＨＰ−１、Ｓｒｃ、の複合体形成を生じさせる。更に、リガンドの結合は、Ｃｂｌ、ＳＴＡＴ３、ＳＴＡＴ５ａ、ｐ８５ＰＩ３Ｋ、ＳＨＰ−１、Ｖａｖのような細胞質タンパク質および細胞骨格形成に関与するタンパク質過剰の迅速なチロシンリン酸化を引き起こす（Ｙｅｕｎｇら、１９９８）。ＣＳＦ−１レセプターは単核食細胞の生存、増殖、分化および形態を制御している（Ｈａｍｐｅら、１９８９）。ＣＳＦ−１レセプターは健康喫煙者に比較してＣＯＰＤ喫煙者においては首尾一貫してダウンレギュレーション（４５．２％）していることが見いだされる。このことは、”ａｖｇ ｄｉｆｆ”値（表１４）によって示される。ＣＯＰＤ喫煙者と健康喫煙者との間の比較に関するｐ値は０．００２である。
【００５３】
【表１３】
表１４．　ＣＳＦ−１レセプターの発現レベル：各患者についての”ａｖｇ ｄｉｆｆ”値は３つの患者グループについての平均値及びメジアン値と共に示してある。；ＯＳは閉塞症喫煙者、ＨＳは健康な喫煙者、ＮＳは非喫煙者を表す。

【００５４】
１．６．　 ＤＮＡ チップデータの確認および診断のための ＴａｑＭａｎ 解析の使用
ＤＮＡチップによって得られたｍＲＮＡ発現プロファイルは同じＲＮＡ調製物によりＴａｑＭａｎ解析によって確認される。さらに、この方法は培養細胞株およびヒトから単離した細胞中のＦＰＲＬ−１レセプターのｍＲＮＡレベルを測定して疾病の進行をモニターするためにも応用される。
１０ｎＭのレチノイン酸で３日間処理したＵ９３７細胞から単離したＲＮＡを用いてＦＰＲＬ−１レセプターのｍＲＮＡレベルを測定するための反応条件を最適化しＦＰＲＬ−１レセプターおよびハウスキーピング遺伝子としてＧＡＰＤＨ（グリセルアルデヒド−３−リン酸デヒドロゲナーゼ）のための標準曲線を設定する。ＦＰＲＬ−１レセプターの定量は以下のプライマーを用いて行う：フォワードプライマー（ＦＰ）（配列番号１７参照、逆プライマー（ＲＰ）、配列番号１８参照、およびレポーター色素ＦＡＭで５’を、消光色素ＴＭＡＲＡで３’末端を標識されたＴａｑＭａｎプローブ（ＴＰ）、配列番号１９参照。
【００５５】
ＧＡＰＤＨのｍＲＮＡレベルを測定するために、市販のキット”ＴａｑＭａｎ ＧＡＰＤＨ ＣＯｎｔｒｏｌ Ｒｅａｇｅｎｔ”（Ｐ／Ｎ ４０２８６９）（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使用する。ＧＰＰＤＨプローブはＪＯＥをレポーター色素として、ＴＭＡＲＡを消光色素として標識する。ＲＴ−ＰＣＲ反応は”ＴａｑＭａｎ ＥＺ ＲＴ−ＰＣＲ Ｃｏｒｅ Ｒｅａｇｅｎｔｓ”（Ｐ／Ｎ Ｎ８０８−０２３６） （Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）キットを使用して行う。ＦＰＲＬ−１およびＧＡＰＤＨのための標準曲線は１μＭのレチノイン酸で処理したＵ９３７細胞からのＲＮＡ濃度を、各アッセイについて０，５、１０、２５、５０、１００ｎｇの範囲で増加させて行う。反応混合物は１ｘＴａｑＭａｎ ＥＺ−バッファー、３ｍＭ Ｍｎ（Ｏａｃ）_２、３００μＭ ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、および６００μＭ ｄＵＴＰ、２．５Ｕ ｒＴｔｈ ＤＮＡポリメラーゼ、０．２５Ｕ ＡｍｐＥｒａｓｅ ＵＮＧを総体積２５μｌ中に含む。ＦＰＲＬ−１レセプタ−の解析のためには、反応混合物は３００ｎＭのＦＰおよびＲＰおよび１００ｎＭのＴＰを含む。ＧＡＤＰＨレベルを測定するためのプライマー濃度は各プライマーについて２００ｎＭでありＧＡＰＤＨ ＴａｑＭａｎプローブについては１００ｎＭである。
【００５６】
ＦＰＲＬ−１レセプターおよびＧＡＰＤＨのヒト患者および細胞株におけるｍＲＮＡレベルを測定するため、反応当たり１６〜５０ｎｇのＲＮＡを使用する。全てのサンプルは３つ組で走らせる。反応はＭｉｃｒｏＡｍｐ Ｏｐｔｉｃａｌ キャップでシールした”ＭｉｃｒｏＡｍｐ Ｏｐｔｉｃａｌ ９６−穴反応プレート“（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ） により、ＡＢＩ ＰＲＩＳＭ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）中で行う。ＰＣＲ条件は、５０℃２分間、６０℃３０分間、９５℃５分間、続いて９４℃２０秒間および５９℃１分間を４０サイクルである。データ解析はＦＰＲＬ−１レセプターおよびＧＡＰＤＨのｍＲＮＡレベルを標準曲線に従って決定するか、または、ＦＰＲＬ−１レセプターのＣ_Ｔ値をＧＡＰＤＨのＣ_Ｔ値に直接関連づけるかして行う。後者は、どちらの反応の効率も９５％付近なのでこれらの遺伝子について行うことができる。同じ方法を用いてＣＯＰＤ患者から単離したｍＲＮＡレベルを調べ疾病の診断または想定される活性薬剤で治療後にその治療の成功性をモニターする。
実施例１．５において述べた他のレセプターはそれぞれに適切なプライマーを用いてしかるべく調べられる。
【００５７】
１．７．　細胞系
ヒト単球／マクロファージ細胞株ＨＬ−６０、Ｕ９３７、ＴＨＰ−１およびＭｏｎｏＭａｃ６を細胞モデル系として使用する。１０％ＦＣＳを含み１００Ｕ／ｍｌのペニシリン、１００μｇ／ｍｌのストレプトマイシン、２ｍＭグルタミンおよび１ｘ非必須アミノ酸を添加したＲＰＭＩ １６４０培地で細胞を増殖させる。ＭｏｎｏＭａｃ６細胞用の培地は５ｍｌ／ｌ ＯＰＩ培地サプリメント（Ｓｉｇｍａ）も含む。ＭｏｎｏＭａｃ６はもっぱら２４穴プレートで培養する。全ての細胞は５％ＣＯ_２を含む湿大気中で３７℃にて維持し、定期的にマイコプラズマによる汚染についてテストする。１０ｎＭ ＰＭＡ（ホルボル１２ミリステート−１３アセテート）を培地に添加して分化を行わせる。
【００５８】
１．８．　 ＦＰＲＬ−１ レセプターのクローニング
ＦＰＲＬ−１レセプターは１μＭのレチノイン酸で３日間処理したＵ９３７細胞から抽出した総ＲＮＡからクローン化する。５μｇのＲＮＡを５ｎｇのオリゴ（ｄｔ）_１８プライマー、１ｘ第１鎖バッファー、１０ｍＭ ＤＴＴ、０．５ｍＭ ｄＮＴＰｓおよび２ＵのＳｕｐｅｒｓｃｒｉｐｔＩＩ（Ｇｉｂｃｏ　ＢＲＬ Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）で４２℃にて５０分間逆転写してｃＤＮＡにする。次に、反応を７０℃、１５分間で停止させｃＤＮＡ濃度をＵＶ分光計で測定する。ＦＰＲＬ−１レセプターの増幅には１００ｎｇのｃＤＮＡおよび１０ｐｍｏｌのＦＰＲＬ−１レセプターについて配列特異的なプライマー（フォワードプライマーａｔｔＢ１；配列番号１５、および逆プライマーａｔｔＢ２；配列番号１６）をＰＣＲに使用する。反応条件は以下の通り：Ｔａｑポリメラーゼを用いて、９４℃２分間、続いて３０サイクルの（９４℃３０秒間、５３℃３０秒間、７２℃９０秒間）、続いて７２℃にて７分間。反応混合物を２％アガロースゲル上で分離し、約１０００ｂｐのバンドを切り出しＱＩＡＥＸ ＩＩ抽出キット（Ｑｉａｇｅｎ）で精製する。精製したバンドの濃度を測定し、約１２０ｎｇを３００ｎｇのｐＤＯＮＲ２０１（Ｇａｔｅｗａｙシステム用のドナーベクター）（Ｇｉｂｃｏ ＢＲＬ Ｌｉｆｅ Ｔｅｃｈｏｎｏｌｏｇｉｅｓ）、１ｘＢＰクロナーゼ反応バッファー、ＢＰクロナーゼ酵素混合物と総体積２０μｌにて２５℃、６０分間インキュベーションする。次に反応混合物を２μｌのプロテイナーゼＫと３７℃にて１０分間インキュベーションする。反応混合物を次にコンピテントＤＢ３．１細胞にエレクトロポレーションし、カナマイシン含有プレートにプレーティングする。配列番号１に記載の核酸配列を保持するｐＤＯＮＲ−ＨＭ６３と命名したクローンを更なる実験に使用する。
実施例１．５で述べた他のレセプターも同様な方法を用いてクローニングする。
【００５９】
１．９．　 ＦＰＲＬ−１ レセプターのトランスフェクション
１．８に記載のＦＰＲＬ−１を含むベクターを使用してＦＰＲＬ−１レセプターのｃＤＮＡをＧａｔｅｗａｙクローニングシステム（Ｇｉｂｃｏ ＢＲＬ Ｌｉｆｅ Ｔｅｃｈｏｎｏｌｏｇｉｅｓ）のａｔｔＲ１およびａｔｔＲ２組換え部位を有する発現ベクターｐｃＤＮＡ３．１（＋）／ａｔｔＲに移す。ここで、ＦＰＲＬ−１レセプターはＣＭＶプロモーターの制御下で発現される。１５０ｎｇの「エントリー」ベクターｐＤＯＮＲ−ＨＭ６３を１５０ｎｇの「デスティネーションベクター」ｐｃＤＮＡ３．１（＋）／ａｔｔＲ、４μｌのＬＲクロナーゼ酵素ミックス、４μｌのＬＲクロナーゼ反応バッファー、と共にＴＥ（Ｔｒｉｓ／ＥＤＴＡ）によって２０μｌにして混合し、２５℃にて６０分間インキュベーションする。２μｌのプロテイナーゼＫ溶液を加え、３７℃にて１０分間インキュベーションする。１μｌの反応混合物を５０μｌのＤＨ５αへ形質転換する（氷上で３０分間ＤＮＡと共に細胞をインキュベーションした後４２℃にて３０秒間の熱ショックをかける）。細胞の熱ショック後４５０μｌのＳ．Ｏ．Ｃを添加し、細胞を３７℃にて６０分間インキュベーションする。細胞（１００μｌ）を１００μｇ／ｍｌのアンピシリンを含むＬＢプレートにプレーティングし、一晩インキュベーションする。
ＦＰＲＬ−１レセプターをインサートとして有するｐｃＤＮＡ３．１（＋）／ａｔｔＲを含むコロニーをｐｃＤＮＡ／ＦＰＲＬ１と命名しトランスフェクション研究に使用する。
１．５に記載した他のレセプターをコードする核酸配列を保持する１．８で得られるベクターを含む細胞クローンは類似の方法で調製する。
【００６０】
実施例２：細胞系および ＦＰＬＲ−１ レセプターの表現型効果
ＦＰＲＬ−１レセプターについて実施例２に記載したのと類似の方法を１．５に記載した他のレセプターを用いて行うことができる。
２．１．　細胞系
ヒト単球／マクロファージ細胞株ＨＬ−６０、Ｕ９３７、ＴＨＰ−１およびＭｏｎｏＭａｃ６を細胞モデル系として使用する。細胞を１０％ＦＣＳを含み１００Ｕ／ｍｌのペニシリン、１００μｇ／ｍｌのストレプトマイシン、２ｍＭグルタミンおよび１ｘ非必須アミノ酸を添加したＲＰＭＩ １６４０培地で細胞を増殖させる。ＭｏｎｏＭａｃ６細胞用の培地は５ｍｌ／ｌ ＯＰＩ培地サプリメント（Ｓｉｇｍａ）も含む。ＭｏｎｏＭａｃ６はもっぱら２４穴プレートで培養する。全ての細胞は５％ＣＯ_２を含む湿大気中で３７℃にて維持し、定期的にマイコプラズマによる汚染についてテストする。１０ｎＭ ＰＭＡ（ホルボル１２ミリステート−１３アセテート）を培地に添加して分化を行わせる。
ＦＰＲＬ−１レセプターの表現型効果（２．２−２．９）
２．２．　リガンド結合アッセイ
３００ｍｌの細胞培養物をＥＤＴＡ溶液で集め、懸濁液を用いて１１０〜１２０ｘｇにて細胞を遠心して落とし、１０ｍＭのＴｒｉｓ／ＨＣｌ、ｐＨ７．４、２．５ｍＭ ＣａＣｌ_２、１．２ｍＭ ＭｇＣｌ_２、４０μｇ／ｍｌバシトラシン、４μｇ／ｍｌロイペプチン、４μｇ／ｍｌキモスタチン、１０μｇ／ｍｌペファブロック、２μＭのホスホアミドンおよび０．１ｍｇ／ｍｌのウシ血清アルブミン（ＢＳＡ Ｆｒａｋｔｉｏｎ Ｖ， ＢＩ Ｂｉｏｐｒｏｄｕｃｔｓ）に再懸濁し、２ｘ１０^６細胞／ｍｌに希釈する。
【００６１】
０．５ｍｌアリコートを０．３ｎＭ ３^Ｈ−リポキシンＡ４（比活性〜１０Ｃｉ／ｍｍｏｌ）と共にまたはトリチウムを含まないリポキシンＡ４の濃度を増加させて（３〜３００ｎＭ）、４℃にて３０分間インキュベーションする。ＧＦ／Ｂフィルターを備えたＣｅｌｌ−Ｈａｒｖｅｓｔｅｒ（Ｓｋａｔｒｏｎ）により細胞を集めてインキュベーションを終了させ、５０ｍＭのＴｒｉｓ／ＨＣｌ、１００ｍＭ ＮａＣｌ、１０ｍＭ ＭｇＣｌ_２、ｐＨ７．４からなる３ｍｌの冷却したバッファーで３回洗浄し、フィルター片をバイアルに移す。２ｍｌのシンチレーションカクテルを加え、シンチレーション計測器（ＬＫＢ）で放射活性を測定する。非特異的結合は１００ｎＭの非標識リポキシンＡ４存在下で測定する。トリチウム化リポキシンＡ４を置き換えるため、ペプチドリガンドＭＭＫ−１（Ｋｌｅｉｎら、１９９８）を含む一連のペプチド及び低分子量化合物を濃度０．５〜３００ｎＭの範囲で同じ反応条件下で使用する。
結合した放射活性（フィルター片上）を計測器で評価し、値をオンラインで記録し、モデルにフィッティングする。^３Ｈ−リポキシンＡ４の結合を阻害する物質は全てそのＩＣ_５０値を計算する。
【００６２】
２．３．　 ＦＬＩＰＲ アッセイによって測定される Ｃａ ^２＋ − 放出
ＦＰＲＬ−１レセプターを用いたＦＬＩＰＲアッセイ（蛍光イメージングプレートリーダー）をＧ−プロテインαサブユニットα１６またはキメラＧ−プロテインＧｑｉ５若しくはＧｑｏ５（これらはＧα（ｉ）またはＧα（ｏ）の最後の５残基を有する２つのＧα（ｑ）キメラである）およびＦＰＲＬ−１レセプターを構成的に発現する種々のＣＨＯ細胞株で行う。Ｇα１６、Ｇｑｉ５若しくはＧｑｏ５を構成的に発現する細胞株ＣＨＯ／Ｇａｌｐｈａ１６、ＣＨＯ／ＧａｌｐｈａＧｑｉ５およびＣＨＯ／ＧａｌｐｈａＧｑｏ５をＦＰＲＬ−１レセプター発現ベクターでトランスフェクションする。これらの細胞株を１０％ＦＣＳ（子牛胎仔血清）、２ｍＭグルタミン、２００ｎｇ／ｍｌハイグロマイシン、１００Ｕ／ｍｌペニシリンおよび１００μｇ／ｍｌストレプトマイシンを含むＨａｍのＦ１２培地（Ｂｉｏ Ｗｈｉｔｔａｋｅｒ）中で、５％ＣＯ_２を含む湿大気中で３７℃にて培養する。３〜７ｘ１０^５細胞を６０ｍｍペトリ皿に播き一晩培養する。細胞を５０〜８０％コンフルエントまで増殖させ、トランスフェクションに使用する。６μｌのＦｕＧｅｎｅ６（Ｒｏｃｈｅ Ｂｉｏｃｈｅｍｉｃａｌｓ）を血清を含まない１００μｌの培地に添加し、室温にて５分間平衡化させる。
【００６３】
次に、２μｇの精製ｐｃＤＮＡ／ＦＰＲＬ−１レセプターを予め希釈したＦｕＧｅｎｅ６溶液に加え、緩やかに混合し、更に室温にて１５分間インキュベーションする。培地を細胞から吸引し、４ｍｌの新しい培地を細胞に加える。ＦｕＧｅｎｅ６溶液を滴下して細胞に加え、培地を揺すって均等に配分する。４８時間後培地を吸引し、培地をＨａｍのＦ１２培地、１０％ＦＣＳ（子牛胎仔血清）、２ｍＭグルタミン、２００ｎｇ／ｍｌハイグロマイシン、１００Ｕ／ｍｌペニシリンおよび１００μｇ／ｍｌストレプトマイシンおよび２００μｇ／ｍｌ Ｇ４１８と置換する。その後５日間の間、死んだ細胞及び細胞破砕物が洗浄され細胞の単一コロニーが見えるまで培地を毎日交換する。単一コロニーをクローニングシリンダーで分離することによって単離し、１００μｌの１ｘトリプシン／ＥＤＴＡを加えて表面から遊離させる。
【００６４】
クローニングシリンダーから細胞を４ｍｌの培地に移し６穴プレートにプレーティングする。単一コロニーをエクスパンジョンし、いくつかのコロニーにおけるＦＰＲＬ−１レセプターの発現レベルをリガンド結合アッセイ（２．２）によってテストする。ＣＨＯ／Ｇａｌｐｈａ１６／ＦＰＲＬ−１レセプター、ＣＨＯ／ＧａｌｐｈａＧｑｉ５／ＦＰＲＬ−１レセプターまたは、ＦＰＲＬ−１レセプターを最も高発現するＣＨＯ／ＧａｌｐｈａＧｑｏ５／ＦＰＲＬ−１レセプターと命名される細胞クローンを用いてＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ）によって細胞内Ｃａ^２＋を測定する。細胞（ＣＨＯ／Ｇａｌｐｈａ１６／ＦＰＲＬ−１レセプター、ＣＨＯ／ＧａｌｐｈａＧｑｉ５／ＦＰＲＬ−１レセプターまたは、ＣＨＯ／ＧａｌｐｈａＧｑｏ５／ＦＰＲＬ−１レセプター）を３８４−ブラックウェルプレート（Ｃｏｒｎｉｎｇ）にウェルあたり２５００〜５０００細胞にて４０μｌの体積で播き、５％ＣＯ_２を含む湿大気中３７℃にて一晩増殖させる。
【００６５】
陰性対照として、ＣＨＯ／Ｇａｌｐｈａ１６、ＣＨＯ／ＧａｌｐｈａＧｑｉ５または、ＣＨＯ／ＧａｌｐｈａＧｑｏ５細胞を使用する。次に４０μｌのＦｌｕｏ−４（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ）染色溶液を各ウェルに最終濃度２μＭで加え、細胞をＦｌｕｏ−４で標識する。Ｆｌｕｏ−４染色溶液は、１０．５ｍｌの上述の細胞培地、４２０μｌのプロベニシド（Ｐｒｏｂｅｎｉｃｉｄ）溶液（１．４２ｇプロベニシド（Ｓｉｇｍａ）、１０ｍｌ １Ｍ ＮａＯＨ、１０ｍｌ Ｈａｎｋｓバッファー）、４２μｌのＦｌｕｏ−４ストック溶液（５０μｇのＦｌｕｏ−４、２３μｌ ＤＭＳＯ、２３μｌ プルロニックＦ−１２７（２０％ＤＭＳＯ溶液）（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）および４２０μｌ １Ｍ ＨＥＰＥＳからなっている。５％ＣＯ_２を含む湿大気中３７℃、４５分間のインキュベーション後２０ｍｌのプロベニシド溶液を含む２０００ｍｌのハンクスバッファーを洗浄溶液として、ＥＭＢＬＡ−洗浄機（４洗浄工程、プログラム０３）で細胞を洗浄し、各ウェルに２５μｌの洗浄バッファーを残す。次に、染色ウェルについてＦＬＩＰＲを１００００計測、非染色細胞と染色細胞との差を１：５に設定する。次に、ハンクスバッファー／０．１％ＢＳＡで希釈した２５μｌのリポキシンＡ４およびペプチドリガンドＭＭＫ−１を含む一連のリガンド、ペプチドおよび低分子量化合物を濃度を増加させつつ（０．５〜３００ｎＭ）ウェルに加える。本発明の物質は濃度を増加させて（０．５〜３００ｎＭ）リポキシンＡ４（５０ｎＭ）と競合させてテストし、それらのアンタゴニスト能を測定する。リガンドの添加から開始して６０秒まで毎秒記録し、更にその後の６０秒間について５秒ごとに記録する。
【００６６】
２．４．　サイトカインまたはマトリックスメタロプロテアーゼの産生及び放出
単球／マクロファージ細胞株細胞を２．５〜５ｘ１０^５細胞／ｍｌの細胞密度にてリポキシンＡ４で処理する。細胞を０、１、３、６、１２、２４、４８および７２時間後に集め、上清をさらなる調査のために凍結し、細胞をＰＢＳで洗浄し、１４３ｍＭのβ−メルカプトエタノールを含む４００ｍｌのＲＬＴバッファー（Ｑｉａｇｅｎ ＲＮｅａｓｙ Ｔｏｔａｌ ＲＮＡ Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ）に再懸濁し、ＤＮＡを２０ｇの針で少なくとも５回剪断力をかけ、−７０℃に保存する。
総ＲＮＡを製造業者のプロトコルに従ってＱｉａｇｅｎ ＲＮｅａｓｙ Ｔｏｔａｌ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ）で単離する。ＴａｑＭａｎ解析には精製ＲＮＡを用いる。サイトカインＴＮＦα、ＩＬ−１β、ＩＬ−８、ＩＬ−６およびヒトマトリックスメタロプロテアーゼＭＭＰ−１、ＭＭＰ−７、ＭＭＰ−９、ＭＭＰ−１２を適当なプライマー配列を用いて測定する。
【００６７】
２．４．１．　分泌されたサイトカインの検出
培養および刺激された細胞の上清中のタンパク質をＴＣＡ（トリクロロ酢酸）を最終濃度１０％になるように加えることにより沈殿させる。沈殿物を８０％エタノールで２回洗浄し、ペレットを５０ｍＭ Ｔｒｉｓ／ＨＣｌ、ｐＨ７．４、１０ｍＭ ＭｇＣｌ_２、１ｍＭ ＥＤＴＡに再懸濁する。タンパク質濃度はＢｒａｄｆｏｒｄ法によって測定し、各サンプルの５０μｇを１２％ＳＤＳポリアクリルアミドゲル上に乗せる。ゲルをＰＶＤＦ−膜にブロットし、ＴＢＳＴ中の５％ＢＳＡで１時間ブロッキングし、商業的に入手可能なヒトＴＮＦα（腫瘍壊死因子α）、ＩＬ−１β（インターロイキン−１β）、ＩＬ−８（インターロイキン８）およびＩＬ−６（インターロイキン６）に対する抗体と１時間インキュベーションする。ＴＢＳＴで洗浄した後、ブロットをホースラディッシュペルオキシダーゼ結合抗−ヒトＩｇＧとインキュベーションし、再度洗浄し、ＥＣＬ化学発光キット（Ａｍｅｒｓｈａｍ）で現像する。バンドの強度をＢｉｏＭａｘ　Ｘ−線フィルム（Ｋｏｄａｋ）で視覚化し、デンシトメーターで定量する。
【００６８】
２．４．２．　分泌されたマトリックスメタロプロテアーゼの検出及び解析
本方法は２．４．１に記載したのと同一である。ウェスタンブロッティングに使用する抗体はヒトＭＭＰ−１、ＭＭＰ−７、ＭＭＰ−９およびＭＭＰ−１２に対するものである。
プロテアーゼ活性は、蛍光基質で測定する。刺激したおよび刺激しない細胞から単離した上清（上述）を、総体積５０μｌで、１μＭの基質（ＤＡＢ−Ｇａｂａ−Ｐｒｏ−Ｇｌｎ−Ｇｌｙ−Ｌｅｕ−Ｇｌｕ（ＥＤＡＮＳ）−Ａｌａ−Ｌｙｓ−ＮＨ２（Ｎｏｖａｂｉｏｃｈｅｍ））と室温にて５分間インキュベーションする。陽性対照は各反応あたり１２５ｎｇの精製ＭＭＰ−１２で行う。プロテアーゼ活性は３２０ｎｍの励起および４０５ｎｍの光放射により蛍光分光計で測定する。
【００６９】
タンパク質分解活性および細胞遊走を測定する別のアッセイでは走化チャンバーを使用する。チャンバーの上部部分のウェル中、８μｍのマトリゲル（Ｍａｔｒｉｇｅｌ）（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）層で被覆したフィルター上に細胞をプレーティングする（ウェルあたり１０^５個の細胞）。下部区画においてはリポキシンＡ４（１００ｎＭ）、ＭＣＰ−１（単球走化性タンパク質１）（１０ｎｇ／ｍｌ）のような化学誘引物質を培地に加える。５日後、フィルタを取り出し、マトリゲルを通過した下層表面上の細胞をメタノールで固定し、Ｄｉｆｆ−Ｑｕｉｋ染色キット（Ｄａｄｅ Ｂｅｈｒｉｎｇ）で染色し、光学顕微鏡により、３つの高出力フィールド中（４００ｘ）で計測する。
【００７０】
２．５．　走化性アッセイ
走化性を測定するために、４８穴走化（Ｂｏｙｄｅｎ）チャンバー（Ｎｅｕｒｏｐｒｏｂｅ）を使用する。細胞をＦＣＳを含まないＲＰＭＩ培地中で２４時間飢餓させる。化学誘引物質（５０ｎｇ／ｍｌ ＩＬ−８、１０ｎｇ／ｍｌ ＭＣＰ−１、１０ｎＭリポキシンＡ４、１０ｎＭ ＭＭＫ−１ペプチド（２．３．））をＦＣＳを含まないＲＰＭＩ培地で希釈し、３０μｌをウェルの下部区画に置く。上部区画は下部区画とポリカーボネートフィルター（孔細部８μｍ）によって隔てられている。５０μｌの細胞懸濁液（５ｘ１０^４）を上部区画のウェルに置く。チャンバーを５％ＣＯ_２を含む湿大気中で３７℃にて５時間インキュベーションする。次に、フィルターを取り出し、上側の細胞をこすり落とし、下側の細胞をメタノール中で５分間固定し、Ｄｉｆｆ−Ｑｕｉｋ染色セット（Ｄａｄｅ Ｂｅｈｒｉｎｇ）で染色する。遊走した細胞を光学顕微鏡により３つの高出力フィールドで計測する。
【００７１】
２．６．　付着性アッセイ
細胞を集め、ＰＢＳで洗浄し、ＰＢＳおよび１μＭのＢＣＥＣＦ（（２’−７’−ビス−（カルボキシエチル）−５（６’）−カルボキシフルオレセインアセトキシメチル）エステル、Ｃａｌｂｉｏｃｈｅｍ）中に再懸濁し（４ｘ１０^６／ｍｌ）、３７℃にて２０分間インキュベーションする。細胞をＰＢＳで洗浄し、０．１％ＢＳＡを含むＰＢＳに再懸濁する（３．３ｘ１０^６／ｍｌ）。３ｘ１０^５細胞（９０μｌ）をラミニン（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）で被覆した９６穴平底プレートの各ウェルに加える。１０μｌのアゴニスト（１００ｎＭリポキシンＡ４＋リポキシンＡ４アンタゴニスト）を加え、プレートを３７℃にて２０分間インキュベーションする。次に、細胞を０．１％ＢＳＡを含むＰＢＳで洗浄し、付着細胞を１００μｌの０．０２５ＭのＮａＯＨと０．１％ＳＤＳで可溶化する。定量は蛍光測定によって行う。
【００７２】
２．７．　食細胞活動
細胞懸濁液（２．５ｘ１０^４細胞／ｍｌ）を、５ｍｌのＵ９３７またはＴＨＰ−１として６穴プレートに、または２ｍｌのＭｏｎｏＭａｃ６として２４穴プレートに播き、アゴニスト（１００ｎＭリポキシンＡ４、５０ｎＭ ＭＭＫ−１ペプチド（２．３．））およびアゴニスト効果を打ち消すために本発明の低分子量化合物の存在下で、５％ＣＯ_２を含む湿大気中で３７℃にて１時間インキュベーションする。熱不活性化サッカロミセス・ボウラルディィ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｂｏｕｌａｒｄｉｉ）（２０酵母／細胞）の分散懸濁液４０μｌを各ウェルに添加する。細胞を更に３時間インキュベーションし、ＰＢＳで２回洗浄し、スピンする。細胞スピン調製物をＭａｙ−Ｇｒｕｎｗａｌｄ−Ｇｉｅｍｓａで染色し、食細胞作用を受けた粒子を光学顕微鏡で計測する。
【００７３】
実施例３： ＣＯＰＤ 患者から単離されたマクロファージのための細胞培養モデル
ＦＰＲＬ−１について実施例３に記載したのと類似の方法を１．５に記載したレセプターを用いて行うことができる。
ＣＯＰＤ患者から単離されたマクロファージのための細胞培養モデルとして、我々は単球細胞細胞株ＭｏｎｏＭａｃ６およびＴＨＰ−１を選択する。これらの細胞株の過活性化状態を模倣するために、細胞をＰＭＡで処理する。ＣＯＰＤにおける状況と類似した条件を模倣する筈の刺激に更に細胞を曝露する。これらの刺激は喫煙またはＬＰＳに対する曝露である。
【００７４】
ＭｏｎｏＭａｃ６細胞のＰＭＡ、喫煙およびＬＰＳ刺激後のＦＰＲＬ−１の発現
ＭｏｎｏＭａｃ６細胞を１０％ＦＣＳ（低エンドトキシン）、２ｍＭグルタミン、１ｘ非必須アミノ酸、２００Ｕ／ｍｌペニシリン、２００μｇ／ｍｌストレプトマイシンおよび５ｍｌ ＯＰＩ培地サプリメント（Ｓｉｇｍａ）を補充したＲＰＭＩ １６４０培地で２４穴プレート中で培養する。細胞をウェルあたり６０００００細胞の密度（２ｍｌ培地）まで増殖させ１０ｎＭ ＰＭＡ（ホルボル１２−ミリステート１３−アセテート）（Ｓｉｇｍａ）または２０ｎｇ／ｍｌ ＬＰＳ（サルモネラミネソタＲｅ５９５からのリポポリサッカライド）（Ｓｉｇｍａ）で刺激する。煙曝露のため、細胞を煙を多く含ませた培地中で３７℃、５％ＣＯ_２において１ｘ１０^６細胞／ｍｌの密度でインキュベーションする。
【００７５】
ＲＰＭＩ １６４０培地に煙を多く含ませるのは２本のタバコで行った。タバコの煙を５０ｍｌシリンジに吸い込み（タバコ１本あたり５０ｍｌシリンジ約２０容）、次に補填物を含まない１００ｍｌのＲＰＭＩ １６４０培地に灌流する。その後、煙富裕化培地のｐＨを７．４に調整し、培地を０．２μｍフィルターで滅菌して使用する。煙曝露後、細胞をＲＰＭＩ １６４０で少なくとも２回洗浄し残存する煙粒子を除去する。次に、細胞を上記補充物を含む新しい２ｍｌのＲＰＭＩ １６４０培地を満たした２４穴プレートにウェルあたり４０００００〜６０００００細胞となるように播く。
【００７６】
ＴＨＰ−１細胞を１０％ＦＣＳ（低エンドトキシン）、２００Ｕ／ｍｌペニシリン、２００μｇ／ｍｌストレプトマイシンを補充したＲＰＭＩ １６４０ Ｇｌｕｔａｍａｘ中、７５ｃｍ^２フラスコ内で増殖させる。細胞を１０ｎＭのＰＭＡで４８時間、３７℃、５％ＣＯ_２にて処理し、細胞をマクロファージ様細胞に分化させる。次に、培地を２０ｎｇ／ｍｌのＬＰＳを含む新しいＰＭＡフリーの培地に交換する。
両方の細胞型とも３７℃、５％ＣＯ_２湿大気中で培養し、細胞をいろいろな時点で集め、経時的効果を監視する。細胞をスピンダウンし、ＰＢＳで洗浄し、１４３ｍＭのβ−メルカプトエタノールを含む４００μｌのＲＬＴバッファー（Ｑｉａｇｅｎ ＲＮｅｗａｓｙ Ｔｏｔａｌ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ）に再懸濁し、ＤＮＡに２０ｇの針で少なくとも５回剪断力をかけ、−７０℃に保存する。
総ＲＮＡをＱｉａｇｅｎ Ｎｅｗａｓｙ Ｔｏｔａｌ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ）で製造業者のプロトコルに従って単離する。精製ＲＮＡをＲＮａｓｅフリーのＤＮａｓｅ（Ｑｉａｇｅｎ）で消化し、ＴａｑＭａｎ解析に使用する。
【００７７】
ＴａｑＭａｎ解析
種々の時点で刺激した、および刺激しない細胞株におけるＦＰＲＬ−１のｍＲＮＡレベルを決定するためにＴａｑＭａｎ解析を使用する。１０ｎＭのレチノイン酸で３日間処理したＵ９３７細胞から単離した総ＲＮＡを用いてＦＰＲＬ−１のｍＲＮＡレベルを決定するための条件を最適化し、およびＦＰＲＬ−１およびハウスキーピング遺伝子としてのＧＡＰＤＨ（グリセルアルデヒド−３−リン酸デヒドロゲナーゼ）のための標準曲線を設定する。ＦＰＲＬ−１の定量は以下のプライマーを用いて行う：フォワードプライマー（ｒｈＨＭ６３ ６６８（−）ＦＰ、配列番号２２）、リバースプライマー（ｈＨＭ６３ ５２５（＋）ＲＰ、配列番号２３）、および５’末端をレポーター色素ＦＡＭで、３’末端を消光色素ＴＡＭＲＡで標識したＴａｑＭａｎプローブ（ｒｈＨＭ６３ ６２９（−）ＴＰ、配列番号２４）。ＧＡＰＤＨのｍＲＮＡレベルは、市販のＧＡＰＤＨ用キット”ＴａｑＭａｎ ＧＡＰＤＨ Ｃｏｎｔｒｏｌ Ｒｅａｇｅｎｔ”（Ｐ／Ｎ ４０２８６９）（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）で測定する。ＧＡＰＤＨプローブはレポーター色素としてＪＯＥで、消光色素としてＴＡＭＲＡで標識する。
【００７８】
ＲＴ−ＰＣＲ反応はＰｅｒｋｉｎ Ｅｌｍｅｒの”ＴａｑＭａｎ ＥＺ ＲＴ−ＰＣＲ Ｃｏｒｅ Ｒｅａｇｅｎｔ”（Ｐ／Ｎ Ｎ８０８−０２３６）キットで行う。ＦＰＲＬ−１およびＧＡＰＤＨの標準曲線は１μＭのレチノイン酸で処理したＵ９３７細胞のＲＮＡを濃度を各アッセイについて０、５、１０、２５、５０から１００ｎｇまでの範囲で増加させて作成する。反応混合物は、１ｘＴａｑＭａｎ ＥＺ−バッファー、３ｍＭ Ｍｎ（Ｏａｃ）_２、３００μＭのｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰおよび６００μＭのｄＵＴＰ、２．５Ｕ ｒＴｔｈ ＤＮＡポリメラーゼ、０．２５Ｕ ＡｍｐＥｒａｓｅ ＵＮＧを総体積２５μｌ中に含む。ＦＰＲＬ−１用には反応混合物は、３００ｎＭのｒｈＨＭ６３ ６６８（−）ＦＰおよびｈＨＭ６３ ５２５（＋）ＲＰ、および１００ｎＭのｒｈＨＭ６３ ６２９（−）ＴＰを含む。ＧＡＰＤＨレベルを測定するためのプライマーの濃度は各プライマーについて２００ｎＭであり、ＴａｑＭａｎプローブについて１００ｎＭである。ヒト患者および細胞株のＦＰＲＬ−１およびＧＡＰＤＨのｍＲＮＡレベルを測定するために、反応あたり１６〜５０ｎｇのＲＮＡを使用する。すべてのサンプルは３つ組で走らせる。反応はＭｉｃｒｏＡｍｐ ＯｐｔｉｃａｌキャップでシールしたＭｉｃｒｏＡｍｐ Ｏｐｔｉｃａｌ ９６−穴プレート（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）でＡＢＩ ＰＲＩＳＭ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）にて行う。ＰＣＲ条件は、５０℃２分間、６０℃３０秒間、９５℃５分間、続いて、４０サイクルの（９４℃２０秒間、５９℃１分間）である。データ解析はＦＰＲＬ−１およびＧＡＰＤＨのｍＲＮＡレベルを標準曲線に従って決定するか、ＦＰＲＬ−１のＣ_Ｔ値を直接ＧＡＰＤＨのＣ_Ｔ値に関連づけるかによって行う。後者の方法は、どちらの反応の効率も互いによく一致するので（９５％程度）これらの遺伝子に適用することができる。
【００７９】
【表１４】
表１：１０ｎＭのＰＭＡで刺激したＭｏｎｏＭａｃ６細胞におけるＦＰＲＬ−１の発現

【００８０】
【表１５】
表２：１０ｎＭ ＰＭＡによる分化および２０ｎｇ／ｍｌ ＬＰＳによる刺激後のＭｏｎｏＭａｃ６細胞におけるＦＰＲＬ−１の発現

【００８１】
【表１６】
表３：１０ｎＭ ＰＭＡによる分化および煙による刺激後のＭｏｎｏＭａｃ６細胞におけるＦＰＲＬ−１の発現

【００８２】
【表１７】
表４：ＰＭＡによる分化およびＬＰＳによる刺激後のＴＨＰ−１細胞におけるＦＰＲＬ−１の発現

【００８３】
ＦＰＲＬ−１のリガンドの効果を調べるため、ＭｏｎｏＭａｃ６細胞を２４穴プレートに２５００００細胞／ｍｌの密度で（ウェルあたり２ｍｌ）播き、２４時間、３７℃、５％ＣＯ_２湿大気中で増殖させ、２００ｎＭリポキシンＡ４（Ｂｉｏｉｍｏｌ）、Ｗ−ペプチド（１μＭ）（Ｍｅｔａｂｉｏｎ、Ｍａｒｔｉｎｓｒｉｅｄによって合成）、および陽性対照としてＬＰＳ（Ｓｉｇｍａ）で刺激する。細胞をいろいろな時点で集め、総ＲＮＡを上述したようにＱｉａｇｅｎ ＲＮｅａｓｅｙ Ｔｏｔａｌ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ）を用いて単離する。
Ｗ−ペプチド（Ｂａｅｋら、１９９６， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ ２７１， ８１７０−８１７５）の配列は以下の通りである。
Ｗ−Ｋ−Ｙ−Ｍ−Ｖ−ｍ
このＲＮＡをＴａｑＭａｎ解析に用いてＴＮＦα、ＩＬ−８およびＭＭＰ−１２のような炎症マーカーの発現を調べる。
【００８４】
【表１８】
表５：リポキシンＡ４およびＷ−ペプチド刺激後のＭｏｎｏＭａｃ６細胞におけるＴＮＦαの発現

【００８５】
【表１９】
表６：リポキシンＡ４およびＷ−ペプチド刺激後のＭｏｎｏＭａｃ６細胞におけるＩＬ−８の発現

【００８６】
【表２０】
表７：リポキシンＡ４およびＷ−ペプチド刺激後のＭｏｎｏＭａｃ６細胞におけるＭＭＰ−１２の発現

【００８７】
ＣＯＰＤ患者において末梢気道へのマクロファージの侵襲増加がみられるので、肺胞マクロファーの培養細胞モデルとして働くＭｏｎｏＭａｃ６の走化能をテストした。ＭｏｎｏＭａｃ６の走化性はＦＰＲＬ−１の種々のリガンドを投与することによって決定した。
ＭｏｎｏＭａｃ６細胞をＰＭＡで２４〜３０時間処理して細胞の活性化状態を誘導する。細胞を集め、補充物を含まないＲＰＭＩ １６４０で２回洗浄し、１０ｎＭ ＰＭＡ存在下で５０００００細胞／ウェル（２４穴プレート）の密度で播く。２４〜３０時間後、ピペットで繰り返しリンスすることにより細胞を基層から遊離させ、スピンダウンし、計測し、補充物を含まないが１０ｎＭのＰＭＡを含むＲＰＭＩ １６４０培地中に１ｘ１０^６細胞／ｍｌの密度に調整する。走化性は４８穴走化チャンバー（Ｎｅｕｒｏｐｒｏｂｅ Ｉｎｃ．）および８μｍの孔サイズを有するポリカーボネート膜で行う。チャンバーの下部ウェルを２８μｌの種々の濃度のリポキシンＡ４、Ｗ−ペプチド、陽性対照としてＭＣＰ−１、および陰性対照として補充物を含まないＲＰＭＩ １６４０培地（１０ｎＭのＰＭＡを含む）で満たす。下部ウェルはポリカーボネート膜で被覆し、チャンバーの上部区画は５０μｌの細胞懸濁液（５００００細胞／ウェル）で満たす。
【００８８】
３７℃、５％ＣＯ_２にて４時間の遊走後、膜の上側部分にある細胞をこすり落とし、膜の下側部分に付着している細胞をＤｉｆｆ Ｑｕｉｋ染色キット（Ｄａｄｅ Ｂｅｈｒｉｎｇ）により製造業者のプロトコルに従って染色する。染色された細胞を光学顕微鏡で２５０倍の倍率で６〜８の高出力フィールドにて計測する。遊走指数は対照培地に対する、化学誘引物質に応答して遊走した細胞の数の増加倍数を表す。
【００８９】
【表２１】
表８：リポキシン Ａ４、Ｗ−ペプチド、およびＭＣＰ−１に応答したＭｏｎｏＭａｃ６の遊走

【００９０】
上記実施例および上記各培養細胞モデルの細胞は、テストすべき物質を添加し、続いて上述の効果を測定することによって、ある物質がマクロファージにおいて本発明に従って脱調節された本発明のＩＬＭレセプターの阻害因子であるかどうかを決定するために使用される。
【００９１】
参考文献
文献 （ＦＰＲＬ−１ レセプター ， ＨＭ７４ レセプター ）：
Ｋｉｍ， Ｓ．Ｊ． （１９８８）． Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． １５０， ８７０−８７６．
Ｌｅｅ， Ｔ．Ｈ．， Ｃｒｅａ， Ａ．Ｅ．Ｇ．， Ｇａｎｔ， Ｖ．， Ｓｐｕｒ， Ｂ．Ｗ．， Ｍａｒｒｏｎ， Ｂ．Ｅ．， Ｎｉｃｏｌａｏｕ， Ｋ．Ｃ．， Ｒｅａｒｄｏｎ， Ｅ．， Ｂｒｅｚｉｎｓｋｉ， Ｍ．， ａｎｄ Ｓｅｒｈａｎ， Ｃ． （１９９０）． Ａｍ． Ｒｅｖ． Ｒｅｓｐｉｒ． Ｄｉｓ． １４１， １４５３−１４５８．
Ｂａｏ， Ｌ．， Ｇｅｒａｒｄ， Ｎ．Ｐ．， Ｅｄｄｙ， Ｒ．Ｌ．， Ｓｈｏｗｓ， Ｔ．， ａｎｄ Ｇｅｒａｒｄ， Ｃ． （１９９１）． Ｇｅｎｏｍｉｃｓ １３， ４３７−４４０．
Ｃｈｒｉｓｔｉｅ， Ｐ．Ｅ．， Ｓｐｕｒ， Ｂ．Ｗ．， ａｎｄ Ｌｅｅ， Ｔ．Ｈ． （１９９２）． Ａｍ Ｒｅｖ． Ｒｅｓｐｉｒ． Ｄｉｓ． １４５， １２８１−１２８４．
Ｙｅ， Ｒ．Ｄ．， Ｃａｖａｎａｇｈ， Ｓ．Ｌ．， Ｑｕｅｈｅｎｂｅｒｇｅｒ， Ｏ．， Ｐｒｏｓｓｎｉｔｚ， Ｅ．Ｒ．， ａｎｄ Ｃｏｃｈｒａｎｅ， Ｃ．Ｇ． （１９９２）． Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． １８４， ５８２−５８９．
Ｐｅｒｅｚ， Ｈ．Ｄ．， Ｈｏｌｍｅｓ， Ｒ．， Ｋｅｌｌｙ， Ｅ．， ＭｃＣｌａｒｙ， Ｊ．， ａｎｄ Ａｎｄｒｅｗｓ， Ｗ．Ｈ． （１９９２）． Ｇｅｎｅ， １１８， ３０３−３０４．
Ｍｕｒｐｈｙ， Ｐ．Ｍ．， Ｏｚｃｅｌｉｋ， Ｔ．， Ｋｅｎｎｅｙ， Ｒ．Ｔ．， Ｔｉｆｆａｎｙ， Ｈ．Ｌ．， ＭｃＤｅｒｍｏｔｔ， Ｄ．， ａｎｄ Ｆｒａｎｃｋｅ， Ｕ． （１９９２）． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２６７， ７６３７−７６４３．
Ｎｏｍｕｒａ， Ｈ．， Ｎｉｅｌｓｅｎ， Ｂ．Ｗ．， ａｎｄ Ｍａｔｓｕｓｈｉｍａ， Ｋ． （１９９３）． Ｉｎｔｅｒｎａｔ． Ｉｍｍｕｎｏｌ． ５， １２３９−１２４９．
Ｌｅｖｙ， Ｂ．Ｄ．， Ｒｏｍａｎｏ， Ｍ．， Ｃｈａｐｍａｎ， Ｈ．Ａ．， Ｒｅｉｌｌｙ， Ｊ．Ｊ．， Ｄｒａｚｅｎ， Ｊ．， ａｎｄ Ｓｅｒｈａｎ， Ｃ．Ｎ． （１９９３）． Ｊ． Ｃｌｉｎ． Ｉｎｖｅｓｔ． ９２， １５７２−１５７９．
Ｄｕｒｓｔｉｎ， Ｍ．， Ｇａｏ， Ｊ．−Ｌ．， Ｔｉｆｆａｎｙ， Ｈ．Ｌ．， ＭｃＤｅｒｍｏｔｔ， Ｄ．， ａｎｄ Ｍｕｒｐｈｙ， Ｐ．Ｍ． （１９９４）． Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． ２０１， １７４−１７９．
Ｍａｄｄｏｘ， Ｊ．Ｆ． ａｎｄ Ｓｅｒｈａｎ， Ｃ．Ｎ． （１９９６）． Ｊ． Ｅｘｐ． Ｍｅｄ． １８３， １３７−１４６．
Ｍａｄｄｏｘ， Ｊ．Ｆ．， Ｈａｃｈｉｃｈａ， Ｍ．， Ｔａｋａｎｏ， Ｔ．， Ｐｅｔａｓｉｓ， Ｎ．Ａ．， Ｆｏｋｉｎ， Ｖ．Ｖ．， ａｎｄ Ｓｅｒｈａｎ， Ｃ．Ｎ． （１９９７）． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７２， ６９７２−６９７８．
Ｋｌｅｉｎ， Ｃ．， Ｐａｕｌ， Ｊ．Ｉ．， Ｓａｕｖｅ， Ｋ．， Ｓｃｈｍｉｄｔ， Ｍ．Ｍ．， Ａｒｃａｎｇｅｌｉ， Ｌ．， Ｒａｎｓｏｍ， Ｊ．， Ｔｒｕｅｌｈｅａｒｔ， Ｊ．， Ｍａｎｆｒｅｄｉ， Ｊ．Ｐ．， Ｂｒｏａｃｈ， Ｊ．Ｒ．， ａｎｄ Ｍｕｒｐｈｙ， Ａ．Ｊ． （１９９８）． Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈ． １６， １３３４−１３３７．
Ｓｅｒｈａｎ， Ｊ．Ｎ． （１９９９）． １３３−１４９ ｉｎ Ｌｉｐｏｘｙｇｅｎａｓｅｓ ａｎｄ Ｔｈｅｉｒ Ｍｅｔａｂｏｌｉｔｅｓ， ｅｄ． Ｎｉｇａｍ ａｎｄ Ｐａｃｅ−Ａｓｃｉａｋ， Ｐｌｅｎｕｍ Ｐｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ．
文献 （ＡＩＣＬ レセプター ）：
Ｏｄａ， Ｓ．， Ｓａｔｏ， Ｍ．， Ｔｏｙｏｓｈｉｍａ， Ｓ．， ａｎｄ Ｏｓａｗａ， Ｔ． （１９８８）． Ｊ． Ｂｉｏｃｈｅｍ． １０４， ６００−６０５．
Ｈａｍａｎｎ， Ｊ． Ｆｉｅｂｉｇ， Ｈ．， ａｎｄ Ｓｔｒａｕｓｓ， Ｍ． （１９９３）． Ｊ． Ｉｍｍｕｎｏｌ． １５０， ４９２０−４９２７．
Ｈａｍａｎｎ， Ｊ．， Ｍｏｎｔｇｏｍｅｒｙ， Ｋ．Ｔ．， Ｌａｕ， Ｓ．， Ｋｕｃｈｅｒｌａｐａｔｉ， Ｒ．， ａｎｄ ｖａｎ Ｌｉｅｒ， Ｒ．Ａ．Ｗ． （１９９７）． Ｉｍｍｕｎｏｇｅｎｅｔｉｃｓ ４５， ２９５−３００．
文献 （ＩＬＴ１ レセプター ）：
Ｓａｍａｒｉｄｉｓ， Ｊ．， ａｎｄ Ｃｏｌｏｎｎａ， Ｍ． （１９９７）． Ｅｕｒ． Ｊ． Ｉｍｍｕｎｏｌ． ２７， ６６０−６６５．
Ｎａｋａｊｉｍａ， Ｈ．， Ｓａｍａｒｉｄｉｓ， Ｊ．， Ａｎｇｍａｎ， Ｌ．， ａｎｄ Ｃｏｌｏｎｎａ， Ｍ． （１９９９）． Ｊ． Ｉｍｍｕｎｏｌ． １６２， ５−８．
文献 （ＳＨＰＳ−１ レセプター ）：
Ｆｕｊｉｏｋａ， Ｙ． ｅｔ ａｌ． （１９９６）． Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ． １６， ６８８７−６８９９．
Ｋｈａｒｉｔｏｎｅｎｋｏｖ， Ａ．， Ｃｈｅｎ， Ｚ．， Ｓｕｒｅｓ， Ｉ．， Ｗａｎｇ， Ｈ．， Ｓｃｈｉｌｌｉｎｇ， Ｊ．， ａｎｄ Ｕｌｌｒｉｃｈ， Ａ． （１９９７）． Ｎａｔｕｒｅ ３８６， １８１．１８６．
Ｂｒｏｏｋｅ， Ｇ．Ｐ．， Ｐａｒｓｏｎｓ， Ｋ．Ｒ．， ａｎｄ Ｈｏｗａｒｄ， Ｃ．Ｊ． （１９９８）． Ｅｕｒ． Ｊ． Ｉｍｍｕｎｏｌ． ２８， １−１１．
Ｖｅｉｌｌｅｔｔｅ， Ａ．， Ｔｈｉｂａｕｄｅａｕ， Ｅ．， ａｎｄ Ｌａｔｏｕｒ， Ｓ． （１９９８）． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７３， ２２７１９−２２７２８．
文献 （ＫＤＥＬ レセプター １）：
Ｔｏｗｎｓｌｅｙ， Ｆ．Ｍ．， Ｗｉｌｓｏｎ， Ｄ．Ｗ．， ａｎｄ Ｐｅｌｈａｍ， Ｄ．Ｒ． （１９９３）． ＥＭＢＯ Ｊ． １２，２８２１−２８２９．
Ａｏｅ， Ｔ．， Ｃｕｋｉｅｒｍａｎ， Ｅ．， Ｌｅｅ， Ａ．， Ｃａｓｓｅｌ， Ｄ．， Ｐｅｔｅｒｓ， Ｐ．Ｊ．， ａｎｄ Ｈｓｕ， Ｖ．Ｗ． （１９９７）． ＥＭＢＯ Ｊ． １６， ７３０５−７３１６．
文献 （ＣＳＦ−１ レセプター ）：
Ｈａｍｐｅ， Ａ．， Ｓｈａｍｏｏｎ， Ｂ．Ｍ．， Ｇｏｂｅｔ， Ｍ．， Ｓｈｅｒｒ， Ｃ．Ｊ．， ａｎｄ Ｇａｌｉｂｅｒｔ， Ｆ． （１９８９）． Ｏｎｃｏｇｅｎｅ Ｒｅｓ． ４， ９−１７．
Ｙｅｕｎｇ， Ｙ．−Ｇ．， Ｗａｎｇ， Ｙ．， Ｅｉｎｓｔｅｉｎ， Ｄ．Ｂ．， Ｌｅｅ， Ｐ．Ｓ．Ｗ．， ａｎｄ Ｓｔａｎｌｅｙ， Ｅ．Ｒ． （１９９８）． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７３， １７１２８−１７１３７．[0001]
(Field of the Invention)
The present invention belongs to the field of inflammatory processes in which macrophages play an important role, in particular the regulation of inflammatory processes in inflammatory airway diseases. According to the present invention, inflammatory processes can be regulated by affecting the function of receptors on macrophages that have been shown to be involved in inflammatory processes.
[0002]
(Background of the Invention)
The inflammatory process involves a cascade of multiple reactions. A variety of factors are involved in the inflammatory process, and single therapies that circumvent those factors have been unsuccessful. In particular, it is especially true for inflammatory processes of the airways such as chronic inflammatory airway disease.
Chronic inflammatory airway diseases include chronic bronchitis and chronic obstructive pulmonary disease (COPD). For example, COPD is a complex disease that includes a variety of abnormalities: chronic bronchitis characterized by cough and mucus hypersecretion, peripheral airway disease including inflammation and peribronchial fibrosis, and emphysema. COPD is characterized by an accelerated and irreversible decrease in lung function. A major risk factor for developing COPD is ongoing tobacco smoking. Genetic predisposition may also contribute to the disease, as only about 20% of all smokers have COPD.
[0003]
The first event in the early stages of COPD onset is inflammation, which affects the small and airways. The stimuli caused by smoking attract macrophages and neutrophils, increasing their number in smokers' sputum. Constant smoking causes an inflammatory response by releasing mediators that recruit inflammatory cells to the site of injury from macrophages, neutrophils and epithelial cells. To date, there is no treatment available to reverse the course of COPD. Smoking cessation may reduce the decline in lung function. Only a few drugs relieve patient suffering. Long-lasting β2-agonists and anticholinergics are applied to achieve temporary bronchodilation. LTB₄A variety of antagonists against inflammatory events are being studied, such as inhibitors, IL-8 inhibitors, TNFα-inhibitors.
Chronic inflammatory airway disease can be characterized by activated inflammatory immune cells, such as macrophages. It is desirable to modulate the function of macrophages to eliminate the basis of the inflammatory process.
[0004]
Description of the invention
In the present invention, macrophages are involved in inflammatory processes, preferably chronic inflammatory airway diseases, more preferably chronic bronchitis or COPD, and are macrophages from healthy or stimulated donors (actually including activated macrophages). It has been found to exhibit a pattern of nucleic acid sequences and proteins with differential expression that is different from the pattern of gene expression. Thus, macrophages exhibit different levels of activation under different inflammatory conditions, and the hyperactive state may be involved in inflammatory processes, preferably chronic inflammatory airway disease, more preferably chronic bronchitis or COPD. Shown in the present invention. The present invention provides for inhibiting macrophage hyperactivation or reducing the hyperactivated state by allowing the identification of substances that modulate receptors involved in macrophage hyperactivation or maintaining the hyperactivated state. .
[0005]
The present invention relates to differentially expressed, involved in causing and / or maintaining the induction of a hyperactivated state of macrophages involved in an inflammatory process, preferably chronic inflammatory airway disease, more preferably bronchitis or COPD. Based on the identification of nucleic acid sequences or proteins that Such differentially expressed nucleic acid sequences or proteins are hereinafter referred to as differentially expressed nucleic acid sequences or proteins, respectively, of the present invention. In particular, the present invention teaches the link between alteration of macrophage phenotype by differentially expressed nucleic acid sequences and protein expression patterns and the involvement of macrophages in the inflammatory process, and thus provides a basis for various applications. For example, the present invention provides methods and test systems for determining the expression levels of the differentially expressed nucleic acid sequences or proteins of the present invention in macrophages, such as, for example, mammals, preferably humans, especially inflammatory processes, Provide a method for diagnosing or monitoring an inflammatory process involving overactivated macrophages in a patient suffering from chronic inflammatory airway disease, more preferably chronic bronchitis or COPD.
[0006]
The present invention also relates to a method of regulating a differentially expressed nucleic acid sequence or protein of the present invention by the differentially expressed nucleic acid sequence or protein of the method of the present invention, that is, a method of identifying a substance acting as an inhibitor or an activator thereto. Thereby, a method of positively affecting a chronic inflammatory process by inhibiting or reducing the overactivation state of macrophages, and a mammal thereby affected by such a disease, preferably A method for treating a human. The present invention also relates to a method for selectively modulating the differentially expressed nucleic acids and proteins of the present invention in macrophages, the method comprising administering a substance determined to be a modulator of the protein or the differentially expressed nucleic acid sequence. Including. The present invention includes the use of said substance for treating a patient in need of treatment for an inflammatory process, preferably a chronic inflammatory airway disease, more preferably chronic bronchitis or COPD.
[0007]
In a first step of the invention, differentially expressed nucleic acid sequences and proteins having a different expression pattern in the overactivated macrophages as compared to the non-activated macrophages are identified. For simplicity, this description deals specifically with the study of macrophages involved in COPD, but comparable results will be observed in examples of patients with other chronic inflammatory airway diseases, such as chronic bronchitis. Examining the various expression patterns, as exemplified in the Examples below, is based on a series of differentially expressed nucleic acid sequences that are differentially expressed in macrophages depending on the activation state of the macrophages involved in the inflammatory process. Results in identification.
[0008]
Briefly, such differentially expressed nucleic acid sequences include overactivated macrophages or cellular extracts thereof, such as macrophage cells or cellular extracts from inflammatory sites of COPD, and tobacco cells that are not afflicted with the disease, Are identified by comparative expression profiling experiments using macrophage cells or cell extracts thereof from corresponding control sites that are in a stimulatory state, such as being exposed to smoke.
Among the differentially expressed macrophage genes, to identify a differentially expressed nucleic acid sequence class encoding a macrophage surface receptor class characterized by being expressed at a lower or higher level than a control level in non-activated macrophages. Therefore, the differentially expressed nucleic acid sequence or protein of the present invention can be easily detected by the method described above. Such a macrophage surface receptor of the present invention is hereinafter referred to as an ILM receptor. However, the present invention not only relates to naturally occurring ILM receptors, but also includes those receptors that are functionally equivalent to the ILM receptor, ie, have a binding ability and a cell function that are common to the ILM receptor.
[0009]
Examples of ILM receptors of the present invention are FPRL-1 receptor type receptors, including the FPRL-1 receptor (SEQ ID NO: 2). In the context of the present invention, a “receptor type receptor”, for example a FPRL-1 receptor type receptor, is “functionally equivalent” to the respective receptor, eg the FPRL-1 receptor of SEQ ID NO: 2, ie, SEQ ID NO: 2. Is a receptor that shares binding ability and cellular function with the FPRL-1 receptor of the present invention; the term also refers to naturally occurring receptors, such as the FPRL-1 receptor, or naturally occurring receptor-type receptors, such as the FPRL-1 receptor-type receptor, Wherein the variant, variant or fragment is one that is functionally equivalent to a receptor, such as the FPRL-1 receptor.
[0010]
Further examples of ILM receptors include HM74 receptor type receptors, including the HM74 receptor (SEQ ID NO: 21); AICL receptor type receptors, including the AICL receptor (SEQ ID NO: 6); ILT1 receptor type receptors, including the ILT1 receptor (SEQ ID NO: 12) A PHPS-1 receptor type receptor containing the SHPS-1 receptor (SEQ ID NO: 4); a KDEL receptor 1 type receptor containing the KDEL receptor 1 (SEQ ID NO: 8); and CSF-1 containing the CSF-1 receptor (SEQ ID NO: 10). Receptor type receptors are included. Each receptor shown in the sequence listing or a variant, mutant or fragment thereof having the same function is preferred, and each receptor shown in SEQ ID NOs: 21, 6, 12, 4, 8, and 10 in the sequence listing is more preferred. . In a further preferred embodiment, the receptor is encoded by a nucleic acid sequence having the sequence of SEQ ID NO: 20, 5, 11, 3, 7 or 9.
[0011]
A preferred embodiment of the ILM receptor in the context of the present invention is a FPRL-1 receptor type receptor. The term FPRL-1 receptor type receptor is a naturally occurring FPRL-1 receptor or a variant, variant or fragment of the FPRL-1 receptor type receptor (these variants, variants or fragments are functionally equivalent to the FPRL-1 receptor). Thing). In describing embodiments of the present invention, a more preferred embodiment is the FPR-1 receptor of SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function, and a more preferred embodiment is the FPRL-1 receptor of SEQ ID NO: 2. In a most preferred embodiment, the FPRL-1 receptor is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1.
[0012]
According to the present invention, the ILM receptor expressing at a lower level than the control level is preferably activated to inhibit macrophage overactivation or reduce the macrophage hyperactivation state, while the control level Preferably, ILM receptors expressed at higher levels are inhibited to inhibit macrophage overactivation or reduce macrophage hyperactivation. In the context of the present invention, receptor function refers to any function of the receptor of the present invention that can affect the inflammatory process. For example, certain receptors of the present invention activate inflammation in that they are activated by ligands (any substance capable of binding to at least one domain exposed on the cell surface of the receptor), leading to intracellular signals involved in the inflammatory process. Mediate.
[0013]
In one embodiment, the present invention relates to a method of determining whether a substance is an activator or an inhibitor of an ILM receptor, said receptor preferably being overexpressed in macrophages involved in chronic inflammatory activation diseases. It is expressed or downregulated and deregulated, characterized in that said receptor is a receptor that plays an important role in mediating inflammation. The method of the invention involves subjecting the substance of interest to a test system that produces a measurable readout in the event of modulation of the ILM receptor or modulation of ILM receptor function. Test systems useful for performing the methods of the present invention include cell-based or cell-free systems. For example, in one embodiment of the invention, the system is designed to allow for the testing of substances that affect the expression level of a differentially expressed nucleic acid sequence; in another embodiment, the system interacts directly with the receptor. It allows the testing of substances that act, or substances that inhibit the binding of natural or artificial but suitable ligands. Later systems are designed in such a way that the substance to be tested can physically contact the receptor of the invention, the direct interaction of which gives rise to a measurable readout indicative of a change in said receptor function. And includes the receptor of the present invention.
[0014]
The method of the present invention comprising a cell line is, for example, a method wherein MonoMac6 or THP-1 cells are used, said cells being stimulated with phorbol 12-myristate 13-acetate and selected from LPS and smoke. It may be a method stimulated by.
The present invention provides a test system for determining whether a substance, according to the present invention, is an activator or an inhibitor of the function of the ILM receptor of the present invention, wherein the receptor comprises a chronic inflammatory airway disease. And an expression vector capable of expressing an ILM receptor or an ILM receptor in a cell, or an expression vector capable of expressing an ILM receptor. A system comprising a transformed host cell.
[0015]
Cellular and cell-free systems can be used to perform the methods of the present invention for determining whether a substance is an activator or an inhibitor of receptor function. A test system for performing the method can be designed and assembled using, for example, elements and methods well known in the art. For example, a cell-free system may include, for example, a cell compartment or vesicle containing a receptor of the invention. Suitable cell lines include, for example, suitable prokaryotic or eukaryotic cells, such as cells containing the respective receptor of the present invention. Cells suitable for performing the methods of the invention can be obtained by recombinant techniques, i.e., after transformation or transfection with a vector suitable for expressing the desired receptor of the invention, or It may be isolated from cell lines or natural sources expressing the desired receptor of the invention. The test system of the invention, including cell lines, uses, for example, MonoMac6 or THP-1 cells, which are stimulated with phorbol 12-myristate 13-acetate and stimulated with a substance selected from LPS and smoke. Test system. The test system of the invention may include a natural or artificial ligand for the receptor, if desired or necessary to test whether the substance of interest is an inhibitor or activator of the receptor of the invention.
[0016]
The test method of the invention involves measuring the read data, i.e. measuring the phenotypic change in the test system, for example, if a cell line is used, the phenotypic change of the cell. Such an alteration may be a naturally occurring response or an artificial response of the cell to activation or inhibition of the receptor, for example, as described in detail in the Examples below.
The test method of the present invention is useful for a high-throughput test suitable for determining whether a substance is an inhibitor or an activator of the present invention. It is also useful for secondary testing or evaluation of hits or leads.
[0017]
The present invention also relates to substances identified by the method of the present invention as inhibitors or activators of the receptor of the present invention. A substance according to the invention is any compound which can activate, preferably inhibit, the function of the receptor according to the invention. An example of a method of activating or inhibiting the function of a receptor is by affecting the expression level of said receptor. Another example of a method of activating or inhibiting a receptor is to act on a substance that binds directly to the receptor, thereby activating or inhibiting a functional domain of said receptor, which depends on the properties of the acted on substance. It can be performed reversibly or irreversibly depending on.
Thus, substances useful for activating or inhibiting receptor function include substances that act on the expression of the differentially expressed nucleic acid sequence, but also substances that act on the receptor itself. Thus, according to the present invention, what is meant by "substance of the present invention" is a nucleic acid sequence encoding the gene of the receptor of the present invention, or a fragment or variant thereof, which can affect the level of gene expression. Such as, but not limited to, nucleic acid molecules suitable as antisense nucleic acids, ribozymes, or those suitable for triple helix formation. Other examples of substances of the invention include, for example, antibodies or organic or inorganic compounds which bind directly to the receptor of the invention or inhibit the binding of the receptor of the invention to a suitable ligand, thereby affecting its function. It is.
[0018]
In a further aspect, the invention relates to a method for determining the level of an ILM receptor in macrophages, comprising measuring the expression level of a differentially expressed ILM receptor nucleic acid sequence or protein according to the invention. Such methods include, for example, determining whether an agent can affect the expression level of a differentially expressed nucleic acid sequence in the methods described above for determining whether an agent is an activator or an inhibitor. Available to test. However, methods for measuring the expression levels of differentially expressed ILM receptor nucleic acid sequences or proteins are useful for testing the activation status of macrophages, for example, for diagnostic purposes or for the treatment of diseases caused by overactivated macrophages. Can also be used to find out. Accordingly, the present invention requires a method of diagnosing a chronic inflammatory disease or monitoring such a disease, including, for example, treating such a disease, comprising measuring the level of a receptor expressed in macrophages according to the present invention. A method of monitoring the success of a treatment in a patient having a disease. Said macrophages are preferably mammalian cells, more preferably human cells. Accordingly, the macrophages of the invention are preferably obtained from a site of inflammation in a mammal, more preferably from a site of inflammation in a human.
[0019]
The method of measuring the expression level of the receptor according to the present invention comprises, depending on the purpose of measuring the expression level, measuring the concentration of each RNA transcript by a hybridization technique or a reporter gene-driven assay such as a luciferase assay, or The determination can be performed by a known method such as measuring the protein concentration of the receptor using each antibody to confirm the protein.
The present invention relates to the use of the substances according to the invention for the treatment of chronic inflammatory airway diseases. Another embodiment of the present invention is directed to a method of determining whether an agent is an activator or an inhibitor, wherein at least one of the at least one agent determined to be an activator or an inhibitor using the method of the present invention. A pharmaceutical composition comprising the substance of the present invention, wherein each of the receptors of the present invention is overexpressed in the macrophage of the present invention involved in the inflammatory airway disease of the present invention. The composition can be produced by a method known per se, for example, by a conventional mixing method, granulation method, sugar-coated tablet method, fine particle method, emulsification method, encapsulation method, entrapping method or freeze-drying method. .
[0020]
In order to use the activators or inhibitors of the invention as agents for the treatment of chronic inflammatory airway diseases, these agents may be used in animal models, such as animals with inflammatory airway disease, or expressing the receptors of the invention. Can be tested on transgenic animals. The toxicity and therapeutic efficacy of the substances of the present invention can be determined by standard pharmaceutical procedures, including cell culture and animal studies to determine IC₅₀, LD₅₀And ED₅₀Measuring is included. The data obtained is used to determine the dosage in animals, and more preferably in humans, which determines the dosage form (tablet, capsule, aerosol spray, ampoule, etc.) and route of administration (eg, dermal, oral, buccal) Nasal, enteral, parenteral, inhalation, endotracheal, or rectal).
[0021]
Pharmaceutical compositions containing at least one of the substances of the present invention as an active ingredient can be formulated in a conventional manner. Methods for making such formulations can be found in several manuals, for example, "Remington Pharmaceutical Science". Examples of ingredients useful for formulating at least one substance of the invention can also be found in WO 99/18193, which is incorporated herein by reference.
In a further aspect, the present invention teaches a method for treating a chronic inflammatory disease according to the present invention, which method comprises providing a patient, preferably a human, in need of such treatment with an ILM receptor according to the present invention in accordance with the present invention. Administering an appropriate amount of a pharmaceutical composition comprising at least one substance determined to be an activator or inhibitor, wherein said receptor is overexpressed in a macrophage of the invention and is chronically in accordance with the invention. It is characterized by having a role in mediating inflammation related to inflammatory airway diseases.
In another aspect, the invention relates to a method of selectively modulating ILM receptor levels in macrophages, comprising administering a substance determined to be an activator or inhibitor of a receptor of the invention. .
The following examples are intended to illustrate the invention and should not be construed as limiting. However, these examples describe the best mode of the invention.
[0022]
(Example)
Embodiment 1 FIG. Comparative expression profiling and FPLR-1 Cloning
The following is an example of how comparative expression profiling to identify the receptor of the present invention can be performed.
1.1. Patient selection
Examine three groups of patients: healthy non-smokers, healthy smokers and patients with COPD
Patients perform spirometry to assess lung function. Analyze the results using simple calculations based on age and height. COPD patients have their predicted FEV₁Include if% is less than 70%. Healthy smokers are elderly and have a similar smoking history to COPD patients but have normal lung function. Healthy nonsmokers have normal lung function and have never smoked. The last group receives a methacholine attack to eliminate asthma. This technique requires increasing the dose of methacholine given to the patient and measures spirometry between each dose. FEV₁Stop testing when PC drops by 20%, PC₂₀Is calculated. This is FEV₁Is a dose of methacholine that produces a 20% reduction in the value of greater than 32 as evidence of the absence of asthma. All patients must perform a skin puncture test for common allergens and obtain negative results. This eliminates atopic individuals. Monitor and examine the patient's clinical history to rule out concomitant illnesses.
[0023]
1.2. BAL (Bronchoalveolar lavage) Procedure
Patients are sedated with midazolam before BAL. A local anesthetic spray is used to anesthetize the back of the throat. Use a 7 mm Olympus bronchoscope. The washing area is the right middle lobe. Inject 250 ml of sterile saline and aspirate immediately. The resulting aspirate contains macrophages.
[0024]
1.3. BAL processing
Filter BAL through sterile gauze to remove residue. The cells are washed twice with HBSS, resuspended in 1 ml HBSS (Hank's balanced salt solution) and counted. The macrophages were spun and pelleted using 15 ml of Falcon blue-capped polypropylene and added to Trizol reagent (Gibco @ BRL Life Technologies) for 10 minutes.⁷Resuspend at a concentration of 1 ml Trizol reagent per cell and freeze at -70 ° C.
[0025]
1.4. Differential gene expression analysis
Total RNA is extracted from the macrophage sample obtained according to Example 1.3. The cell suspension in Trizol is homogenized by pipetting and incubated at room temperature for 5 minutes. 200 μl of chloroform is added per 1 ml of Trizol, the mixture is carefully mixed for 15 seconds and incubated for a further 3 minutes at room temperature. Centrifuge the sample at 10,000 g for 15 minutes at 4 ° C. Transfer the upper layer to a new reaction tube and add 0.5 ml of isopropanol per 1 ml of Trizol to precipitate the RNA for 10 minutes at room temperature. Next, the precipitate is pelleted at 10000 g at 4 ° C. for 10 minutes using a microcentrifuge. The pellet is washed twice with 75% ethanol, air-dried, and DEPC-H₂Resuspend in O.
[0026]
RNA is generated using Qiagen RNeasy Total RNA Isolation Kit (Qiagen) to improve RNA purity. RNA purity is determined by agarose electrophoresis, and concentration is measured by UV absorption at 260 nm.
5 μl of each RNA is used for cDNA synthesis. First and second strand synthesis is performed on the SuperScript Choise system (Gibco BRL Life Technologies). A total of 11 μl of RNA and 1 μl of 100 μM T7- (dt)₂₄The primer (the sequence is set forth in SEQ ID NO: 13) is heated to 70 ° C. for 10 minutes and cooled on ice for 2 minutes. Add a final concentration of 1x first strand buffer, 10 mM concentration of DTT and a final concentration of 0.5 mM dNTP mixture to a final volume of 18 μl. The reaction mixture is incubated at 42 ° C. for 2 minutes and 2 μl of Superscript II reverse transcriptase (200 U / μl) is added. 130 μl containing 1.15 × second strand buffer, 230 μM dNTPs, 10 U E. coli DNA ligase (10 U / μl), E. coli DNA polymerase (10 U / μl), RNaseH (2 U / μl) for second strand synthesis Add the mixture to the first strand synthesis reaction and mix carefully with a pipette. Second strand synthesis was performed at 16 ° C. for 2 hours, then 2 μl of T4 DNA polymerase (5 U / μl) was added, incubated at 16 ° C. for 5 minutes, and the reaction was performed by adding 10 μl of 0.5 M EDTA. Stop.
[0027]
The double-stranded cDNA is purified before cRNA synthesis. The cDNA is mixed with an equivalent amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) and spun through a gel matrix of phase lock gel (Eppendorf) in a microcentrifuge to separate the cDNA from unbound nucleotides. The aqueous phase is precipitated with ammonium acetate and ethanol. Subsequently, in vitro transcription is performed using this cDNA. cRNA synthesis is performed using the ENZO BioAray @ High Yield RNA @ Transscript labeling kit according to the manufacturer's protocol (ENZO Diagnostics). Briefly, cDNA is incubated with 1 × HY reaction buffer, 1 × biotin-labeled ribonucleotide, 1 × DTT, 1 × RNase inhibitor mix, and 1 × T7 RNA polymerase for a total of 40 μl at 37 ° C. for 5 hours. Next, the reaction mixture is purified by an RNeasy column (Quiagen) and the cRNA is precipitated with ammonium acetate and ethanol and finally resuspended in DEPC-treated water. The concentration is determined by UV spectroscopy at 260 nm. The remaining cRNA is incubated for 35 minutes at 94 ° C. in 1 × fragmentation buffer (5 × fragmentation buffer: 200 mM Tris acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc).
[0028]
For DNA chip hybridization, 15 μg of cRNA was used and 50 pM of a biotin-labeled control B2 oligonucleotide (sequence is set forth in SEQ ID NO: 14), 1 × cRNA cocktail, 0.1 mg / ml herring sperm DNA, 0.5 mg / Ml acetylated BSA, 1 × MES (2- [N-morpholino] -ethanesulfonic acid) hybridization buffer in a total volume of 300 μl. The hybridization mixture is heated to 99 ° C. for 5 minutes, cooled to 45 ° C. for 10 minutes, and 200 μl of the mixture is used to fill the probe array. Hybridization is performed at 45 rpm and 60 rpm for 16 hours.
After hybridization, the hybridization mixture on the chip is replaced with 300 μl of non-stringent wash buffer (100 mM MES, 100 mM NaCl, 0.01% Tween 20). Insert the chip into the Affymetrix Fluidics station and perform washing and staining according to the EukGE-WS2 protocol. The staining solution per chip consisted of 600 μl of 1 × staining buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg / ml BSA, 10 μg / ml SAPE (streptavidin phycoerythrin) (Dianova), and the antibody solution was 1 × Staining buffer consists of 2 mg / ml BSA, 0.1 mg / ml goat IgG, 3 μg / ml biotinylated antibody. After the washing and staining steps, the chips are scanned with an HP Gene Array Scanner (Hewlett Packard). Data analysis is performed by comparison between duplicates of a chip hybridized with RNA isolated from a COPD smoker and a chip hybridized with RNA isolated from a healthy smoker.
[0029]
One of the identified differentially expressed nucleic acid sequences is the FPRL-1 (formyl peptide receptor-like-1) receptor (LXA₄R, HM63, FPR2, FPRH2, FMLP-R-II, also called Lipoxin A4 receptor); see SEQ ID NOS: 1 and 2. It belongs to the chemoattractant peptide receptor family including fMLP (N-formyl-methionyl-leucyl-phenylalanine), IL-8 or C5a. These receptors display seven transmembrane helix motifs and heterotrimeric G-protein mediated signals. FPRL-1 receptor is lipoxin A₄(LXA₄) (Murphy et al., 1992).
Alveolar macrophages have been shown to produce lipoxins, which are synthesized by 15-lipoxygenase (Kim 1988). Lipoxin A₄(LXA₄) Stimulates chemotaxis, adhesion and calcium release in monocytes. In neutrophils, LXA₄Inhibits chemotaxis and adhesion and down-regulates migration from epithelial cells (Maddox and Serhan 1996). LXA₄Was found to be elevated in BAL from asthmatic patients (Lee et al., 1990, Serhan 1999). In particular, LXA₄Has been found to cause dose-dependent atrophy of the human bronchus (Christie et al., 1992). LXA₄Is thought to be a global regulator of inflammation in the lung.
[0030]
1.5 . 　 COPD Overexpressed in macrophages FPRL-1 Receptor
The FPRL-1 receptor is found to be consistently up-regulated in COPD smokers compared to healthy smokers (66.7%). This is shown as the "fold change" calculated from the 42 sets of comparisons, and the average difference ("avg diff") (Tables 1, 2). The relative expression levels of non-smokers and healthy smokers are similar, with elevated expression levels being restricted to COPD patients. Thus, COPD-specific effects cause up-regulation.
[0031]
Table 1: Expression pattern of FPRL-1 receptor: fold change calculated for 42 sets compared between COPD and healthy smokers. Only values higher than 2 times and values lower than -2 times are considered deregulated control. Thus, the FPRL-1 receptor is up-regulated in 28 cases and unregulated in 14 cases.
[0032]
[Table 1]

[0033]
Table 2: FPRL-1 receptor expression levels: "avg diff" values, relative indices of the intensity of the hybridization signal on the chip, given for each patient; OS is an obstructive smoker, HS is a healthy smoker , NS represent non-smokers.
[0034]
[Table 2]

P value for comparison between COPD smokers and healthy smokers: 0.02
[0035]
Chip data for the FPRL-1 receptor is confirmed by TaqMan analysis (Perkin Elmer Applied Biosystems). The fold change obtained with TaqMan is very similar to the data from the gene chip (Table 3).
[0036]
[Table 3]
Table 3: Up-regulation of FPRL-1 receptor in COPD smokers as determined by gene chip and TaqMan. Fold change measurement of FPRL-1 by chip data in six comparisons between COPD smokers and healthy smokers is confirmed by TaqMan in the same sample, and relative up-regulation calculates GAPDH as the housekeeping gene.

[0037]
Another differentially expressed nucleic acid sequence identified encodes the HM74 receptor (see SEQ ID NOs: 20 and 21), which belongs to the family of G-protein coupled receptors. The HM74 receptor was cloned from a human monocyte library (Nomura et al., 1993). To date, the ligand has not been identified. The HM74 receptor is found to be consistently up-regulated in COPD smokers compared to healthy smokers (54.8%). This is indicated by the "fold change" value (Table 5) and the "avg diff" value from the 42 sets of comparisons (Table 6).
[0038]
[Table 4]
Table 5. HM74 receptor expression pattern: fold change calculated for 42 sets compared between COPD and healthy smokers. Only values higher than 2 times and values lower than -2 times are considered deregulated. Thus, the HM74 receptor is up-regulated in 23 cases and unregulated in 17 cases.

[0039]
[Table 5]
Table 6: HM74 receptor expression levels. The "avg diff" value, a relative measure of the intensity of the hybridization signal on the chip, is shown for each patient; OS represents obstructive smoker, HS represents healthy smoker, NS represents non-smoker.

[0040]
Chip data for the HM74 receptor is confirmed by TaqMan analysis (Perkin Elmer Applied Biosystems). The fold change obtained with TaqMan is very similar to the data from the gene chip (Table 7).
[0041]
[Table 6]
Table 7. Upregulation of HM74 receptor in COPD smokers as determined by gene chip and TaqMan. Fold change measurement of HM74 by chip data in six comparisons between COPD smokers and healthy smokers is confirmed by TaqMan in the same sample, and relative up-regulation calculates GAPDH as the housekeeping gene.

[0042]
Another differentially expressed nucleic acid sequence that has been identified is the AICL receptor (activation-induced C-type lectin), a type II membrane protein that recognizes and binds N-acetylgalactosamine or N-acetylglucosamine (Oda et al. 1988) (see SEQ ID NOs: 5 and 6). It is expressed on lymphoid tissues and hematopoietic cells and NK and T cells. This expression is induced during lymphocyte activation and after PMA stimulation (Hamann et al., 1997). Since homologs of the AICL receptor are involved in lymphocyte signaling and lymphocyte proliferation, it is plausible to assume that the AICL receptor is also involved in these processes (Hamann et al., 1993).
The AICL receptor is found to be consistently upregulated (66.7%) in COPD smokers compared to healthy smokers. This is indicated by the "fold change" value (Table 8) and the "avg diff" value from a comparison of 42 sets (Table 9).
[0043]
[Table 7]
Table 8. AICL receptor expression pattern: fold change calculated for 42 sets compared between COPD and healthy smokers. Only values higher than 2 times and values lower than -2 times are considered deregulated. Thus, the AICL receptor is up-regulated in 28 cases and unregulated in 14 cases.

[0044]
[Table 8]
Table 9. AICL receptor expression level: "avg diff" value, a relative indicator of the intensity of the hybridization signal on the chip, shown for each patient; OS is an obstructive smoker, HS is a healthy smoker, NS is no smoking Person.

[0045]
Another differentially expressed nucleic acid sequence identified encodes the ILT1 receptor (immunoglobulin-like transcript 1) (see SEQ ID NOs: 11 and 12). The ILT1 receptor belongs to the Ig superfamily, which relates to a subset of activating receptors similar to NK cell receptors for MHC class I molecules. The ILT1 receptor is a 69 kDa glycosylated transmembrane receptor, which is mainly expressed in lung and liver and monocytes, granulocytes, macrophages and dendritic cells (Samaridis and Colonna 1997). When cross-linked with the antibody, the ILT1 receptor interacts with the γ-chain of the Fc receptor (Fc_εRI_γ) (Nakajima et al., 1999). The ILT1 receptor is found to be consistently upregulated (59.5%) in COPD smokers compared to healthy smokers. This is indicated by the "avg diff" values from the 42 sets of comparisons (Table 10). The p-value for the comparison between COPD smokers and healthy smokers was 0.01.
[0046]
[Table 9]
Table 10. ILT1 receptor expression levels: "avg diff" values for each patient are shown, along with the mean and median values for the three patient groups. OS stands for obstructive smoker, HS stands for healthy smoker, NS stands for non-smoker.

[0047]
The identified differentially expressed nucleic acid sequence is a SHPS-1 receptor (SIRP-α1, MYD1, MFR) that is known to be highly expressed on macrophages (Fujioka et al., 1996, Kharitonenkov et al., 1997, Brooke et al. 1998). (See SEQ ID NOs: 3 and 4). SHPS-1 receptor is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. It contains three extracellular Ig-like domains, a cytoplasmic tail with predicted tyrosine phosphorylation sites, and an immunoreceptor tyrosine-based inhibitory motif (ITIM). Activation of the receptor tyrosine kinase results in phosphorylation of the SHPS-1 receptor, resulting in association with SHP-1 (in macrophages) or with SHP-2 (in non-hematopoietic cells) (Veillette et al., 1998). . In addition, other proteins have been found to associate with the cytoplasmic domain of the SHPS-1 receptor, and it is therefore plausible to assume that the SHPS-1 receptor functions as a scaffold protein.
The SHPS-1 receptor is found to be consistently down-regulated (73.8%) in COPD smokers compared to healthy smokers. This is indicated by the "fold change" value (Table 11) and the "avg diff" value (Table 12) from a 42 set comparison. The p-value for the comparison between COPD smokers and healthy smokers is 0.005.
[0048]
[Table 10]
Table 11. SHPS-1 receptor expression pattern: fold change calculated for 42 sets compared between COPD and healthy smokers. Only values higher than 2 times and values lower than -2 times are considered deregulated. Thus, the SHPS-1 receptor is down-regulated in 29 cases and unregulated in 13 cases.

[0049]
[Table 11]
Table 12. SHPS-1 receptor expression levels: "avg diff" values for each patient are shown along with the mean and median values for the three patient groups. OS stands for obstructive smoker, HS stands for healthy smoker, NS stands for non-smoker.

[0050]
Another differentially expressed nucleic acid sequence identified encodes KDEL receptor 1 (see SEQ ID NOs: 7 and 8), a receptor that has important functions in protein folding and assembly in the endoplasmic reticulum. It recognizes soluble proteins having the amino acid sequence KDEL and recovers them after binding to the endoplasmic reticulum (Townsley et al., 1993). KDEL receptor 1 may be involved in controlling protein transport in the Golgi complex. Upon binding of the ligand, the KDEL receptor dimerizes and interacts with ARF GAP, the GTPase activating protein of the ADP ribosylation factor (Aoe et al., 1997). This is found to be consistently down regulated (71.4%) in COPD smokers compared to healthy smokers. This is indicated by the "avg diff" value (Table 13). The p-value for the comparison between COPD smokers and healthy smokers is 0.003.
[0051]
[Table 12]
Table 13. KDEL receptor expression levels: "avg diff" values for each patient are shown along with the mean and median values for the three patient groups. OS stands for obstructive smoker, HS stands for healthy smoker, NS stands for non-smoker.

[0052]
Another differentially expressed nucleic acid sequence identified encodes macrophage colony stimulating factor-1 receptor precursor (CSF-1 receptor, c-fms); see SEQ ID NOs: 9 and 10. The CSF-1 receptor belongs to the subfamily of receptor tyrosine kinases. Activation of the CSF-1 receptor results in the formation of a complex of multiple proteins, for example, the CSF-1 receptor, Shc, PI3K, Grb2, Cbl, SHP-1, Src. In addition, ligand binding causes rapid tyrosine phosphorylation of cytoplasmic proteins such as Cbl, STAT3, STAT5a, p85PI3K, SHP-1, Vav and excess proteins involved in cytoskeletal formation (Yeung et al., 1998). The CSF-1 receptor regulates the survival, proliferation, differentiation and morphology of mononuclear phagocytes (Hampe et al., 1989). The CSF-1 receptor is found to be consistently downregulated (45.2%) in COPD smokers compared to healthy smokers. This is indicated by the "avg diff" value (Table 14). The p-value for the comparison between COPD smokers and healthy smokers is 0.002.
[0053]
[Table 13]
Table 14. CSF-1 receptor expression level: “avg diff” values for each patient are shown along with the mean and median values for the three patient groups. OS stands for obstructive smoker, HS stands for healthy smoker, NS stands for non-smoker.

[0054]
1.6. DNA For checking and diagnosing chip data TaqMan Use of analysis
The mRNA expression profile obtained by the DNA chip is confirmed by TaqMan analysis with the same RNA preparation. In addition, the method is applied to monitor disease progression by measuring FPRL-1 receptor mRNA levels in cultured cell lines and cells isolated from humans.
Using RNA isolated from U937 cells treated with 10 nM retinoic acid for 3 days, the reaction conditions for measuring the FPRL-1 receptor mRNA level were optimized, and GAPDH (glyceraldehyde) was used as the FPRL-1 receptor and housekeeping gene. -3-phosphate dehydrogenase). Quantification of the FPRL-1 receptor is performed using the following primers: forward primer (FP) (see SEQ ID NO: 17, reverse primer (RP), see SEQ ID NO: 18, and 5 'with the reporter dye FAM and the quenching dye TMARA. See TaqMan probe (TP) labeled at the 3 'end, SEQ ID NO: 19.
[0055]
To measure GAPDH mRNA levels, a commercially available kit "TaqMan GAPDH Control Reagent" (P / N 402869) (Perkin Elmer Applied Biosystems) is used. The GPPDH probe labels JOE as a reporter dye and TMARA as a quenching dye. The RT-PCR reaction is performed using a "TaqMan EZ RT-PCR Core Reagents" (P / NN808-0236) (Perkin Elmer Applied Biosystems) kit. Standard curves for FPRL-1 and GAPDH are performed with increasing RNA concentrations from U937 cells treated with 1 μM retinoic acid in the range of 0, 5, 10, 25, 50, 100 ng for each assay. The reaction mixture was 1x TaqMan EZ-buffer, 3 mM Mn (Oac)₂, 300 μM dATP, dCTP, dGTP, and 600 μM dUTP, 2.5 U rTth DNA polymerase, 0.25 U AmpErase UNG in a total volume of 25 μl. For analysis of the FPRL-1 receptor, the reaction mixture contains 300 nM FP and RP and 100 nM TP. Primer concentrations for measuring GADPH levels are 200 nM for each primer and 100 nM for the GAPDH TaqMan probe.
[0056]
To measure mRNA levels in FPRL-1 receptor and GAPDH human patients and cell lines, 16-50 ng of RNA is used per reaction. All samples are run in triplicate. Reactions are performed in an ABI PRISM 7700 Sequence Deterministics System with a "MicroAmp Optical 96-Well Reaction Plate" (Perkin Elmer Applied Biosystems) sealed with a MicroAmp Optical Cap. The PCR conditions were 50 ° C for 2 minutes, 60 ° C for 30 minutes, 95 ° C for 5 minutes, followed by 40 cycles of 94 ° C for 20 seconds and 59 ° C for 1 minute. Data analysis determines mRNA levels of FPRL-1 receptor and GAPDH according to a standard curve, or_TValue is GAPDH C_TThis is done by directly associating with the value. The latter can be performed on these genes since the efficiency of both reactions is around 95%. The same method is used to examine mRNA levels isolated from COPD patients to monitor the success of the treatment after diagnosis of the disease or treatment with the putative active agent.
The other receptors mentioned in Example 1.5 are each probed accordingly using the appropriate primers.
[0057]
1.7. Cell line
The human monocyte / macrophage cell lines HL-60, U937, THP-1 and MonoMac6 are used as cell model systems. Cells are grown in RPMI 1640 medium containing 10% FCS and 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM glutamine and 1 × non-essential amino acids. The medium for MonoMac6 cells also contains a 5 ml / l OPI medium supplement (Sigma). MonoMac6 is cultured exclusively in 24-well plates. All cells have 5% CO₂Are maintained at 37 ° C. in a humid atmosphere containing and periodically tested for contamination by mycoplasma. 10 nM PMA (phorbol 12 myristate-13 acetate) is added to the medium to allow differentiation.
[0058]
1.8. FPRL-1 Receptor cloning
The FPRL-1 receptor is cloned from total RNA extracted from U937 cells treated with 1 μM retinoic acid for 3 days. 5 μg of RNA is replaced with 5 ng of oligo (dt)₁₈Primers, 1x first strand buffer, 10mM DTT, 0.5mM dNTPs and 2U Superscript II (Gibco @ BRL Life Technologies) are reverse transcribed at 42 ° C for 50 minutes to cDNA. Next, the reaction is stopped at 70 ° C. for 15 minutes, and the cDNA concentration is measured with a UV spectrometer. For amplification of the FPRL-1 receptor, 100 ng of cDNA and 10 pmol of primers specific to FPRL-1 receptor (forward primer attB1; SEQ ID NO: 15 and reverse primer attB2; SEQ ID NO: 16) are used for PCR. Reaction conditions are as follows: 94 ° C. for 2 minutes using Taq polymerase, followed by 30 cycles (94 ° C. for 30 seconds, 53 ° C. for 30 seconds, 72 ° C. for 90 seconds) followed by 7 minutes at 72 ° C. The reaction mixture is separated on a 2% agarose gel, a band of about 1000 bp is excised and purified with a QIAEX II extraction kit (Qiagen). The concentration of the purified band was measured, and about 120 ng of 300 ng of pDONR201 (donor vector for Gateway system) (Gibco BRL Life Technologies), 1 × BP clonase reaction buffer, BP clonase enzyme mixture and a total volume of 20 μl at 25 ° C. for 60 minutes Incubate. The reaction mixture is then incubated with 2 μl of proteinase K at 37 ° C. for 10 minutes. The reaction mixture is then electroporated into competent DB3.1 cells and plated on plates containing kanamycin. The clone designated pDONR-HM63 carrying the nucleic acid sequence as set forth in SEQ ID NO: 1 is used for further experiments.
Other receptors described in Example 1.5 are cloned using similar methods.
[0059]
1.9. FPRL-1 Receptor transfection
Using the vector containing FPRL-1 described in 1.8, cDNA of the FPRL-1 receptor was transformed into an expression vector pcDNA3.1 (+) / having an attR1 and attR2 recombination site of a Gateway cloning system (Gibco BRL Life Technologies). Transfer to attR. Here, the FPRL-1 receptor is expressed under the control of the CMV promoter. 150 ng of the “entry” vector pDONR-HM63 is brought to 20 μl with TE (Tris / EDTA) together with 150 ng of the “destination vector” pcDNA3.1 (+) / attR, 4 μl of LR clonase enzyme mix, 4 μl of LR clonase reaction buffer. And incubate at 25 ° C. for 60 minutes. Add 2 μl of proteinase K solution and incubate at 37 ° C. for 10 minutes. Transform 1 μl reaction mixture into 50 μl DH5α (incubate cells with DNA for 30 minutes on ice followed by heat shock at 42 ° C. for 30 seconds). After heat shock of cells, 450 μl of S. O. C is added and the cells are incubated at 37 ° C. for 60 minutes. Cells (100 μl) are plated on LB plates containing 100 μg / ml ampicillin and incubated overnight.
Colonies containing pcDNA3.1 (+) / attR with the FPRL-1 receptor as insert are designated pcDNA / FPRL1 and used for transfection studies.
Cell clones containing the vector obtained in 1.8 carrying the nucleic acid sequences encoding the other receptors described in 1.5 are prepared in a similar manner.
[0060]
Example 2: Cell lines and FPLR-1 Phenotypic effects of receptors
A method similar to that described in Example 2 for the FPRL-1 receptor can be performed using the other receptors described in 1.5.
2.1. Cell line
The human monocyte / macrophage cell lines HL-60, U937, THP-1 and MonoMac6 are used as cell model systems. Cells are grown in RPMI 1640 medium containing 10% FCS, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM glutamine and 1 × non-essential amino acids. The medium for MonoMac6 cells also contains a 5 ml / l OPI medium supplement (Sigma). MonoMac6 is cultured exclusively in 24-well plates. All cells have 5% CO₂Are maintained at 37 ° C. in a humid atmosphere containing and periodically tested for contamination by mycoplasma. 10 nM PMA (phorbol 12 myristate-13 acetate) is added to the medium to allow differentiation.
Phenotypic effect of FPRL-1 receptor (2.2-2.9)
2.2. Ligand binding assay
300 ml of cell culture was collected in EDTA solution and the suspension was used to centrifuge cells down at 110-120 xg to remove 10 mM Tris / HCl, pH 7.4, 2.5 mM CaCl2.₂, 1.2 mM MgCl₂Resuspended in 40 μg / ml bacitracin, 4 μg / ml leupeptin, 4 μg / ml chymostatin, 10 μg / ml pefablock, 2 μM phosphoamide and 0.1 mg / ml bovine serum albumin (BSA Fraction V, BI Bioproducts), 2x10⁶Dilute to cells / ml.
[0061]
Aliquot 0.5 ml to 0.3 nM 3^H-Incubate with lipoxin A4 (specific activity 〜１０10 Ci / mmol) or with increasing concentrations of lipoxin A4 without tritium (3-300 nM) at 4 ° C. for 30 minutes. The cells were collected by a Cell-Harvester (Skatron) equipped with a GF / B filter to terminate the incubation, and then to 50 mM Tris / HCl, 100 mM NaCl, 10 mM MgCl.₂Wash 3 times with 3 ml of chilled buffer, pH 7.4, and transfer filter pieces to vials. Add 2 ml of scintillation cocktail and measure radioactivity with a scintillation counter (LKB). Non-specific binding is measured in the presence of 100 nM unlabeled lipoxin A4. To replace tritiated lipoxin A4, a series of peptides and low molecular weight compounds containing the peptide ligand MMK-1 (Klein et al., 1998) are used under the same reaction conditions at concentrations ranging from 0.5 to 300 nM.
The bound radioactivity (on the filter strip) is assessed with a meter, the values recorded online and fitted to the model.³All substances that inhibit the binding of H-lipoxin A4 have their IC₅₀Calculate the value.
[0062]
2.3. FLIPR Measured by the assay Ca ²⁺ − release
The FLIPR assay (fluorescence imaging plate reader) using the FPRL-1 receptor was performed using G-protein α subunit α16 or chimeric G-protein Gqi5 or Gqo5 (these were the last five residues of Gα (i) or Gα (o)). And two different CHO cell lines that constitutively express the FPRL-1 receptor. Cell lines CHO / Galpha16, CHO / GalphaGqi5 and CHO / GalphaGqo5, which constitutively express Gα16, Gqi5 or Gqo5, are transfected with the FPRL-1 receptor expression vector. These cell lines were transformed with 5% CO2 in Ham's F12 medium (Bio Whittaker) containing 10% FCS (fetal calf serum), 2 mM glutamine, 200 ng / ml hygromycin, 100 U / ml penicillin and 100 μg / ml streptomycin.₂Culture at 37 ° C in a humid atmosphere containing 3-7x10⁵Cells are seeded in 60 mm Petri dishes and cultured overnight. Cells are grown to 50-80% confluence and used for transfection. Add 6 μl of FuGene 6 (Roche Biochemicals) to 100 μl of medium without serum and equilibrate for 5 minutes at room temperature.
[0063]
Next, 2 μg of the purified pcDNA / FPRL-1 receptor is added to the previously diluted FuGene6 solution, mixed gently, and further incubated at room temperature for 15 minutes. The medium is aspirated from the cells and 4 ml of fresh medium is added to the cells. The FuGene6 solution is added dropwise to the cells and the medium is shaken to distribute evenly. After 48 hours, the medium was aspirated and the medium was replaced with Ham's F12 medium, 10% FCS (fetal calf serum), 2 mM glutamine, 200 ng / ml hygromycin, 100 U / ml penicillin and 100 μg / ml streptomycin and 200 μg / ml G418. I do. During the next 5 days, the medium is changed daily until the dead cells and cell debris are washed and a single colony of cells is visible. Single colonies are isolated by separation on a cloning cylinder and released from the surface by adding 100 μl of 1 × trypsin / EDTA.
[0064]
Transfer cells from the cloning cylinder to 4 ml of medium and plate on 6-well plates. Single colonies are expanded and several colonies are tested for FPRL-1 receptor expression levels by a ligand binding assay (2.2). FLIPR (Molecular Devices) using a cell clone named CHO / Galpha16 / FPRL-1 receptor, CHO / GalphaGqi5 / FPRL-1 receptor or CHO / GalphaGqo5 / FPRL-1 receptor which most highly expresses the FPRL-1 receptor. Intracellular Ca²⁺Is measured. Cells (CHO / Galpha16 / FPRL-1 receptor, CHO / GalphaGqi5 / FPRL-1 receptor or CHO / GalphaGqo5 / FPRL-1 receptor) were placed in a 384-black well plate (Corning) at 2500-5000 cells per well at 40 μl. Seed by volume, 5% CO₂Grow overnight at 37 ° C. in a humid atmosphere containing
[0065]
CHO / Galpha16, CHO / GalphaGqi5 or CHO / GalphaGqo5 cells are used as negative controls. Next, 40 μl of Fluo-4 (Molecular Probes) staining solution is added to each well at a final concentration of 2 μM, and the cells are labeled with Fluo-4. Fluo-4 staining solution consisted of 10.5 ml of the above cell culture medium, 420 μl probenicid solution (1.42 g probenicid (Sigma), 10 ml 1 M NaOH, 10 ml Hanks buffer), 42 μl Fluo-4 stock solution (50 μg). Fluo-4, 23 μl DMSO, 23 μl Pluronic F-127 (20% DMSO solution) (Molecular Probes) and 420 μl 1 M HEPES.₂After incubation for 45 minutes at 37 ° C. in a humid atmosphere containing 20 ml of Hannic's buffer containing 20 ml of probenicid solution, the cells were washed with an EMBLA-washer (4 washing steps, program 03), and 25 μl was added to each well. Leave wash buffer. Next, the FLIPR of the stained well is measured at 10,000, and the difference between the unstained cell and the stained cell is set to 1: 5. Next, a series of ligands, peptides and low molecular weight compounds, including 25 μl lipoxin A4 and peptide ligand MMK-1, diluted in Hanks buffer / 0.1% BSA, were added to the wells at increasing concentrations (0.5-300 nM). Add. The substances of the invention are tested in increasing concentrations (0.5-300 nM) in competition with lipoxin A4 (50 nM) to determine their antagonist potential. Record every second up to 60 seconds starting from the addition of the ligand and then every 5 seconds for the subsequent 60 seconds.
[0066]
2.4. Production and release of cytokines or matrix metalloproteases
Monocyte / macrophage cell line cells 2.5-5 x 10⁵Treat with lipoxin A4 at a cell density of cells / ml. Cells were collected after 0, 1, 3, 6, 12, 24, 48 and 72 hours, supernatants were frozen for further investigation, cells were washed with PBS, 400 ml RLT containing 143 mM β-mercaptoethanol. Resuspend in buffer (Qiagen RNeasy Total RNA Isolation @ Kit), shear the DNA at least 5 times with a 20 g needle and store at -70 ° C.
Total RNA is isolated with the Qiagen RNeasy Total RNA Isolation Kit (Qiagen) according to the manufacturer's protocol. Purified RNA is used for TaqMan analysis. The cytokines TNFα, IL-1β, IL-8, IL-6 and human matrix metalloproteases MMP-1, MMP-7, MMP-9, MMP-12 are measured using appropriate primer sequences.
[0067]
2.4.1. Detection of secreted cytokines
The protein in the supernatant of the cultured and stimulated cells is precipitated by adding TCA (trichloroacetic acid) to a final concentration of 10%. The precipitate is washed twice with 80% ethanol, and the pellet is washed with 50 mM Tris / HCl, pH 7.4, 10 mM MgCl₂Resuspend in 1 mM EDTA. The protein concentration is measured by the Bradford method, and 50 μg of each sample is loaded on a 12% SDS polyacrylamide gel. The gel was blotted onto PVDF-membrane, blocked with 5% BSA in TBST for 1 hour, and commercially available human TNFα (tumor necrosis factor α), IL-1β (interleukin-1β), IL-8 ( Incubate for 1 hour with antibodies to interleukin 8) and IL-6 (interleukin 6). After washing with TBST, blots are incubated with horseradish peroxidase-conjugated anti-human IgG, washed again, and developed with an ECL chemiluminescence kit (Amersham). Band intensities are visualized with BioMax @ X-ray film (Kodak) and quantified with a densitometer.
[0068]
2.4.2. Detection and analysis of secreted matrix metalloproteases
The method is the same as described in 2.4.1. The antibodies used for western blotting are against human MMP-1, MMP-7, MMP-9 and MMP-12.
Protease activity is measured with a fluorescent substrate. Supernatants isolated from stimulated and unstimulated cells (described above) were combined with 1 μM substrate (DAB-Gaba-Pro-Gln-Gly-Leu-Glu (EDANS) -Ala-Lys-NH2 (Novabiochem) in a total volume of 50 μl. )) And incubated for 5 minutes at room temperature. Positive controls are performed with 125 ng of purified MMP-12 per reaction. Protease activity is measured on a fluorescence spectrometer with 320 nm excitation and 405 nm light emission.
[0069]
Another assay for measuring proteolytic activity and cell migration uses a chemotaxis chamber. Plate cells in 8 μm Matrigel (Becton Dickinson) coated filters in wells in upper part of chamber (10 per well)⁵Cells). In the lower compartment, a chemoattractant such as lipoxin A4 (100 nM), MCP-1 (monocyte chemoattractant protein 1) (10 ng / ml) is added to the medium. Five days later, the filter was taken out, the cells on the lower surface that had passed through Matrigel were fixed with methanol, stained with Diff-Quik staining kit (Dade Behring), and measured with an optical microscope in three high-power fields (400 ×). I do.
[0070]
2.5. Chemotaxis assay
To measure chemotaxis, use a 48-well Boyden chamber (Neuroprobe). Cells are starved for 24 hours in RPMI medium without FCS. A chemoattractant (50 ng / ml IL-8, 10 ng / ml MCP-1, 10 nM lipoxin A4, 10 nM MMK-1 peptide (2.3.)) Was diluted in FCS-free RPMI medium, and 30 μl was added to the bottom of the well. Place in parcel. The upper compartment is separated from the lower compartment by a polycarbonate filter (pore details 8 μm). 50 μl of cell suspension (5 × 10⁴) In the wells of the upper compartment. 5% CO chamber₂Incubate for 5 hours at 37 ° C. in a humid atmosphere containing Next, the filter is removed, the upper cells are scraped off, the lower cells are fixed in methanol for 5 minutes, and stained with a Diff-Quik staining set (Dade Behring). The migrated cells are measured in three high power fields by light microscopy.
[0071]
2.6. Adhesion assay
Cells are collected, washed with PBS, and resuspended in PBS and 1 μM BCECF ((2′-7′-bis- (carboxyethyl) -5 (6 ′)-carboxyfluorescein acetoxymethyl) ester, Calbiochem) ( 4x10⁶/ Ml) at 37 ° C for 20 minutes. The cells are washed with PBS and resuspended in PBS containing 0.1% BSA (3.3 × 10⁶/ Ml). 3x10⁵Cells (90 μl) are added to each well of a 96-well flat bottom plate coated with laminin (Becton Dickinson). 10 μl of agonist (100 nM lipoxin A4 + lipoxin A4 antagonist) is added and the plate is incubated at 37 ° C. for 20 minutes. Next, the cells are washed with PBS containing 0.1% BSA, and the adherent cells are solubilized with 100 μl of 0.025 M NaOH and 0.1% SDS. Quantification is performed by fluorescence measurement.
[0072]
2.7. Phagocyte activity
Cell suspension (2.5 × 10⁴Cells / ml) were seeded on a 6-well plate as 5 ml U937 or THP-1 or on a 24-well plate as 2 ml MonoMac6, agonist (100 nM lipoxin A4, 50 nM MMK-1 peptide (2.3.)) And agonist 5% CO2 in the presence of low molecular weight compounds of the invention to counteract the effect₂Incubate at 37 ° C. for 1 hour in a humid atmosphere containing Add 40 μl of a dispersion suspension of heat-inactivated Saccharomyces boulardii (20 yeast / cell) to each well. The cells are incubated for a further 3 hours, washed twice with PBS and spun. Cell spin preparations are stained with May-Grunwald-Giemsa and phagocytosed particles are counted with a light microscope.
[0073]
Example 3 COPD Cell culture model for macrophages isolated from patients
A method similar to that described in Example 3 for FPRL-1 can be performed using the receptor described in 1.5.
As cell culture models for macrophages isolated from COPD patients, we select the monocytic cell lines MonoMac6 and THP-1. Cells are treated with PMA to mimic the hyperactivated state of these cell lines. The cells are further exposed to a stimulus that should mimic conditions similar to those in COPD. These stimuli are smoking or exposure to LPS.
[0074]
FPRL-1 expression after PMA, smoking and LPS stimulation of MonoMac6 cells
MonoMac6 cells are cultured in 24-well plates in RPMI 1640 medium supplemented with 10% FCS (low endotoxin), 2 mM glutamine, 1 × non-essential amino acids, 200 U / ml penicillin, 200 μg / ml streptomycin and 5 ml OPI medium supplement (Sigma). . Cells are grown to a density of 600,000 cells per well (2 ml medium) and stimulated with 10 nM PMA (phorbol 12-myristate 13-acetate) (Sigma) or 20 ng / ml LPS (lipopolysaccharide from Salmonella Minnesota Re595) (Sigma). I do. For smoke exposure, cells were grown at 37 ° C., 5% CO 2 in a medium rich in smoke.₂1x10 at⁶Incubate at a density of cells / ml.
[0075]
Enrichment of the RPMI 1640 medium with smoke was carried out with two cigarettes. The tobacco smoke is drawn into a 50 ml syringe (about 20 volumes of 50 ml syringe per cigarette) and then perfused into 100 ml of RPMI 1640 medium without supplements. Thereafter, the pH of the smoke-enriched medium is adjusted to 7.4, and the medium is sterilized with a 0.2 μm filter for use. After smoke exposure, cells are washed at least twice with RPMI 1640 to remove residual smoke particles. The cells are then seeded into 24-well plates filled with fresh 2 ml RPMI 1640 medium containing the above supplements at 400,000 to 600,000 cells per well.
[0076]
THP-1 cells were 75 cm in RPMI 1640 Glutamax supplemented with 10% FCS (low endotoxin), 200 U / ml penicillin, 200 μg / ml streptomycin.²Grow in flask. Cells are incubated with 10 nM PMA for 48 hours at 37 ° C., 5% CO 2.₂To differentiate the cells into macrophage-like cells. The medium is then replaced with a new PMA-free medium containing 20 ng / ml LPS.
37 ° C., 5% CO for both cell types₂Culture in a humid atmosphere, collect cells at various time points, and monitor effects over time. The cells are spun down, washed with PBS, resuspended in 400 μl of RLT buffer (Qiagen RNeasy Total RNA Isolation Kit) containing 143 mM β-mercaptoethanol, and the DNA is sheared at least 5 times with a 20 g needle, Store at -70 ° C.
Total RNA is isolated with the Qiagen Newly Total RNA Isolation Kit (Qiagen) according to the manufacturer's protocol. The purified RNA is digested with RNase-free DNase (Qiagen) and used for TaqMan analysis.
[0077]
TaqMan analysis
TaqMan analysis is used to determine FPRL-1 mRNA levels in stimulated and unstimulated cell lines at various time points. Total RNA isolated from U937 cells treated with 10 nM retinoic acid for 3 days was used to optimize the conditions for determining FPRL-1 mRNA levels and to determine FPRL-1 and GAPDH (Glycel as a housekeeping gene) A standard curve is set up for (aldehyde-3-phosphate dehydrogenase). Quantification of FPRL-1 is performed using the following primers: forward primer (rHM63668 (-) FP, SEQ ID NO: 22), reverse primer (hHM63525 (+) RP, SEQ ID NO: 23), and a reporter at the 5 'end. TaqMan probe (rhHM63629 (-) TP, SEQ ID NO: 24) labeled with the quenching dye TAMRA at the 3 'end with the dye FAM. GAPDH mRNA levels are measured with a commercially available kit for GAPDH "TaqMan GAPDH Control Reagent" (P / N 402869) (PE Applied Biosystems). The GAPDH probe is labeled with JOE as a reporter dye and TAMRA as a quenching dye.
[0078]
The RT-PCR reaction is performed with Perkin Elmer's "TaqMan EZ RT-PCR Core Reagent" (P / NN808-0236) kit. Standard curves for FPRL-1 and GAPDH are generated with increasing concentrations of RNA from U937 cells treated with 1 μM retinoic acid for each assay ranging from 0, 5, 10, 25, 50 to 100 ng. The reaction mixture was 1x TaqMan EZ-buffer, 3 mM Mn (Oac)₂, 300 μM dATP, dCTP, dGTP and 600 μM dUTP, 2.5 U rTth DNA polymerase, 0.25 U AmpErase UNG in a total volume of 25 μl. For FPRL-1, the reaction mixture contains 300 nM rhHM63 668 (-) FP and hHM63 525 (+) RP, and 100 nM rhHM63629 (-) TP. The concentration of primers for measuring GAPDH levels is 200 nM for each primer and 100 nM for TaqMan probe. To measure FPRL-1 and GAPDH mRNA levels in human patients and cell lines, 16-50 ng of RNA is used per reaction. All samples are run in triplicate. Reactions are performed on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystem) in a MicroAmp Optical 96-well plate (PE Applied Biosystems) sealed with a MicroAmp Optical Cap. The PCR conditions are 50 ° C. for 2 minutes, 60 ° C. for 30 seconds, 95 ° C. for 5 minutes, followed by 40 cycles (94 ° C. for 20 seconds, 59 ° C. for 1 minute). Data analysis determines FPRL-1 and GAPDH mRNA levels according to a standard curve, or_TValue directly to GAPDH C_TIt depends on whether it is associated with a value. The latter method can be applied to these genes because the efficiency of both reactions agrees well with each other (about 95%).
[0079]
[Table 14]
Table 1: Expression of FPRL-1 in MonoMac6 cells stimulated with 10 nM PMA

[0080]
[Table 15]
Table 2: Expression of FPRL-1 in MonoMac6 cells after differentiation with 10 nM PMA and stimulation with 20 ng / ml LPS

[0081]
[Table 16]
Table 3: Expression of FPRL-1 in MonoMac6 cells after differentiation with 10 nM PMA and stimulation with smoke

[0082]
[Table 17]
Table 4: Expression of FPRL-1 in THP-1 cells after differentiation with PMA and stimulation with LPS

[0083]
To examine the effect of FPRL-1 ligand, MonoMac6 cells were seeded at a density of 250,000 cells / ml (2 ml per well) in a 24-well plate and incubated at 37 ° C., 5% CO 2 for 24 hours.₂Grow in humid atmosphere and stimulate with 200 nM lipoxin A4 (Bioimol), W-peptide (1 μM) (synthesized by Metabion, Martinsried), and LPS (Sigma) as a positive control. Cells are harvested at various time points and total RNA is isolated using the Qiagen RNeasy Total RNA Isolation Kit (Qiagen) as described above.
The sequence of the W-peptide (Baek et al., 1996, J. Biol. Chem 271, 8170-8175) is as follows.
WKYMYMVm
This RNA is used for TaqMan analysis to examine the expression of inflammatory markers such as TNFα, IL-8 and MMP-12.
[0084]
[Table 18]
Table 5: Expression of TNFα in MonoMac6 cells after lipoxin A4 and W-peptide stimulation

[0085]
[Table 19]
Table 6: Expression of IL-8 in MonoMac6 cells after lipoxin A4 and W-peptide stimulation

[0086]
[Table 20]
Table 7: Expression of MMP-12 in MonoMac6 cells after lipoxin A4 and W-peptide stimulation

[0087]
Because of the increased invasion of macrophages into the peripheral airways in COPD patients, the chemotactic ability of MonoMac6, which serves as a cultured cell model of alveolar macrophages, was tested. MonoMac6 chemotaxis was determined by administering various ligands of FPRL-1.
MonoMac6 cells are treated with PMA for 24-30 hours to induce the activated state of the cells. Cells are collected, washed twice with RPMI 1640 without supplements, and seeded at a density of 500,000 cells / well (24-well plate) in the presence of 10 nM PMA. After 24-30 hours, the cells are released from the substratum by repeated rinsing with a pipette, spun down, counted and 1x10 in RPMI 1640 medium without supplements but with 10 nM PMA.⁶Adjust to a density of cells / ml. Chemotaxis is performed with a 48-well chemotaxis chamber (Neuroprobe Inc.) and a polycarbonate membrane with a pore size of 8 μm. The lower well of the chamber is filled with 28 μl of various concentrations of lipoxin A4, W-peptide, MCP-1 as a positive control, and RPMI 1640 medium without supplements as a negative control (containing 10 nM PMA). The lower well is coated with a polycarbonate membrane and the upper compartment of the chamber is filled with 50 μl of the cell suspension (50,000 cells / well).
[0088]
37 ° C, 5% CO₂After migration for 4 hours at, the cells in the upper part of the membrane are scraped off and the cells adhering to the lower part of the membrane are stained with the Diff Quik staining kit (Dade Behring) according to the manufacturer's protocol. The stained cells are counted with a light microscope at 250 × magnification in a 6-8 high power field. The migration index represents the fold increase in the number of cells that have migrated in response to the chemoattractant over the control medium.
[0089]
[Table 21]
Table 8: Migration of MonoMac6 in response to lipoxin A4, W-peptide, and MCP-1

[0090]
The cells of the above Examples and each of the cultured cell models described above were prepared by adding the substance to be tested, and then measuring the above-mentioned effects to determine whether the substance was deregulated according to the present invention in macrophages. Used to determine if it is an inhibitor.
[0091]
References
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Lee, T.M. H. Crea, A .; E. FIG. G. FIG. Gant, V .; Spur, B .; W. , Marron, B .; E. FIG. Nicolaou, K .; C. , Reardon, E.A. , Brezinski, M .; , And Serhan, C.I. (1990). Am. Rev .. Respir. Dis. 141, 1453-1458.
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Claims (61)

A method for determining whether a substance is an activator or an inhibitor of an ILM receptor, comprising subjecting the substance to a test system that produces a measurable output when the ILM receptor or ILM receptor function is modulated. The method comprising: 2. The method of claim 1, wherein the ILM receptor is a mammalian receptor. 3. The method according to claim 2, wherein the ILM receptor is a human receptor. The method according to claim 1, wherein the analysis using a cell line is performed. 5. The method of claim 4, wherein MonoMac6 or THP-1 cells are used, wherein the cells are stimulated with phorbol 12-myristate 13-acetate and stimulated with a substance selected from the group consisting of LPS and smoke. The method according to claim 1, wherein the analysis is performed using a cell-free system. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) The method according to claim 1, wherein the method is selected from the group consisting of CSF-1 receptor type receptors comprising 10). The method of claim 1, wherein the receptor is a FPRL-1 receptor type receptor. 9. The method according to claim 8, wherein the FPRL-1 receptor type receptor set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. A method for measuring an expression level of an ILM receptor, wherein the level of an ILM receptor expressed in a macrophage is measured. 11. The method according to claim 10, wherein the macrophages are mammalian cells. The method according to claim 11, wherein the macrophages are human cells. 13. The method according to claim 12, wherein the analysis is performed using macrophages or a part thereof obtained from the site of inflammation. 14. The method according to claim 13, wherein the analysis is performed using macrophages or a part thereof obtained from a site of inflammation in a mammal. 15. The method according to claim 14, wherein the analysis is performed using macrophages or a part thereof obtained from a site of human inflammation. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) The method according to claim 10, wherein the method is selected from the group consisting of CSF-1 receptor type receptor comprising 10). The method according to claim 10, wherein the receptor is a FPRL-1 receptor type receptor. 18. The method according to claim 17, wherein the FPRL-1 receptor type receptor set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. 11. The method according to claim 10, for the diagnosis or monitoring of chronic inflammatory airway disease. 20. The method of claim 19, wherein the chronic inflammatory airway disease is selected from the group consisting of chronic bronchitis and COPD. 20. The method according to claim 19, wherein the analysis is performed using macrophages or a part thereof obtained from the site of inflammation. 22. The method according to claim 21, wherein the analysis is performed using macrophages or a part thereof obtained from a site of inflammation in a mammal. 23. The method according to claim 22, wherein the analysis is performed using macrophages or a part thereof obtained from a site of human inflammation. A test system for determining whether a substance is an activator or an inhibitor of ILM receptor function, wherein said receptor is involved in chronic inflammatory airway disease and said receptor has a role in mediating inflammation And at least,
a) an ILM receptor, or b) an expression vector capable of expressing the ILM receptor in the cell, or c) a host cell transformed with an expression vector capable of expressing the ILM receptor,
The above method, comprising: FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 25. The test system according to claim 24, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). The method according to claim 24, wherein the receptor is a FPRL-1 receptor type receptor. 27. The test system according to claim 26, wherein the FPRL-1 receptor type receptor as set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. 28. The test system of claim 27, comprising a cell that expresses an ILM receptor. 29. The test system of claim 28, wherein MonoMac6 or THP-1 cells are used, wherein the cells are stimulated with phorbol 12-myristate 13-acetate and stimulated with a substance selected from the group consisting of LPS and smoke. . A substance determined as an activator or inhibitor of the ILM receptor. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 31. The substance according to claim 30, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). The substance according to claim 30, wherein the receptor is a FPRL-1 receptor type receptor. 33. The substance according to claim 32, wherein a FPRL-1 receptor type receptor as set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. A substance for treating a disease which is an activator or an inhibitor of the ILM receptor. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 35. The method according to claim 34, which is a receptor selected from the group consisting of CSF-1 receptor type receptors comprising 10). The substance according to claim 34, wherein the receptor is a FPRL-1 receptor type receptor. 37. The substance according to claim 36, wherein a FPRL-1 receptor type receptor as set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. 35. The substance of claim 34, wherein the disease is a chronic inflammatory airway disease. 39. The substance of claim 38, wherein the chronic inflammatory airway disease is selected from the group consisting of chronic bronchitis and COPD. A pharmaceutical composition comprising at least one substance determined as an activator and an inhibitor of the ILM receptor. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 41. The pharmaceutical composition according to claim 40, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). 41. The pharmaceutical composition according to claim 40, wherein the receptor is a FPRL-1 receptor type receptor. 43. The pharmaceutical composition according to claim 42, wherein the FPRL-1 receptor type receptor set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. Use of a substance determined to be an activator or inhibitor of an ILM receptor for the preparation of a pharmaceutical composition for the treatment of chronic inflammatory airway disease. 45. The use according to claim 44, wherein the chronic inflammatory airway disease is selected from the group consisting of chronic bronchitis and COPD. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) The use according to claim 44, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). The use according to claim 44, wherein the receptor is a FPRL-1 receptor type receptor. 48. The use according to claim 47, wherein the FPRL-I receptor type receptor according to SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. Administering to a patient in need of treatment for chronic inflammatory airway disease a pharmaceutical composition comprising at least one substance determined to be an activator or inhibitor of the ILM receptor. How to treat airway disease. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 50. The method according to claim 49, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). 50. The method of claim 49, wherein the receptor is a FPRL-1 receptor type receptor. 52. The method of claim 51, wherein the FPRL-1 receptor type receptor set forth in SEQ ID NO: 2, or a variant, variant or fragment thereof having the same function is used. 50. The method of claim 49 for treating a mammal. 54. The method of claim 53 for treating a human. 50. The method of claim 49, wherein the chronic inflammatory airway disease is selected from the group consisting of chronic bronchitis and COPD. A method of selectively modulating an ILM receptor in macrophages, comprising administering a substance determined to be an activator or inhibitor of the ILM receptor. 57. The method of claim 56, wherein the macrophages are involved in chronic inflammatory airway disease. 58. The method of claim 57, wherein the chronic inflammatory airway disease is selected from the group consisting of chronic bronchitis and COPD. FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO: 2), HM74 receptor type receptor including HM74 receptor (SEQ ID NO: 21), AICL receptor type receptor including AICL receptor (SEQ ID NO: 6), ILT1 receptor (SEQ ID NO: 12), SHPS receptor type receptor including SHPS-1 receptor (SEQ ID NO: 4), KDEL receptor type receptor including KDEL receptor 1 (SEQ ID NO: 8), and CSF-1 (SEQ ID NO: 8) 57. The method according to claim 56, which is a receptor selected from the group consisting of CSF-1 receptor type receptors including 10). 57. The method of claim 56, wherein the receptor is a FPRL-1 receptor type receptor. 61. The method of claim 60, wherein the FPRL-1 receptor type receptor set forth in SEQ ID NO: 2, or a variant, variant, or fragment thereof having the same function is used.