グラム陰性桿菌の細胞壁外膜の構成物質であるLipopolysaccharide (LPS)は、宿主細胞に強い炎症反応を惹起する作用を持ち、グラム陰性桿菌による敗血症における全身性の炎症および臓器障害を発生させる主な起因物質であると考えられている。これまで、LPSに関わる研究は数多く行われてきているが、LPSが宿主細胞の表面でいかなる分子挙動をとるかについては、全く知られていなかった。そこで、本研究では、生きた細胞の細胞膜における分子の動きを1分子レベルでリアルタイムに観察可能な全反射レーザー顕微鏡を用いて、E.coli 055:B5由来のAlexa594蛍光標識したLPS 20ng/mlをリコンビナント・マウスLPS Binding Protein (LBP) 1000ng/mlとともに培養したマウス腹腔マクロファージに投与し、LPS投与後5-15分、25-35分、55-65分のマクロファージ細胞膜におけるLPSの分子挙動を観察し、その結果を解析した。さらに、LPSが宿主細胞の細胞膜に結合するために必要とされているLBPおよびCD14のマクロファージ細胞膜におけるLPSの分子挙動への関わりを調べるために、LBP非存在下でのwild-typeマウスのマクロファージ細胞膜におけるLPSの分子挙動とCD14遺伝子欠損マウスから採取した腹腔マクロファージの細胞膜におけるLPSの分子挙動を観察した。次に、LPSが宿主細胞を刺激し炎症反応を惹起するうえで重要な働きをしているLPS受容体であるToll-like受容体4(TLR4)とそのアダプター分子であるMyeloid Differentiation Protein-2(MD2)およびTLR4の細胞内ドメインに結合しLPSの刺激を下流に伝達するシグナル分子であるMyeloid Differentiation Factor 88 (MyD88)とTIR-Domain-Containing Adapter-Inducing IFN-beta(TRIF)がLPSのマクロファージ細胞膜における分子挙動にいかに関与しているかを調べるために、TLR4、MD2、MyD88およびTRIF各々の遺伝子欠損マウスの腹腔マクロファージの細胞膜におけるLPSの分子挙動を観察し解析した。また、細胞膜に結合したLPSは細胞内に取り込まれ(LPS internalization)ゴルジ体に集積することが知られているため、上記の各遺伝子欠損マウスの腹腔マクロファージにおけるAlexa594蛍光標識LPSのinternalizationのwild-type腹腔マクロファージとの差異を、共焦点レーザー顕微鏡を用いて観察した。

結果として、まずwild-typeマウスの腹腔マクロファージ細胞膜では、LPS投与後に時間依存的にAlexa594蛍光標識LPSの輝度が高い蛍光点が出現し、全ての輝点における輝度の平均値もLPS投与後5-15分および25-35分に対してLPS投与後55-65分で有意に増加(p<0.01)していた。このことから、マクロファージ細胞膜に結合したLPSは、時間依存的に凝集体(oligomer)を形成するものと考えられた。また、細胞膜上におけるLPSの運動速度をAlexa594蛍光標識LPSの輝点の拡散係数をもとに解析した結果、時間依存的に動きの遅い輝点が出現し、全ての輝点における拡散係数の対数の平均値もLPS投与後5-15分に対して時間依存的に有意な低下(25-35分:p<0.05、55-65分:p<0.01)を認めた。

一方で、LBP非存在下のwild-typeマウスの腹腔マクロファージおよびCD14遺伝子欠損マウスの腹腔マクロファージにおいては、全反射レーザー顕微鏡による観察ではマクロファージ細胞膜におけるLPSの結合および運動をしめす輝点を全く認めず、この結果はLBPおよびCD14がLPSの宿主細胞の細胞膜への結合に不可欠な分子であるというこれまでの知見を初めて生きた細胞においてリアルタイムな観察により証明することができた。

次に、TLR4、MD2、MyD88およびTRIF各々の遺伝子欠損マウスの腹腔マクロファージの細胞膜におけるLPSの分子挙動の観察にもとづく解析の結果、いずれの遺伝子欠損マクロファージにおいても、wild-typeのマクロファージとは異なり、輝度の高い輝点の出現は認められず、LPS投与後55-65分の全ての輝点における輝度の平均値もwild-typeのマクロファージに対して有意に低値(p<0.01)であった。この傾向はTLR4遺伝子欠損マクロファージおよびMD2遺伝子欠損マクロファージにおいて特に顕著であり、両者ともMyD88およびTRIF遺伝子欠損マクロファージに対してもLPS投与後55-65分の輝度の平均値が有意に低下(p<0.01)していた。他方で、細胞膜におけるLPSの運動速度については、TLR4、MD2、MyD88およびTRIF各々の遺伝子欠損マクロファージにおいてwild-typeマクロファージと対比して拡散係数の小さな輝点の出現が少なく、LPS投与後55-65分の全ての輝点における拡散係数の対数の平均値は、いずれの遺伝子欠損マクロファージについてもwild-typeマクロファージに対して有意に高値(p<0.01)を示した。この傾向はMyD88遺伝子欠損マクロファージおよびTRIF遺伝子欠損マクロファージにおいて特に顕著であり、MyD88遺伝子欠損マクロファージではTLR4およびMD2遺伝子欠損マクロファージに対してもLPS投与後55-65分における拡散係数の対数の平均値が有意に高かった(p<0.01)。なお、共焦点レーザー顕微鏡によるLPS internalizationに関する検討では、wild-type腹腔マクロファージとTLR4、MD2、MyD88およびTRIF各々の遺伝子欠損腹腔マクロファージの間に明らかな変化は認められなかった。

本研究の結果から、通常の腹腔マクロファージにおいては、LPSは細胞膜に結合した後、時間依存的に運動速度が低下したLPS分子が出現し、かつ一部は凝集体を形成することが示された。さらに、このマクロファージ細胞膜におけるLPSの運動速度の低下と凝集体形成には、LPS受容体であるTLR4とそのアダプター分子であるMD2およびLPS刺激のシグナル分子であるMyD88とTRIFが、強く関与していることが明らかとなった。特に、LPSの細胞膜上での凝集体形成には細胞膜表面に発現されている分子であるTLR4とMD2が強く関与しており、一方で細胞膜におけるLPSの運動速度低下には細胞内に存在するシグナル伝達分子であるMyD88およびTRIFが強く関わっていた。全反射レーザー顕微鏡を用いた細胞のリアルタイム観察にもとづく本研究により、グラム陰性桿菌感染における敗血症の重要な起因物質であるLPSの宿主細胞における細胞膜での分子挙動と、その分子挙動へのLPS受容体および細胞内シグナル伝達分子の関わりを、初めて明らかにすることができた。本研究で示した細胞膜におけるLPSの分子挙動とLPSの宿主細胞に対する炎症惹起機序との関係については更なる検討を必要とするが、本研究は今後のLPSに関する研究に有意義な知見を提示できたものと考える。

Sepsis describes a complex clinical syndrome that results from dysregulation in host response to infection. The resulting immunosuppression has been suggested to be a major contributing factor in sepsis induced mortality. Lipopolysaccharide (LPS) or endotoxin plays a pivotal role in initiation of sepsis; it maintains the structural and functional integrity of gram negative bacterial outer membrane. LPS is carried into the body with a specific carrier protein-LPS binding protein (LBP). Then it interacts with CD14, a receptor on macrophages/monocytes and neutrophils. TLR4, with the help of another co-receptor MD2 mediates intracellular invasion of LPS. MyD88 and TRIF are known as intracellular signaling molecules which are essential for transcription of numerous genes of pro-inflammatory mediators in response to LPS.

Evanescent wave microscopy, also termed total internal reflection fluorescence microscopy (TIRFM), has shed new light on important cellular processes taking place near the plasma membrane. For example, this technique can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. TIRFM typically involves very thin (less than 100 nm) optical sectioning which means that the signal to noise (S/N) ratio is much better than with confocal images, and cellular photodamage and photobleaching are also minimal. It has proven useful for quantitative analysis of the dynamics and kinetics of cell signaling reactions.

The aim of this study is to reveal the molecular kinetics of LPS on the plasma membrane of wild type and CD14(-/-), TLR4(-/-), MD2(-/-), Myd88(-/-) or TRIF(-/-) murine peritoneal macrophages by total internal reflection fluorescence microscope (TIRFM) which is an ideal means to study the molecular mechanism of biological interaction of cellular molecules in vitro.

The results of this study have been described below:

1. We stimulated mouse peritoneal macrophages with commercially available Alexa 594-labeled LPS (100% fluorescence labeled LPS) and unlabeled LPS of same strain. Our data showed that both fluorescence labeled and unlabeled LPS triggered almost the same amount of TNF-α. It proves that fluorescence labeled LPS that we used in our experiment is immunologically functional.

2. The molecular action of Alexa 594-labeled LPS from E.coli was examined on the living peritoneal macrophages of C57BL/6 mice by TIRFM, and the molecular kinetics of LPS was analyzed. The fluorescence as well as the number of LPS spots gradually increased with time after administration of LPS and accordingly they were easily distinguishable from the background noise. When we analyzed MSD of only 2 Alexa 594-labeled LPS spots against time, our graph showed linear pattern, but when we calculated the MSD of 100 LPS spots the graph was not linear. It is likely that an individual LPS spot can move freely on the surface of the cell, when the spots bind with receptors and adaptor molecules diffusion becomes restricted.

3. Compared with 5-15 mins after LPS administration, the number of spots in the higher fluorescence range progressively increased for the case of 25-35 mins and 55-65 mins after LPS administration. At 5-15 mins the range of FI was 40-70 A.U.. The range extended up to 75 at 25-35 mins along with apparent decrease in the number of spots with FI around 60 A.U. in comparison to the result at 5-15 mins after LPS administration, while many spots with FI below 55 A.U. still could be found at 25-35 mins. Compared with the case of 5-15 mins after LPS administration, the diagram of number of spots vs. FI after 55-65 mins of LPS administration showed a more diffused pattern with several distributed peaks, which indicates formation of larger oligomers at 55-65 mins. This change was accompanied by a decrease in the number of spots in the lower fluorescence range and an increase in the number of spots with the lower diffusion coefficient (DC). The statistical analysis of the data at different time intervals confirmed the apparent time dependent change of both FI and DC. The mean FI of 55.73±0.31 A.U. (N=520) after 10 mins increased to 59.53±0.35 A.U. (N=524) after 60 mins of LPS administration, while the corresponding mean logarithms of DC values expressed in μm2/sec were -0.156±0.026 (N=520) and -0.467±0.036 (N=524), respectively. The number of fluorescence labeled LPS spots/μm2 of wild type macrophages showed gradual increase with time indicating higher binding of LPS spots with its concerning receptors and adaptor molecules.

4. In our experiments, consistent with the previous reports on the importance of LBP and CD14 in the initiation of cellular response to LPS, no motion of LPS on the plasma membrane was observed in absence of LBP or CD14(-/-) cases indicating the essential role of LBP and CD14 receptors for the movement and oligomerization of LPS on the living cells.

5. While after 55-65 mins of LPS administration the fluorescence spots ranged in between 40-87 A.U. in case of wild type macrophage, spots were not found beyond 60 A.U. in case of TLR4(-/-) macrophage. In fact the mean FI of LPS at 60 mins in case of TLR4(-/-) macrophages was the lowest among all the studied deficient macrophages (18% lower than wild type), suggesting the indispensability of TLR4 for the formation of oligomers of LPS on the plasma membrane. On the other hand, the mean logarithm of DC of LPS expressed in μm2/sec after 60 mins in TLR4(-/-) macrophages was higher (-0.283±0.031, N=498) than that of the wild type macrophages (-0.467±0.03, N=524). The number of fluorescence labeled LPS spots/μm2 incase of TLR4(-/-) macrophage was only 3 at 60 mins of LPS administration whereas in wild type macrophages the number was 10 spots/μm2 at the same time point. It is likely that binding of fluorescence labeled LPS spots with its receptors is the lowest incase of TLR4(-/-) among all the studied macrophages.

In our study, as seen in TLR4(-/-) macrophages, a narrower range of FI of LPS spots (40-69 A.U.) in comparison to the case of wild type (40-87 A.U.) was also observed in case of MD2(-/-) macrophages. Among all the studied deficient macrophages, the mean FI of LPS after 60 mins in case of MD2(-/-) macrophages was the second lowest (50.63±0.27 A.U, N=523).

6. A broader range of FI (up to 76 A.U.) as well as DC of LPS spots in comparison to the cases of LPS receptors (TLR4, MD2) was observed in absence of MyD88. As compared with the wild type macrophage, the specific pattern of the diagram of number of spots vs. fluorescence intensity of LPS in MyD88(-/-) macrophages indicated formation of smaller oligomers. On the other hand, the mean logarithm of DC of LPS expressed in μm2/sec in case of MyD88(-/-) macrophages showed the highest value (-0.113±0.033, N=535), which was 76% higher than the mean value in wild type. In our study, the importance of MyD88 was manifested by higher diffusion rate of LPS on the cell surface in case of MyD88(-/-) macrophages.

In case of TRIF(-/-) macrophages the diagram of number of spots vs. fluorescence intensity at 55-65 mins showed a moderate range (wider than the diagram of TLR4(-/-) and MD2(-/-) but narrower than that of wild type) of fluorescence intensities (ranging up to 81 A.U.). Fewer peaks of higher FI in the same diagram indicated lower extent of oligomerization than in the case of wild type macrophage. When compared to the macrophages deficient in LPS receptors (TLR4, MD2), the mean logarithm of DC of LPS expressed in μm2/sec at 60 mins in case of TRIF(-/-) macrophage (-0.175±0.032, N=540) showed significant difference (63% higher than wild type) with the corresponding value in the case of wild type macrophage, indicating the role of TRIF in lowering DC of LPS on the cell surface.

7. Data of our experiment clearly showed that LPS molecules get entry into the golgi apparatus and endosomal vesicles within a few minutes of LPS administration. After 30 mins of stimulation with E.coli LPS, fluorescence labeled LPS molecules co-localized with lysosome marker in wild type and other knock out macrophages.On the other hand, after 20 mins LPS molecules were clearly detectable in Golgi apparatus of wild type as well as other knock out macrophages where Alexa-594 labeled LPS merged with BIODIPY C5 Ceramide stained Golgi complex.

The presence of all the receptors is not obligatory for the internalization of the LPS molecules into the organelles of a living cell.

This is the first comprehensive investigation carried out to observe the differences of kinetics of LPS in a wide variety of receptor and adaptor molecule knock out mice employing TIRFM technique. Based on the findings in this study, it is likely that not only LPS but the receptors with intracellular domain of other various ligands are responsible for the oligomerization of the ligands and the association and activation of intracellular signaling molecules may be involved in lowering the diffusion rate of the ligands on the plasma membrane of living cells.

This study contributes significantly to the knowledge base of molecular interaction of LPS with its concerning receptors as well as the role of adaptor molecules in signal transduction. The current study bears an important significance for future research by revealing the respective roles of the LPS receptors and signaling molecules in the molecular behavior of LPS on the living cell surface by direct visualization.