E-selectin이 인체 진피 미세혈관 내피세포와 T림프구의 유착에 미치는 영향

Abstract

[한글]

염증반응이 발생하려면 혈관내의 염증세포가 항원이 존재하는 주변조직으로 이동해야 하며 첫 단계로서 혈관 내피세포에 유착하여야 한다. 생물학적 반응 조절물질(biological response modifier: BRM)의 자극으로 발현이 유발되거나 변화를 보이는 혈관 내피세포 표면 세포 유착분자(cell adhesion molecules: CAM)가 이 과정을 중개하고, 그에 따른 유착도의 조절은 염증양상을 결정하는데 중요한 역할을 한다. 인체 제대정맥 내피세포(human umbilical vein endothelial cells;HUVEC)로 부터 처음 분리된 E-selectin분자는 혈관 내피세포와 중성구의 유착을 중개하는 유발성 CAM으로서 최근 T림프구의 유주에도 관여하는 것으로 보고된 바 있어, 만성 염증성질환에서 E-selectin의 역할이 매우 중요할 것으로 생각된다.

본 연구에서 배양한 인체 진피 미세혈관 내피세포(human dermal microvascular endothelial cells: HDMEC)를 interleukin-1α(IL-1α), tumor necrosis factor α(TNFα), interferon γ(IFN-γ)와 lipopolysaccharide(LPS) 등의 BRM으로 처리한 후 면역형광 유량 세

포계산 분석(immunofluorescence flow cytometric analysis) 및 효소 면역표지법을 이용하여 E-selectin분자의 발현 변화를 관찰하고, BRM으로 활성화한 HDMEC에 대한 T림프구의 유착에 미치는 E-selectin 분자의 역할을 규명하고자 HDMEC에 대한 T림프구 유착실험과 항 E-selectin 단 클론 항체를 이용한 T림프구-HDMEC 유착저해실험을 시행하여 다음과 같은 결과를 얻었다.

1. 자극하지 않은 배양한 HDMEC과 HUVEC 모두에서 E-selectin분자의 세포표면 발현을 관찰할 수 없었다.

2. HDMEC과 HUVEC 모두에서 IL-1α혹은 TNFa를 첨가하고 1시간 배양한 후 E-selectin 분자의 발현을 관찰할 수 있었으며, 6시간 후 최대치에 이르렀다가 48시간 후에 사라지는 일시적 발현양상을 보였고 HDMEC에 비해 HUVEC표면에서 더 빨리 소실되었다.

3. IFN-γ를 첨가 후 24시간까지는 HDMEC에서 E-selectin분자의 발현을 관찰할 수 없었고, 48시간 후에 유의한 발현증가를 보였으며 72시간까지 E-selectin분자의 지연적 발현을 관찰할 수 있었던 반면, HUVEC에서는 IFN-γ의 자극 후 72시간까지 E-selectin분자의 발현을 관찰할 수 없었다.

4. IFN-γ로 자극한 HDMEC에 LPS를 첨가하였을때 E-selectin분자 발현에 부가효과를 보였다.

5. HDMEC에 대한 CD4**+ T림프구의 유착정도는 자극 전에 비해 HDMEC에 IL-α 또는TNFe를 첨가하고 6시간 및 48시간 배양 후에 모두 증가하였으며, CD4**+ T림프구의 유착에 미치는 IFN-γ의 증가효과는 자극 48시간 후부터 뚜렷이 관찰되었고, 자극 72시간까지 지속되었다. HDMEC에 IL-le, TNFe, 또는 IFN-γ를 처리 후 CD4**+ T림프구의 아형중 주로 CD4**+ C045RO**+ 기억 T림프구의 유착정도가 증가하였고, CD4**+ CD45RA**+ naive T림프구의 유착정도는 변화하지 않았다.

6. IL-1α또는 TNF7를 첨가 후 6시간 배양한 HDMEC과 IFN-r를 첨가 후 72시간 배양한 HDMEC에 대한 CD4**+ C045RO**+ 기억 T림프구의 유착증가율은 항 E-selectin 단클론 항체의 첨가에 의해 감소되었다.

이상의 결과는 혈관 내피세포에서 IFN-γ의한 E-selectin분자의 발현은 조직 특이성있게 조절된다는 것을 시사하며, 만성 염증성 피부질환에서 기억 T림프구가 염증부위로 유주하기 위한 HDMEC과의 유착과정에 염증시기에 따라 BRM에 의해 선별적으로 발현이 유도된 E-selectin분자가 관여할 것으로 사료된다.

The role of E-selectin on the binding of T Iymphocrtes to human dermal

microvascular endothelial cells

Young Jin Koh

Department of Medical Science, The Graduate School Yonsei University

(Directed by Professor kwang Hoon Lee)

The migration of Iymphocytes from the circulation into the tissue sites of

inflammation is an essential process in the inflammation and the adhesion of

leukocytes to endothelial cells is the first step. The binding of leukocytes to

endothelial cells is governed by the expression of cell adhesion molecules which

are regulated by biological response modifiers(BRM) and the regulation of the

binding may be an important role in determining the progression of acute and

chronic in-flammatory responses. E-selectin was initially characterized in human

umbilical endothelial cells(HUVEC) as an inducible ligand for neutrophils.

Recently, E-selectin has been identified as the endothelial cell ligand for memory

T Iymphocytes.

Cell surface expression of E-selectin on HDMEC that was unstimulated or

pretreated with IL-17, TNFα, LPS, and IFN-γ was evaluated by the

immunofluorescence flow cytometry and the enzyme-linked imrnunosorbent assay, and

compared it to the regulated E-selectin expression on HUVEC. For the evaluation of

the ability of E-selectin to mediate T Iymphocytes adhesion to HDMEC, the binding

assay of T lymphocytes to HDMEC in vito and blocking assay using monoclonal

antibody has been performed after stimulation of HDMEC with IL-1α, TNFe, or

IFN-γ.

1. E-selectin was not constitutively expressed on HUVEC or on HDMEC.

2. Minimal induction of E-selectin was seen at 1 hour of incubation after

stimulation with interleukin (IL- lα) or tumor necrosis factor α(TNFα), and

maximal expression was observed at 6 hours after stimulation on both HDMEC and

HUVEC. E-selectin expression was consistently un-detectable at 48 hours after

stimulation with IL-lα or TNFα and more rapidly disappeared on HUVEC.

3. E-selectin expression on HDMEC was induced at 48 hours after stimulation with

interferon γ(IFN-γ), and persisted until 72 hours after stimulation, but

stimulation with IFN-γ did net induce E-selectin expression on HUVEC.

4. HDMEC cotreated with IFN-γ Plus lipopolysaccharides showed at most additive

increases in E-selectin expression.

5. The binding of T Iymphocytes to HDMEC that was incubated for 6 hours after

stimulation with IL-lα or TNFα, and for 48 hours after stimulation with IFN-γ

was increased. The binding of memory T Iymphocyte to HDMEC was greater than that of

naive T Iymphocytes.

6. Anti-E-selectin antibody partially inhibited memory T Iymphocyte binding to

HDMEC that was incubated for 6 hours after stimulation with IL-lα or TNFα, and

for 72 hours after stimulation with IFN-γ.

These data show that E-selectin expression induced by IFN-γ on endothelial cells

are regulated as tissue-specific fashion. Also, these results suggest that

E-selectin which is regulated selectively by BRM according to the state of the

inflammation may be important in vivo in the preferentia migration of memory T

Iymphocytes into inflammatory sites.

[영문]

The migration of Iymphocytes from the circulation into the tissue sites of inflammation is an essential process in the inflammation and the adhesion of leukocytes to endothelial cells is the first step. The binding of leukocytes to endothelial cells is governed by the expression of cell adhesion molecules which are regulated by biological response modifiers(BRM) and the regulation of the

binding may be an important role in determining the progression of acute and chronic in-flammatory responses. E-selectin was initially characterized in human umbilical endothelial cells(HUVEC) as an inducible ligand for neutrophils.

Recently, E-selectin has been identified as the endothelial cell ligand for memory T Iymphocytes.

Cell surface expression of E-selectin on HDMEC that was unstimulated or pretreated with IL-17, TNFα, LPS, and IFN-γ was evaluated by the immunofluorescence flow cytometry and the enzyme-linked imrnunosorbent assay, and compared it to the regulated E-selectin expression on HUVEC. For the evaluation of

the ability of E-selectin to mediate T Iymphocytes adhesion to HDMEC, the binding assay of T lymphocytes to HDMEC in vito and blocking assay using monoclonal antibody has been performed after stimulation of HDMEC with IL-1α, TNFe, or IFN-γ.

1. E-selectin was not constitutively expressed on HUVEC or on HDMEC.

2. Minimal induction of E-selectin was seen at 1 hour of incubation after stimulation with interleukin (IL- lα) or tumor necrosis factor α(TNFα), and maximal expression was observed at 6 hours after stimulation on both HDMEC and HUVEC. E-selectin expression was consistently un-detectable at 48 hours after

stimulation with IL-lα or TNFα and more rapidly disappeared on HUVEC.

3. E-selectin expression on HDMEC was induced at 48 hours after stimulation with interferon γ(IFN-γ), and persisted until 72 hours after stimulation, but stimulation with IFN-γ did net induce E-selectin expression on HUVEC.

4. HDMEC cotreated with IFN-γ Plus lipopolysaccharides showed at most additive increases in E-selectin expression.

5. The binding of T Iymphocytes to HDMEC that was incubated for 6 hours after stimulation with IL-lα or TNFα, and for 48 hours after stimulation with IFN-γ was increased. The binding of memory T Iymphocyte to HDMEC was greater than that of naive T Iymphocytes.

6. Anti-E-selectin antibody partially inhibited memory T Iymphocyte binding to HDMEC that was incubated for 6 hours after stimulation with IL-lα or TNFα, and for 72 hours after stimulation with IFN-γ.

These data show that E-selectin expression induced by IFN-γ on endothelial cells are regulated as tissue-specific fashion. Also, these results suggest that E-selectin which is regulated selectively by BRM according to the state of the inflammation may be important in vivo in the preferentia migration of memory T

Iymphocytes into inflammatory sites.