Nonobese Diabetic 생쥐로 부터 분리한 췌장 베타세포특이 CD8+ T 임파구에 의한 당뇨병내성생쥐에 이식된 췌장선세포의 파괴
Destruction of Islet-Grafts in Diabetes-resistant Mice by Nonobese Diabetic Mouse-Relived Beta Cell-Cytotoxic CD8+T Lymphocytes
소속 상세정보
박병주/Byung-Ju Park
KMID : 0357119950170040359
Abstract
Nonobese diabetic(NOD)생쥐에서 인슐린의존형 당뇨병의 발생은 췌장 베타세포에 대한 T임파구 매개 자가면역반웅에 기인한다. 인슐린의존형 당뇨병에 있어서 CD8+T임파구는 베타세포를 직접 파괴할뿐만 아니라 다른 effector 세포를 췌 장선세포내로 끌어들인다고 가정되어 왔다. 그러나 베타세포특이 CD8+세포독성 T임파구(β-CTL)클론들은 당뇨병에 이환된 생쥐로부터 분리해낸 비장의
CD4+ T 임파구를 함께 주사하여야만 NOD 생쥐의 췌장선세포내로 침윤해 들어올수 있으므로 β-CTL클론들의 독립적인 병적작용은 여전히 불투명한 상태이다. 따라 서 β-CTL클론들이 in vivo에서 CD4+ T 임파구의 존재없이도 베타세포를 파괴할 수 있는지, 췌장선세포내로 naive T 세포를 끌어 들일수 있는지 여부를 확인코자 본
실험을 시행하였다. BALB/c(H-2d)나 B6(H-2b) 생쥐로부터 췌장선세포를 분리한 다음 streptozotocin 처리로 당뇨병에 걸린 (NOD x BALB/c) Fl(H-2Kd,H-2Dd,b) 또는 (NOD x B6) Fl(H-2Kd,b, H-2Db)의 생쥐의 콩팥 피막하에 이식수술하였다. 이
식하여 정상혈당을 되찾 은 생쥐를 대상으로 이식수술후 3일이내 또는 8일이후에 NOD 생
쥐로 부터 분리한 H-2Kd제한적인 β-CTL클론들을 정맥주사하였다.
H-2d췌장선세포를 이식받은 (NOD x BALB/C) Fl 생쥐에 이식후 3일째에 β-CTL클론들을 주사하면 모든 생쥐에서 이식된 췌장선세포내로 β-CIL클론들이 들어가서 많은 수의 숙주 Mac-1+ 세포들, CD4+및 CD8+T임파구들을 끌어들여서 당뇨병을 유발하였다. 이와 대조적으로 H-2b췌장선세포를 이식 받은 (NOD x BALB/c) Fl 생쥐 들에 β-CTL 클론들을 주사하여도 이식된 췌장선세포의 염 증반응이나 당뇨병은 유발되지 아니하였다.
H-2d췌장선세포를 이식받은(NOD x BALB/c) Fl생쥐들을 CD4+에 대한 단클론 항체인 GK1.5를 이식수술전후에 처치하여 CD4+T 임파구를 고갈시켜도 β-CTL.클론들에 의한 이식된 췌장선세포의 파괴는 억제되지 아니하였으나 심화정도는 감약되었다. 대조적으로 β-CTL 클론들을 이 식수술후 8일째에 주사하면 H-2d췌장선세포를 이식받은 (NOD x BALB/C)Fl생쥐들은 전혀 당뇨병에 걸리지 아니할뿐만 아니라 이식된 췌장선세포내로 T 임파구의 침윤도 관찰할 수 없었다.따라서 β-CTL클론들은 CD4+T 임파구 도움없이도 in vivo에서 베타세포를 파괴할수 있으며, 이식된 췌장선세포내로 β-CTL 클론들이 침윤하여 파괴함은 MHC class I 제한적이며, naive CD4+T 임파구를 끌어들여 베타세포 파괴를 가속화
할 것이다. 그러나 이미 혈관 재형성이 끝난 이식된 췌장선세포는 β-CTL 클론들로 부터 차단되었음을 시사하였다.
#초록#
We have previously shown that beta cell-cytotoxic CD8+T lymphocytes
are consistently present in islets of acutely diabetic NOD mice and that β-CTL clones
can transfer diabetes into irradiated NOD mice, if co-injected with non-diabetogenic
CD4+T cells from diabetic NOD mice. These studies suggested that β
-CTL clones can migrate into pancreatic islets with the assistance of
CD4+T cells and selectively destroy beta cells, leading to diabetes.
We have now transferred these β-CTL clones into diabetes-resistant mice that were
rendered diabetic by STB treatment, then treated by transplanting MHC-compatible
islets under the kidney capsule. Since in this model both the islet donors and the
ransplant recipients are insulitis- and diabetes-resistant, we have been able to explore
the independent pathogenic effects of β-CTL clones without the potentially confounding
contribution of previously activated mononuclear cells residing either in the periphery or
in the target tissue.
In initial experiments, we injected H-2Kd-rutstricted β-CTL clones into
STZ-induced diabetic (NOD x BALB/c) Fl or (NOD x B6) Fl mice within 3 days after
transplantation of BALB/c or B6 islets, respectively. At this time, the grafts are
restoring the recipient's blood glucose levels, but are still undergoing re-vascurarization.
The β-CTL clones migrated into the islet grafts within 2 days and caused diabetes in
H-2d islet-grafted (NOD x BALB/c) Fl mice, but not in
H-2b islet-grafted (NOD x B6) Fl mice. In H-2d
islet-grafted (NOD x BALB/c) Fl mice, beta cell destruction became evident within 2
days after β-CTL clone injection and was CD4+T cell-independent,
because it also occurred in CD4+T cell-depleted mice. Futhermore, (NOD x
BALB/c) Fl mice which received H-2d grafts and either a Nβ-CTL clone
or PBS alone did not develop hyperglycemia or oral glucose intolerance. Thus, β-CTL
clones have a cytopathic effect on grafted beta cells in vivo, and this effect is, like that
observed in vitro, antigen-specific and restricted by the MHC class I molecule
recognized by the β-CTL clone.
These β-CTL clones not only homed into the grafts and destroyed beta cells, but
also recruited large numbers of Mac-1+cells and host
CD4+and CD8+T cells to the site.
Although the mechanisms by which the β-CTL clones triggered the recruitment of
host mononuclear cells to the grafts are unknown, it is probable that they did so by
secreting TNF-α and INF-γ in situ, as they did in vitro when they were stimulated
with islets. It is worth noting that TNF-α and INF-γ can upregulate expression of
cell adhesion molecules, such as ICAM-1, MadCAM-1 and VCAM-1, on vascular
endothelial cells and/or islet cells in vitro and that, When expressed in beta cells of
transgenic mice, these cytokines trigger islet inflammation.
Although all CD4+T cell-depleted mice developed hyperglycemia and/or
oral glucose intolerance upon β-CTL clone injection, with kinetics similar to those
observed in non-CD4+T cell-depleted mice, the average blood glutcose
levels of the CD4+T-cell-depleted mice were decreased compared to those
of the nondepleted animals, particularly at 5-8 days after β-CTL clone injection. Local
recruitment of CD4+T cells by the β-CTL clones therefore appears to
have contributed to the progression of the β-CTL clone-induced beta cell destruction.
We entertain the following possibilities to explain this phenomenon. First, some of the
locally recruited CD4+T cells may have difierentiated into effector cells in
situ upon recognition of shed beta cell antigens processed and presented by local APCs,
leading to the secretion of beta cell-toxic cytokines and to further beta cell destruction
Second, these locally activated CD4+T cells may have promoted β-CTL
clone survival, or may have granted additional cloned β-CTLs access to islets upon
revascularization.
And third, these CD4+T cells may have fostered the differentiation of
host CD8+T cells into effector β-CTLs in situ, or triggered the
recruitment of additional effector cells, including cytotoxic macrophages, to the lesion.
This ability of β-CTL clones to home into and destroy islet of undergoing
re-vascailarization contrasts with the negative results of adoptive transfer experiments
of β-CTL clones into mice carrying fully re-vascularized grafts or into irradiated adult
NOD mice, as reported earlier, where the β-CTL clones neigher migrated into the
target, nor caused detectable beta cell damage.
Furthermore, since these results are similar to those obtained upon adoptive transfer
of unmanipulated CD8+spleen T cells from diabetic NOD mice into
irradiated recipients or nude and acid-NOD mice, and could be reproduced upon injection
of a freshly isolated pclyclonal beta cell-cytotoxic CD8+T cell line derived
from a diabetic NOD mouse, we think it is unlikely that our results are a peculiarity of
the β-CTL clones used here or a result of phenotypic/functional changes resulting from
the β-CTL clones'in vitro expansion. Forthermore, given that these β-CTL clones
expressed very low levels of the α4-integrin, which is important for the tight binding
of lymphocytes to endothelial cells and in the homing of peripheral CD8+
and/or CD4+T cells to islets in IDDM, it is not surprising that they are
unable to home into re-vascularized islet grafts or into pancreatic islets in the absence
of insulitogenic CD4+T cells.
Such apparent self-insuffciency of CD8+T cells or cloned β-CTLs to
migrate into their target tissue in the absence of local microvascular and/or
inflammatory changes (e.g., those mediated by insulitogenic CD4+T cells)
is not unique to β-CTLs or to cells derived from NOD mice. For example, renal
tubular basement membrane antigen-specific CTLs were unable to home into the kidney
interstitium when injected i.v, in the absence of Ag-specific CD4+T cells,
and in hepatitis B surface Ag (HBsAg)-transgenic mice, HBsAg-specific CTL clones
were able to migrate into the liver through the discontinuous ondothelium of the hepatic
sinusoid, but not into most other HBsAg+ tissues. Since in hamsters islet
grafts only become impermeable to fluorescent macromolecules 8-10 days after
transplantation, it is possible that the ondothelia of capillaries of grafts undergoing
revascularization form a fenestrated barrier, in a manner analogous to that observed in
the normal liver, allowing migration of β-CTL clones. Finally, it is worth noting that
the outcome of these β-CTL clone-transfer experiments contrast with those of
experiments using islet-reactive CD4+T cell clones and uncloned splenic
CD4+T cells from NOD mice, which are insulitogenic in the absence of
CD8+T cells, both in NOD mice and in animals carrying re-vascularized
grafts (even >30 days after transplantation). Thus, beta cell-specify
CD4+T cells and β-CTLs appear to have dirtferent islet-homing potential.
In conclusion, accumulation of β-CTL clones into islets is an MHC-restricted and
antigen-specific phenomenon, but requires local inflammatory changes and/or previous
disruption of the structural integrity of the islet microvasculature. In addition, once these
β-CTL clones access islets, they rapidly engage in beta cell destruction and trigger the
recruitment of naive CD4+T cells and other mononuclear cells to the
lesion, leading to further destruction of beta cells and, ultimately, to clinical diabetes.
키워드
IDDM; NOD mouse; STL; Islet-Grafts.;
원문 및 링크아웃 정보
등재저널 정보