在小鼠骨髓移植中CCR5与急性移植物抗宿主病的相关性研究

【摘要】  本研究评价供者CCR5在经过强化预处理的骨髓移植动物模型受者体内的作用，为今后的异基因造血干细胞移植的临床应用提供依据。经过致死剂量照射的BALB/c小鼠接受异基因C57BL/6小鼠的骨髓移植。根据回输的细胞不同实验分为4组：B6 CCR5 KO组，受者接受C57BL/6 CCR5-/-小鼠骨髓和脾脏细胞；B6 WT组，受者接受野生型C57BL/6小鼠骨髓和脾脏细胞；B6 CCR5 KO BMC组，受者只接受C57BL/6 CCR5-/-小鼠骨髓细胞；B6 WT BMC组，受者只接受野生型C57BL/6小鼠骨髓细胞。结果表明：较之B6 WT组，B6 CCR5 KO组小鼠以更快的速度死于急性GVHD；其受者体内的CD8+T细胞更大量的增殖；其 T细胞恢复后产生更多的INF-γ和TNF-α并且由于其T细胞有丝分裂原刀豆素水平处于较高水平，从而进一步促进T细胞的增殖，提示CCR5的作用之一是下调参与排异反应的供者CD8+T细胞的增殖。组织学评价提示，移植剔除CCR5基因受者细胞的小鼠肾脏出现了病理损伤并且肝脏存在有更为严重的病理变化。结论：剔除CCR5基因的异基因骨髓移植使GVHD发病率的增加，供者CD8+T细胞在受者体内增殖增加以及肝肾损害加重，这提示CCR5在异基因骨髓移植中起着重要作用。

【关键词】  CCR5； GVHD； 异基因造血干细胞移植； CD8+细胞

　　Graft-vs-host disease (GVHD) remains a major source of morbidity in patients after allogeneic bone marrow transplantation (BMT). Acute GVHD is a complex pathological process involving numerous cell types and target tissues. At its core, acute GVHD starts out as an immunological recognition of the donor T cells to the genetically disparate recipient. However, the process then amplifies as these T cells expand and, with the help of inflammatory cytokines  (in part produced in response to the intensive condi-tioning in BMT as well as the T cells themselves),  recruit other cells in the generation of organ damage［1,2］.This in part is the“cytokine storm” that  fuels GVHD pathogenesis［3］. Multiple organs are affected in acute GVHD including the skin, gut, lungs, and liver. While many factors can influence the generation of GVHD, it is clear that the extensive conditioning in BMT contributes to the generation and pathology of GVHD in both man and mouse［4,5］, emphasizing the importance of models that can reflect the conditioning regimens used in clinical BMT. Chemokines are a diverse group of cytokines that modulate immune cell trafficking and function［6,7］. Due to the properties of chemokines, it has been postulated that chemokines may play a role in GVHD. Serody et al［8］ demonstrated reduced lethality in recipients of donor T cells from MIP-1α-deficient mice compared to wild-type (WT) T cells in a CD8+ T cell-mediated GVHD model. Recent reports also indicated that blockade of CCR5 using neutralizing antibodies could also protect from GVHD using a parent into F1 GVHD model［9］. This report and another using CCR5 KO mice in the same model indicated that CCR5 on cells played a role in augmenting the GVHD response and that blockade of this interaction could be used to prevent GVHD［9,10］. However, the precise mechanism underlying this protection was not elucidated. Interestingly, there have also been reports that CCR5 may play a role in down regulating T cell function as evidenced by some infectious disease models［11］, suggesting that this receptor-ligand interaction may exert diverse effects. The previous reports assessing the role of CCR5 expression on GVHD used a model in which no conditioning was administered. Chemokine production as well as receptor expression and function have been shown to be markedly influenced by inflammatory cytokines induced after total-body irradiation (TBI)［12,13］. We sought to assess the role of CCR5 on donor cells in models where extensive conditioning of the recipient was applied, thus mimicking the regimens used in conventional clinical BMT. Our results demonstrate that the absence of CCR5 on donor cells results in a significant increase in GVHD morbidity that was associated with an increase in donor CD8+ T-cell expansion in the recipients.

　　Materials and Methods

　　Mice

　　C57BL/6(B6; H2b)and BALB/c(H2d)were purchased from the Animal Production Area (NCI at Frederick,Frederick,MD,USA). C57BL/6 CCR5-/-  mice (B6 CCR5 KO) were provided from two separate colonies by Dr. Jonathan Serody (Univ. North Carolina School of Medicine, Chapel Hill, NC, USA) and Dr. William Kuziel. All recipients were age-matched females and were 2 to 6 months of age at the time of BMT.

　　Cell preparation

　　Bone marrow cell (BMC) suspensions were prepared by gently releasing cells from the backbones, femurs, and tibiae into Dulbecco′s phosphate-buffered saline solution (DPBS) with a mortar and pestle, filtering through amesh filter to remove particulates, and washing the cell suspensions twice. Spleen and lymph node cell preparations were prepared by gently crushing the tissues to release the cells. Preparations were filtered to remove debris and washed twice in DPBS for injection or RPMI 1640 supplemented with 5% fetal bovine serum (FBS) for in vitro assays. Cell counts were performed on a Coulter Z1 cell counter (Coulter Electronics, Hialeah, FL, USA).

　　Induction of GVHD

　　For bone marrow transplants(BMT),recipient BALB/c mice received lethal total-body irradiation of 800 cGy by a 137Cesium source. The mice then received 107 bone marrow cells (BMC) and （7-8）×106 splenocytes and lymph node cells from donor B6 or B6 CCR5 KO mice. Mice were monitored and weighed weekly. All moribund mice were euthanized.

　　Flow cytometry

　　T cell constituency and donor origin of the splenocytes were measured using CD4, CD8, CD3, and H2Db mAbs, respectively, obtained from PharMingen (San Diego,CA,USA).Three-color flow cytometry, using fluorescein isothiocyanate (FITC), phycoerythrin  (PE), or biotin- (along with streptavidin-cychrome) conjugated mAb purchased from PharMingen, was performed. Irrelevant mAb control values were subtracted from values obtained with relevant mAbs. All results were obtained using a FACScan (Becton-Dickinson, San Diego,CA,USA).Forward- and side-scatter settings were gated to exclude red cells and debris. Ten thousand cells were analyzed for each determination. 

　　Histological evaluatian

　　Tissues from the mice were placed in 10% formalin, imbedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue sections were evaluated and graded blind by a veterinary pathologist  (MRA) as previously described［14］. A semi-quantitative scale from 0 to 4 was used where histopathological changes were identified as minimal=1, mild=2, moderate=3, severe=4. A total of 3 to 10 mice per experimental group combined from up to four independent experiments were assessed.

　　Lymphokine production

　　Unfractionated spleen cells from 7 days after BMT were plated in triplicate 96-well plates at 1 million cells per ml in RPMI 1640  (with 10% FBS, 2 mmol/L glutamine, 5×10-5mol/L 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin with or without 10 μg/ml Con A (Sigma Chemical Co.,St.Louis,MO, USA). Cells were incubated at 37℃ with 5% CO2 in a humidified incubator. On day 3 of culture, the number of viable cells per well were determined by trypan blue exclusion. Cell-free supernatants were collected and frozen at 20℃.  Frozen supernatants were thawed and assayed for cytokine production according to manufacturer′s specifications. ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA) for determination of mouse interferon-γ(IFN-γ) and tumor necrosis factor-α (TNF-α).

　　Statistical analysis

　　Survival data were plotted by the Kaplan-Meier method and analyzed by the log-rank test. Comparisons of cell populations and lymphokine production were made with Student′s t-test. Pathology scores were evaluated with the Fisher′s  exact test. A P value of equal to or less than 0.05 was considered  as significant.Results
Increased acute GVHD in recipients receiving cells from CCR5 KO mice after allogeneic BMT
The effects of CCR5 KO donor cells in a model of GVHD  was first to be assessed, where the recipients received lethal TBI followed by a full MHC-mismatched allogeneic BMT. Recipient BALB/c (H2d) mice received lethal TBI (800 cGy) followed by donor cell infusion from CCR5 KO and wild-type control mice on C57BL/6 background. Both BMC and donor T cells were transferred into the irradiated recipients in order to induce GVHD and recipients receiving BMC alone were used as controls. Flow cytometric assessment indicated that both CCR5 KO mice and their wildtype controls had similar numbers of T cells in their spleens (Table 1), indicating that comparable numbers of T cells were administered in these models. GVHD was assessed by monitoring progressive weight loss with subsequent histological assessment of key target organs (skin, gut, lungs, kidney, and liver). Surprisingly, the results demonstrated that use of CCR5 KO donor cells resulted in significant (P<0.001) increases in GVHD morbidity compared to mice receiving wild-type cells (Figure 1). These results suggest that the loss of CCR5 on donor cells results in increased GVHD after allogeneic BMT in models using extensive conditioning.Table 1.T cell subsets in C57BL/6 wild-type  and CCR5 KO mice (略)

　　As CCR5 KO mice have been reported to have increased CD8+ T-cell responses in some infectious disease models［11］, we then assessed the extent of donor cell engraftment and function in the recipients undergoing GVHD. We hypothesized that CCR5 on the donor T cells may play a role in limiting T-cell function and thus increases in donor T cell engraftment and activity may be detected in the mice, which would correlate with the increased GVHD morbidity associated with the use of CCR5 KO donors. BALB/c mice received lethal TBI followed by donor cells from either CCR5 KO or control wild-type recipients. Seven days after BMT, before any mice had succumbed to GVHD, spleens were removed from some mice and assessed for donor cell engraftment. The results demonstrate that the mice receiving cells from CCR5 KO donors had significantly greater donor CD8+ T-cell engraftment in the spleens when compared to recipients of wild-type donors(P<0.01)(Figure 2A).Donor CD4+ T-cell engraftment was not significantly different although slightly increased as well(Figure 2B).The donor CD8+T cells from the CCR5 KO donors

　　Figure 2. Increased numbers of splenic CD8+T cells in CCR5 KO cell recipients.BALB/c recipient mice received B6 wild-type or CCR5 KO bone marrow (107 cells) and (7-8) ×106 splenocytes and lymph node cells after myeloabative TBI. On day 7 post-BMT significant increases (Student′s t test;  P<0.05) in donor (H2Db) CD8+T cells and donor CD8+CD25+ cells but not donor-derived CD4+ T cells were observed. Three mice per group were analyzed for the donor-derived T cell content in spleen by flow cytometry.
 also were significantly greater in their expression of CD25 (P<0.05),  indicating that they were in an activated state. When spleen cells from these mice were then cultured for 72 hours, it was found that splenocytes from the mice receiving cells from the CCR5 KO donors produced significantly(P<0.01) greater amounts of TNF-α and  IFN-γ(Figure 3A). Splenocytes from these mice also proliferated to the T cell mitogen, concanavalin A,to a significantly(P<0.01)greater extent when compared to recipients receiving cells from recipients of wild-type donors (Figure 3B).Taken together,these results indicate that after allogeneic BMT,use of donor cells from CCR5 KO mice resulted in significantly greater donor CD8+ T-cell expansion and function as reflected by proliferation and cytokine production when compared to recipients of wild-type donors. Furthermore, this increase in CD8+ T cell expansion could be correlated with increased morbidity from acute GVHD observed in these recipients.
Figure 3. Enhanced production of TNF-α and IFN-γ in cultures of  splenocytes taken from day 7 post-BMT.  One million splenocytes from day 7 post-BMT BALB/c recipients of B6 WT or B6 CCR5 KO hematopoietic grafts were cultured in media alone or with 10  μg/ml ConA for 3 days. Viable cells were assessed on a hemacytometer and trypan blue exclusion. Supernatants were collected and analyzed by ELISA for lymphokine production (3 mice per group).

　　Increased liver pathological changes in mice undergoing acute GVHD receiving cells from CCR5 KO mice
As opposed to other models of GVHD in which no conditioning is applied, acute GVHD in models where extensive conditioning is applied affects multiple target organs: skin, gut, lungs, and liver. It is believed that the presence of prior conditioning not only predisposes these tissues to further damage by the donor cells, but the conditioning induces potent inflammatory cytokines that add to the “cytokine storm”  associated with acute GVHD［4,5］. Examination of target visceral organs for GVHD revealed diffuse severe hepatocellular vacuolation in the livers of recipients of CCR5 KO donor T cells (Table 1) when compared to mice receiving donor T cells from wild-type controls. Mice undergoing increased acute GVHD after receiving CCR5 KO donor cells also had renal lesions (Table 1), cortical tabular vacuolation, tubular cell necrosis, and cellular/proteinaceous casts. Renal damage did not occur in the recipients of WT grafts or CCR5 KO bone marrow grafts. These results would suggest that the accelerated morbidity from acute GVHD could be correlated with increases in liver and kidney lesions among the recipients receiving the CCR5 KO donor T cells. The intestinal tract is a major target for GVHD. In this model,we did not observe any consistent differ- ences in the degree of GVHD-associated pathological changes in the small or large intestine between recipients of WT or CCR5 KO donor cells (Table 2). No appreciable differences in the extent of tissue damage were detected in the skin, lung, or other organs among the recipients. Thus, increases in GVHD morbidity associated with the use of CCR5 KO donor T cells could be correlated with increases in liver and kidney lesions among the recipients.

　　Discussion

　　Similar to cytokines, the role of chemokines in GVHD are most likely complex and highly dependent on the model used to assess them. This report demonstrated that the absence of CCR5 on donor cells resulted in accelerated morbidity associated with GVHD and heavier  damage in two target organs, the liver and the kidney. The absence of CCR5 on the donor cells resulted in a net acceleration of acute GVHD with increased donor CD8+T-cell expansion. This is in agreement with a recent report by Serody et al that also demonstrated increased GVHD when T cells from CCR5 KO mice were used as donors in models using conditioning［15］. It is important to note that the word “extensive”  is relative. Some GVHD models use considerably higher amounts of conditioning in the study of GVHD pathology, particularly related to the gut and its breakdown［5］. The heavier  GVHD observed with CCR5 KO donors  seems  to be contradictory to reports demonstrating that antibodies to CCR5 or using CCR5 KO mice were protective to GVHD［9,10］. The discrepancy may be due to differences in the models and means to deprive CCR5 interactions. In the aforementioned model, large numbers of parental cells were transferred into F1 recipients and no conditioning was administered to the recipients［9,10］. This GVHD model has  shown to depend largely on the attack of the host lymphoid cells by the donor CD8+ T cells in a one-way immune reaction［16］. The role of conditioning in BMT models has been previously reported to have dramatic effects on the role of cytokines in the pathogenesis.Indeed, at times opposing roles of cytokines in GVHD have been reported depending on the presence of conditioning［17-9］. It has been demonstrated that inflammatory cytokines commonly induced after ex-A semi-quantitative scale from 0-4 was used where histopathological changes were identified as minimal =1, mild= 2, moderate = 3, severe=4. Evaluation of intestinal tract and liver is  from the animals of 4 independent experiments. *: number of mice with graded pathology.Table 2.GVHD-associated pathology in mice receiving CCR5 KO grafts（略）

　　tensive conditioning in conventional BMT models (i.e., TNF) can markedly affect chemokine receptor expression as well as chemokine function［12,13］. It is quite likely that this difference in the models could contribute to the markedly different outcomes. Indeed, recent reports demonstrate that infiltration of CCR5 KO donor cells was increased in lung and liver compared to wild-type donor cells only when extensive pretransplant conditioning was used［20］. Additionally, the survival time of models using extensive conditioning is often shorter, with greater amount of damage being generated affecting multiple organs (being predisposed to immune attack due to damage from the conditioning regimen) in a short time as  compared to models involving no conditioning. The previous model showing protection only demonstrated effects in the liver,  whereas in models involving intensive conditioning multiple organs are attacked［1,4］. Another complicating factor is the use of neutralizing antibodies that can affect both donor and host cells. The divergent roles of CCR5 on these two sources of cells may result in pleiotropic effects. Our study only assessed the effects of CCR5 on the donor cells. It is of interest, though, that the protection observed in the previous model predominantly affected the liver, whereas in our model the liver demonstrated increased histopathological changes. However, the absence of CCR5 resulted in significant protectionof the colon. Thus, protection in the absence of CCR5 can indeed occur but the end result was still increased morbidity from GVHD in BMT models using extensive conditioning. In our studies, the recipients of CCR5 KO donor cells exhibited renal lesions associated with GVHD in addition to increased findings of liver changes. Acute murine GVHD s typically associated with pathological changes in the gut, liver, skin, and mucosal tissues. The finding of renal and liver lesions in recipients of CCR5 KO cells, along with the presence of gut lesions,  are consistent with multi-organ failure. The observation of diffuse vacuolation in the liver is indicative of a systemic effect rather than direct effector cell-mediated damage to the hepatic tissue. It is possible that the histopathological changes observed in CCR5 KO recipients are a result of tissue injury by the cytokine cascade and/or metabolic alterations in fat and muscle resulting in cachexia［3］. Indeed, the appearance of renal necrosis and hepatocellular vacuolation together with histopathological lesions in the colon are consistent with the multi-organ failure from endotoxic shock. The enhanced production of TNF-α by spleen cells from mice with GVHD induced by CCR5 KO donor cells correlates with this observation. Previous studies have demonstrated that induction of TNF-α production by LPS in the early phase of acute murine GVHD leads to rapid mortality［21］. The finding that donor CCR5 KO CD8+ T-cell engraftment and activation is increased would suggest that CCR5 may play a role in suppressing T-cell activation. In agreement with our study,  there are several reports suggesting CCR5 expression can suppress immune responses in murine models［11,22-24］. In a study using a model of GVHD that also targets the lung, it was reported that accelerated idiopathic pneumonia syndrome and GVHD associated mortality occurred in recipients of MIP-1α  KO donor cells, with increased, not decreased, T-cell expansion and recruitment being observed［22］. It has been shown that CCR5 KO mice displayed the ability to overcome defects in IFN-γ production after infection with Leishmania［11］. Additionally, lack of CCR5 expression in tumor-bearing mice can result in delayed tumor growth［23］. Finally, there was a recent report demonstrating that blockade of CCR5 activation with receptor antagonists in a murine model of glomerulonephritis resulted in more tissue damage despite reduction in macrophage infiltration into the glomerulus［24］. The mechanism by which CCR5 modulates immune responses in these studies is not clear. There are several reports demonstrating that CCR5 ligands can stimulate Th1 responses［25,26］. There are differential outcomes with the various ligands which may suggest that other CCR receptors may be critical in these responses. In contrast to the pro-cellular immune responses associated with these ligands, there is also a report that pre-exposure of cells to the CCR5 ligand MIP-1α can inhibit T-cell proliferation and interleukin- 2  (IL-2) production in vitro［27］. Suppressive activity through T regulatory function by CCR5 ligands is consistent with the observation that CD4+CD25+ T cells express CCR5［28］  and the importance of Treg cells in suppressing GVHD has been demonstrated［29,30］. A recent report demonstrates that in a murine model of acute GVHD with pretransplant conditioning, CCR5 KO Treg cells have impaired migration and CCR5 KO effector cells are less susceptible to Treg cell inhibition of GVHD lethality［20］. These studies are in agreement with our data showing increased TNF-α and IFN-γ production from splenocytes of mice receiving CCR5 KO cells and increased donor T-cell engraftment being noted in the spleens. This would suggest that CCR5 may play a role in the suppression or regulation of Th1-type cytokine production and this may be a possible mechanism by which GVHD was increased.In summary, this report demonstrates that the absence of CCR5 on donor cells results in aggravated GVHD in a BMT model using conditioning and two-way immune reactions. This increase in GVHD morbidity was associated with increased donor CD8+ T cells and increased hepatic and renal lesions. Thus, targeting CCR5 may yield divergent effects, and more needs to be understood in ascertaining the pleiotropic roles of  this and other chemokines and their receptors in immune responses before exploiting  clinical applications in a disease with such complex etiologies as GVHD.

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