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Regulation of acute graft-versus-host disease by human umbilical cord blood derived stromal cells in haploidentical stem cell transplantation in mice through very late activation antigen-4
Clinical Immunology (2011) 139, 94–101
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Clinical Immunology www.elsevier.com/locate/yclim
Regulation of acute graft-versus-host disease by human umbilical cord blood derived stromal cells in haploidentical stem cell transplantation in mice through very late activation antigen-4 Cheng Zhang 1 , Xing-Hua Chen ⁎, Xi Zhang 1 , Lei Gao 1 , Li Gao 1 , Pei-Yan Kong 1 , Xian-Gui Peng 1 , Ai-Hua Sun 1 , Qing-Yu Wang 1 Department of Hematology, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, People's Republic of China Received 28 May 2010; accepted with revision 13 January 2011 Available online 20 January 2011 KEYWORDS Very late activation antigen-4; Human umbilical cord blood derived stromal cells; Haploidentical transplantation; Mice; Graft-versus-host disease; Human bone marrow stromal cells
Abstract Human umbilical cord blood derived stromal cells (hUCBDSCs), a novel resource isolated by our laboratory, have been shown to exert an immunologic regulation. Very late activation antigen-4 (VLA-4) has been associated with graft-versus-host disease (GVHD). This study aimed to investigate the possible mechanism by in vitro co-cultured splenocytes of donor mice with hUCBDSCs and in haploidentical stem cell transplantation in mice with acute GVHD. Both hUCBDSCs and human bone marrow stromal cells (hBMSCs) elicited decreased lymphocyte expression of VLA-4, but this decrease was stronger with hUCBDSCs than with hBMSCs (pb 0.05). Cotransplantation of bone marrow with hUCBDSCs significantly decreased the expression of VLA-4 compared with control mice (p b 0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in haploidentical mice cotransplanted with hUCBDSCs. Our study shows that hUCBDSCs may protect mouse recipients of haploidentical stem cell transplantation from aGVHD via downregulating the expression of VLA-4. © 2011 Elsevier Inc. All rights reserved.
Abbreviations: hUCBDSCs, Human umbilical cord blood derived stromal cells; VLA-4, Very late activation antigen-4; GVHD, Graft-versus-host disease; hBMSCs, Human bone marrow stromal cells; HSCT, Hematopoietic stem cell transplantation; MNCs, Mononuclear cells. ⁎ Corresponding author. Fax: + 86 23 6520 0687. E-mail addresses: [email protected] (C. Zhang), [email protected] (X.-H. Chen), [email protected] (X. Zhang), [email protected] (L. Gao), [email protected] (L. Gao), [email protected] (P.-Y. Kong), [email protected] (X.-G. Peng), [email protected] (A.-H. Sun), [email protected] (Q.-Y. Wang). 1 Fax: +86 23 6520 0687. 1521-6616/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2011.01.004
aGVHD regulated by hUCBDSCs through VLA-4
1. Introduction Acute graft-versus-host disease (aGVHD) is still a major hurdle for successful HLA-haploidentical/mismatched hematopoietic stem cell transplantation (HSCT), a proven therapeutic modality for the treatment of various benign and malignant hematopoietic diseases. The use of HLA-haploidentical/mismatched HSCT has been disappointing due to the high incidence of GVHD and infectious complications resulting in high transplant-related mortality, and the fact that there were only a few survivors as a result of this therapy led to the closure of many of these trials [1,2]. Although many immunosuppressive drugs are available, none of them alone or in combination is able to completely abolish aGVHD [3]. The development of new strategies aimed at preventing and/or decreasing GVHD has become critical research areas in HLA-haploidentical/mismatched HSCT. Adhesion molecules are involved in GVHD [4,5], but data on the expression of adhesion molecules in GVHD are limited. The a4β1 integrin very late activation antigen-4 (VLA-4), expressed mainly on lymphocytes and monocytes, is an a4 (CD49d)/β1(CD29) heterodimer that plays a key role in the adhesion of hematopoietic progenitor cells to bone marrow stromal cells. VLA-4 has been associated with GVHD, and anti–VLA-4 treatment could significantly delay the occurrence of aGVHD [6,7]. Cellular therapy for GVHD has attracted much attention. We have previously isolated a novel population of adherent fibroblast-like cells from human umbilical cord blood CD34+ cells, called hUCBDSCs, and confirmed that hUCBDSCs exert identical immunomodulatory effects on T cells in vitro across MHC species barriers as do MSCs [8,9]. Therefore, in this study, the effect of hUCBDSCs on VLA-4 expression was investigated by co-culturing hUCBDSCs with spleen lymphocytes of donor mice and in haploidentical HSCT mice with aGVHD. Our findings could provide indirect insights as to whether and how hUCBDSCs affect human transplant recipients.
2. Materials and methods 2.1. Cell separation of hUCB Cell separation was performed according to our previous report [10]. Briefly, the majority of RBCs in collected hUCB were depleted by a 6% gelatin sedimentation method. The leukocyte-rich fraction was washed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) to remove the gelatin and then loaded onto Percoll density-gradient fractionation columns (density= 1.077 g/l) (Pharmacia Biotech, Uppsala, Sweden). Cells were centrifuged at 400 ×g for 20 min at 4 °C. The mononuclear cells (MNCs) at the interface were washed with PBS and resuspended in PBS. Blood collection was approved by the institutional ethics committee in compliance with national guidelines regarding the use of fetal tissue for research purposes. Informed consent was obtained in all cases.
2.2. CD34+ cell purification from hUCB CD34+ cells were separated using a magnetic cell-sorting (MACS) system (Miltenyi Biotec; Bergisch Gladbach, Germany)
95 according to our previous report [8]. Briefly, 1 × 108 MNCs were mixed with anti-CD34 monoclonal antibody for 20 min at 4 °C. Then, anti-mouse immunomagnetic beads were added and incubated with the MNCs for 15 min at 8 °C. The positive fraction was isolated, and the percent purity of the positive fraction was determined using anti-CD34 fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugate-tagged antibodies (Santa Cruz, CA, USA) by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
2.3. Establishment of hUCBDSCs The hUCB CD34+ cells were cultured in a Dexter system to obtain hUCBDSCs. CD34+ cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 12.5% fetal bovine serum (Hyclone, USA), 12.5% horse serum (HS, Gibco, USA), 10− 6 M hydrocortisone, 10 ng/ml recombinant human stem cell factor (rhSCF) (Sigma, USA), 10 ng/ml recombinant human basic fibroblast growth factor (rhbFGF) (Sigma, USA), 100 U/ml penicillin and 100 ng/ml streptomycin. Fresh medium was replaced after 48 h and then demidepopulated weekly accompanied by the addition of fresh medium. After the cells reached 80% confluence, the hUCBDSCs were subcultured at a 1:2 ratio using the same culture medium and culturing conditions.
2.4. Isolation and cultivation of human bone marrow stroma cells (hBMSCs) The bone marrow samples obtained from healthy donors were diluted with RPMI1640 medium containing 2% newborn calf serum, then loaded on Percoll density-gradient fractionation columns (density= 1.077 g/l). Cells were centrifuged at 400 ×g for 15 min at 4 °C. The MNCs at the interface were washed with PBS and resuspended in PBS. Subsequently, 2–3 ml culture fluid was mixed fully with the sedimented cell clump, and the cell numbers were counted. The cells were cultured at 2–4 × 106 MNCs/ml in a 100-ml plastic culture flask with 5 ml bulk volume in RPMI1640 with 12.5% horse serum, 12.5% calf serum, 100 U/ml penicillin, 100 ng/ml streptomycin, and 10−6 mol/ml hydrocortisone (all from Gibco, USA). The medium was replaced after 48 h. Then the cells were demi-depopulated weekly with fresh medium. After the cells had reached 80% confluence, hBMSCs were subcultured at a 1:2 ratio under the same culture conditions.
2.5. In vitro co-culture The spleen cells of C57BL/6 (H-2b) (termed B6) mice were obtained by passing minced spleen through a 60-mm sterile nylon mesh. Erythrocytes were removed from spleen cell suspensions by density-gradient centrifugation on FicollPaque. The MNCs were harvested from the interface and cultured with DMEM containing 10% fetal bovine serum for 24 h, and then the non-adhesive cells were collected. Some non-adhesive cells were co-cultured with hUCBDSCs or hBMSCs for 7 days, while others were cultured alone for another 7 days as control group.
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2.6. Mice The B6 and F1 (H-2b/d) (C57BL/6 × BALB/c) male mice were purchased from the animal center of the Third Military Medical University, China. Mice were used at 4 to 12 weeks of age and housed in a specific pathogen–free facility in microisolator cages. Experiments adhered to the principles and guidelines for scientific experiments on animals of the Third Military Medical University animal committee.
2.7. Haploidentical bone marrow transplantation and induction of aGVHD Four- to 8-week-old inbred male B6 mice were used as donors. Eight- to 12-week-old male F1 mice were used as the bone marrow transplantation (BMT) recipients. They were given on water containing 250 mg/l of erythromycin and 320 mg/l of gentamicin to prevent infection with Grampositive and Gram-negative bacteria, as well as an ad libitum diet after total body irradiation (TBI). The F1 mice were housed in ventilated cage racks after BMT. The mouse BMT model was established according to previous reports by Li et al. [3]. Recipient F1 mice were anesthetized and subsequently irradiated with 6 MV X-rays from a medical linear accelerator. The total dosage given was 7.0 Gy at a dose rate of 0.5 Gy/min, the day before transplantation. On the day of transplantation, donor mice were killed by cervical dislocation. Their femurs and tibias were removed aseptically and kept on ice in DMEM supplemented with 0.5% fetal calf serum and 1% penicillin–streptomycin. Bones were cut open at both ends, and bone marrow (BM) cells were subsequently flushed out from the shaft by forcing cold DMEM through a 21gauge needle inserted at the proximal end of the bones. Large fragments were removed by filtering the cell suspension through a 60-mm sterile nylon mesh. BM cells were washed twice with PBS and resuspended in DMEM. Donor spleen cells were obtained by passing minced spleen through a 60-mm sterile nylon mesh. Erythrocytes were removed from spleen cell suspensions by density-gradient centrifugation on FicollPaque. The MNCs were harvested from the interface. The BM and spleen cells were counted, and viability was determined by Trypan blue dye exclusion. To induce aGVHD after BMT, 300 ml of medium containing 1 × 107 BM cells and 1 × 107 splenocytes (as the main source of T cells) from B6 donors were injected into each of the irradiated F1 recipients through the tail vein. hUCBDSCs (1 × 106) were injected into the F1 mice of the experimental group. Mice that died within 1 week post-BMT were considered to have succumbed to radiation poisoning and were excluded from the data analysis. In total, 13 recipient mice in the control group and 16 recipient mice in the experimental group were evaluated. Acute GVHD was evident by rapid and sustained weight loss after recovery from irradiation, as well as from features such as hunchback, diarrhea, hair loss and skin thickening.
C. Zhang et al. USA) as isotype control for 20 min and then washed with PBS three times. The stained cells were resuspended in PBS. Ten thousand cells were counted for each preparation on a Coulter flow cytometer (Epics Esp, Coulter Corp., Hialeah, FL, USA), and data analysis was performed with FlowJo software (Treestar, San Carlos, CA, USA). The cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, USA).The co-cultured cells in control group and hUCBDSCs group were incubated on ice with FITC-VLA-4PE-CD4 (PE-CD4 from eBioscience, USA) and FITC-VLA-4PE-CD8 (PE-CD8 from eBioscience, USA) as double labeling and FITC-IgGPE-IgG as isotype control, the following steps were performed as the aforementioned details. Percentages of cells in each group were estimated. Spleen MNCs were obtained from mice 0, 7, 14 and 21 days post-transplantation, and then the level of VLA-4 was analyzed by flow cytometry according to the aforementioned methods.
2.9. Analysis of chimerism Four weeks after transplantation, peripheral WBCs were analyzed to determine the number of recipient- or donortype cells by flow cytometry. Briefly, peripheral blood was incubated with PE-labeled anti-mouse H-2d and FITCconjugated anti-2b mAb, followed by hemolysis using BD PharM Lyse (BD Biosciences Pharmingen, San Jose, CA). The stained cells were analyzed using FACScan (Becton Dickinson, Mountain View, CA). The percent donor chimerism is defined as donor/(donor + host) × 100%.
2.10. Fluorescence immunocytochemistry Immunocytochemical labeling was performed. Briefly, cells cocultured with hUCBDSCs or hBMSCs or the control group were slided and incubated with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience, USA) for 20 min in a humid chamber at room temperature. The slides were washed with PBS and photographed immediately. Sets of slides were processed in duplicate. The extent of positive staining was calculated from the average optical density using Image pro-plus 5.0 software.
2.11. Fluorescence immunohistochemistry Immunohistochemical labeling was performed as previously described [11]. Briefly, Cryostat sections of the liver, intestine and skin obtained from the F1 recipients posttransplantation were incubated with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience, USA) for 20 min in a humid chamber at room temperature. The slides were washed with PBS and photographed immediately. Sets of slides were processed in duplicate. The extent of positive staining was calculated from the average optical density using Image pro-plus 5.0 software.
2.12. Statistical analysis 2.8. Flow cytometry The lymphocytes in co-culture were collected and washed with PBS three times. The cells were incubated on ice with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience,
Data are shown as mean ± standard deviation. Comparisons of two means were determined by t-test, and multiple comparisons of means were determined by the Tukey honestly significant difference test and Tamhane T2 of
aGVHD regulated by hUCBDSCs through VLA-4 post-hoc comparisons. The statistical significance level was set at P-values lower than 0.05. SPSS, version 13.0 (SPSS Inc., Chicago, IL, USA) was used to process the data.
3. Results 3.1. Expression of VLA-4 in lymphocytes in co-culture by flow cytometry To investigate the regulation of lymphocyte VLA-4 by hUCBDSCs in co-culture, the expression of VLA-4 was
97 detected by flow cytometry. Co-culture with either hUCBDSCs or hBMSCs decreased the expression of VLA-4 on the spleen lymphocytes. There was no statistical significance between VLA-4 expression in the hBMSC and control groups (p N 0.05). However, there was a significant difference between VLA-4 in the hUCBDSC and control groups (p b 0.05). The expression of VLA-4 was lower in the presence of hUCBDSCs compared with hBMSCs (p b 0.05) (Fig. 1). We further observed the expression of VLA-4 on CD4 and CD8 T cell subtype after co-cultured with hUCBDSCs , the data showed that the level of VLA-4 decreased on both T cell subtype (p b 0.05), however, the decrease was greater on
Figure 1 Expression of VLA-4 in splenic lymphocytes of donor mice in co-culture by flow cytometry analysis. Both hUCBDSCs and hBMSCs decreased the expression of VLA-4. hUCBDSCs decreased the expression of VLA-4 significantly more than control conditions (p b 0.05). The expression of VLA-4 was lower in the hUCBDSC group than in the hBMSCs group (p b 0.05). The expression of VLA-4 on CD4 and CD8 T cell subtype decreased on both T cell subtype (p b 0.05), however, the decrease was greater on CD8 T cell subtype than that on CD4 T cell subtype. (A) Isotype control, (B) control group, (C) hBMSC group, (D) hUCBDSC group, (E) the expression of VLA-4 in splenic lymphocytes of donor mice in co-culture, (F) the expression of VLA-4 on CD4 and CD8 T cell subtype of donor mice in co-culture.
98 CD8 T cell subtype than that on CD4 T cell subtype (35% versus 28%) (Fig. 1 F).
3.2. Expression of VLA-4 in lymphocytes in co-culture by fluorescence immunocytochemistry To further observe the effect of hUCBDSCs on VLA-4, VLA-4 was detected by fluorescence immunocytochemistry, and the AOD was calculated. The AOD was decreased in both groups, but the decrease was greater in the hUCBDSC group than in the hBMSC group (p b 0.05) (Fig. 2).
3.3. Expression of VLA-4 in spleen cells post-transplantation Our in vitro experiments showed that hUCBDSCs decreased the expression of VLA-4 on spleen lymphocytes, so we further investigated the effect of hUCBDSCs on VLA-4 in haploidentical transplantation mice by flow cytometry. VLA-4 decreased in spleen MNCs of mouse recipients post-transplantation and reached the lowest level on day 14, then increased in both hUCBDSC and control transplant groups. However, the degree of decrease was much sharper in the BMT + hUCBDSC group than in the BMT-only group, and the subsequent rate of increase was slower in the former (p b 0.05) (Fig. 3).
3.4. Expression of VLA-4 in target organs of GVHD post-transplantation Immunohistochemical VLA-4 labeling increased in the target organs of GVHD (skin, liver and intestine) after irradiation, but VLA-4 decreased post-transplantation in both BMT + hUCBDSC and BMT groups. A significant reduction of VLA-4 in the target organs of GVHD occurred in the BMT + hUCBDSC group compared with the BMT group (p b 0.05) (Fig. 4).
3.5. GVHD observation The body weight of all the recipient mice decreased by about 10–40% in the first week after TBI. In the BMT + hUCBDSC group, the body weight of the mice began to increase
Figure 2 Expression of VLA-4 in splenic lymphocytes of donor mice in co-culture by fluorescence immunocytochemistry. The AOD was decreased in the presence of both hUCBDSCs and hBMSCs, but the hUCBDSCs decreased VLA-4 more than the hBMSCs did (pb 0.05).
C. Zhang et al. approximately 2 weeks post-transplantation and continued to increase gradually. In the BMT group, the changes in body weight followed the same general pattern. However, the body weight of mice in the BMT + hUCBDSC group decreased significantly more slowly and increased faster than in the BMT group (P b 0.05).
3.6. Donor chimerism At 28 days post-transplantation, the proportion of chimerism was 75.95 ± 4.20% in the BMT + hUCBDSC group, which was higher than that of the BMT group (62.49 ± 5.48%) (P b 0.05).
4. Discussion Acute GVHD, a complex pathological process involving numerous cell types and target tissues, remains a major hurdle for successful BMT, a proven therapeutic modality for the treatment of various benign and malignant hematopoietic diseases. Although many immunosuppressive drugs are available, none of them alone or in combination is able to completely abolish aGVHD. Most current immunosuppressive protocols in organ/cell transplantation rely on calcineurin inhibitors, such as cyclosporine or FK506, which are nephrotoxic and diabetogenic and which may promote the establishment and growth of virally induced tumors. Furthermore, patients need to take these drugs continuously post-BMT to prevent the occurrence or reoccurrence of aGVHD. This extended immunosuppression profoundly reduces the quality of life of BMT recipients. Therefore, new therapeutic approaches are needed to overcome these side-effects. Adhesion molecules have been shown to play roles in effector cell migration in GVHD after experimental and clinical allo-HSCT. The integrins are a family of heterodimeric transmembrane proteins functioning in cell–cell and cell-extracellular matrix (ECM) adhesion. There are currently 18 known α subunits and β subunits in mammals [12]. The integrins relevant to the immune system include those in the β1, β2, and β7 families.VLA-4 belongs to the β1 family and LFA-1 belongs to the β2 family. Adhesion molecule–integrin interactions are critical in target organ inflammation in multiple specific transplantation settings. For example, targeting receptors important in migration after reducedintensity allo-BMT may exacerbate GVHD after a fully myeloablative transplantation. Thus, the tools with which to prevent target organ inflammation through inhibition of adhesion molecules may already be in place, but they currently await a more thorough understanding of the function of this complex system during GVHD [13]. Both ICAM-1 and VCAM-1 belong to the receptors of β2integrins, which are members of the immunoglobulin superfamily. ICAM-1 is expressed on the cell surface of cytokine-stimulated cells (e.g., leukocytes, endothelial cells, melanocytes, and dermal fibroblasts) and certain types of carcinomas. It binds to LFA-1 and Mac-1 on T cells, neutrophils, and macropages and provides a mechanism for selective recruitment of leukeocytes in different pathologic situations. Many cytokines, such as INF-γ, IL-1 and TNF-α, induce expression of ICAM-1 on the surface of endothelial cells. VCAM-1 is expressed on activated endothelial cells and
aGVHD regulated by hUCBDSCs through VLA-4 contains six or seven Ig domains of the H-type. VCAM-1 regulates adhension of monocytes, lymphocytes, eosinophils, and basophils to activated endothelial cells; the activation is induced by lipopolysaccharide, TNF, or IL-1. VCAM-1 binds to VLA-4 and has a role in the localization of B and T cells in the humoral immune response, which possibly providing a mechanism for selective recruitment of granulocytes in different pathologic conditions, such as rheumatoid arthritis, transplant-rejection, and allergy [14]. The expression of ICAM-1 and VCAM-1 plays an important role in GVHD. It has become a fundamental basis to understand the mechanism of LFA-1 and VLA-4 binding to their ligands in the developing therapeutic agents for immune diseases [14].
99 Data on expression of adhesion molecules in human GVHD are limited. In murine models of GVHD, however, enhanced expression of ICAM-1 on biliary and portal vein endothelial cells and sinusoidal cells is commonly observed [4]. Also, the number of T cells expressing the VCAM-1 ligand, VLA-4, is increased [15]. The expression of VLA-4 is significantly increased after allogenic transplantation, and all mice develop chronic GVHD. VLA-4 on donor CD4+ and CD8+ T cells is strongly up-regulated in GVHD mice [16], and antiLFA-1 mAb and anti-VLA-4 mAb have been shown to reduce the severity and incidence of murine GVHD [17,18]. In an aGVHD model using SCID mice as recipients, incubating donor spleen cells with mAb directed at CD49d and CD62L
Figure 3 Expression of VLA-4 in splenic MNCs of recipients at different times post-transplantation by flow cytometry analysis. VLA-4 decreased post-transplantation and reached its nadir on day 14, then increased in both groups. However, the degree of decrease was sharper in the BMT + hUCBDSC group than in the BMT group, and the rate of increase was slower in the former (p b 0.05). (A) Isotype control, (B) expression of VLA-4 in normal mice, (C) expression of VLA-4 on day 7 in the BMT group, (D) expression of VLA-4 on day 7 in the BMT + hUCBDSC group, (E) expression of VLA-4 on day 14 in the BMT group, (F) expression of VLA-4 on day 14 in the BMT + hUCBDSC group, (G) expression of VLA-4 on day 21 in the BMT group, (H) expression of VLA-4 on day 21 in the BMT + hUCBDSC group.
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C. Zhang et al.
Figure 3 (continued).
significantly delays the occurrence of aGVHD. We have found that hUCBDSCs play a role in immune regulation [9]. Here, we detected the effect of hUCBDSCs on VLA-4 in mice haploidentical transplantation. We found that in vitro hUCBDSCs significantly downregulated the expression of VLA-4 on the spleen lymphocytes of donor mice. It has been reported that Anti-VLA-4 antibodies inhibited CD8+ mediated primed CTL response, a critical parameter in
Figure 4 Expression of VLA-4 in the target organs of GVHD (skin, liver and intestine) on day 21 post-transplantation. Immunohistochemical VLA-4 labeling increased in skin, liver and intestine after irradiation, but VLA-4 decreased posttransplantation in both BMT + hUCBDSC and BMT groups. The post-transplantation VLA-4 level was lower in all three tissues in the BMT + hUCBDSC than in the BMT group (p b 0.05).
GVHD. We further detected that the level of VLA-4 decreased on both CD4 and CD8 T cell subtype and the decrease was greater on CD8 T cell subtype than that on CD4 T cell subtype. Also, we observed that hUCBDSCs more strongly downregulated lymphocyte VLA-4 than did hBMSCs, which shows that umbilical cord blood, which is easily obtained and contains many hematopoietic progenitor cells, is a potential source of stromal cells that regulate GVHD. As anticipated, treatment of mice during GVHD induction with anti-ICAM-1 mAb was ineffective. Others have reported successful inhibition of hepatic lesions using anti-ICAM-1 mAb, but not anti–LFA-1 mAb. Inhibition is optimal using a combination of the two mAbs. Blocking the interaction between VCAM and VLA-4 with anti-VLA-4 antibody leads to a significant reduction in the incidence and severity of hepatic lesions. We found that in vivo, hUCBDSCs significantly downregulated the expression of VLA-4 in spleen and target organs of haploidentical transplantation mice with aGVHD. Further, the lesions of GVHD organs were much less severe in the BMT + hUCBDSC group than in the BMT group (data not shown), suggesting that hUCBDSCs alleviate GVHD in part through the downregulation of VLA-4. However, There was no blocking of VCAM-1 in this study, this is only a speculative conclusion, further study should be done with the anti-VCAM1 to understand this phenomenon. It has been reported that antibodies against VLA-4 have been shown to mobilize HSPCs to the peripheral circulation [19,20]. In this study, hUCBDSCs significantly decreased the expression of VLA-4, which may be correlated with increased proportion of chimerism for BMT + hUCBDSCs transplantation. Altogether, our study shows that hUCBDSCs may protect haploidentical transplantation mice from aGVHD through
aGVHD regulated by hUCBDSCs through VLA-4 downregulating the expression of VLA-4, which could provide indirect insight as to whether and how hUCBDSCs protect human transplant recipients from GVHD.
Acknowledgments This work was funded by grants from the National Natural Science Foundation (No. 30971109) and Natural Science Foundation Project of CQ “CSTC” (CSTC, 2009BA5011), and by the Innovation Foundation for Young Scientists of the Third Military Medical University (No. 2009D226).
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Regulation of acute graft-versus-host disease by human umbilical cord blood derived stromal cells in haploidentical stem cell transplantation in mice through very late activation antigen-4 Cheng Zhang 1 , Xing-Hua Chen ⁎, Xi Zhang 1 , Lei Gao 1 , Li Gao 1 , Pei-Yan Kong 1 , Xian-Gui Peng 1 , Ai-Hua Sun 1 , Qing-Yu Wang 1 Department of Hematology, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, People's Republic of China Received 28 May 2010; accepted with revision 13 January 2011 Available online 20 January 2011 KEYWORDS Very late activation antigen-4; Human umbilical cord blood derived stromal cells; Haploidentical transplantation; Mice; Graft-versus-host disease; Human bone marrow stromal cells
Abstract Human umbilical cord blood derived stromal cells (hUCBDSCs), a novel resource isolated by our laboratory, have been shown to exert an immunologic regulation. Very late activation antigen-4 (VLA-4) has been associated with graft-versus-host disease (GVHD). This study aimed to investigate the possible mechanism by in vitro co-cultured splenocytes of donor mice with hUCBDSCs and in haploidentical stem cell transplantation in mice with acute GVHD. Both hUCBDSCs and human bone marrow stromal cells (hBMSCs) elicited decreased lymphocyte expression of VLA-4, but this decrease was stronger with hUCBDSCs than with hBMSCs (pb 0.05). Cotransplantation of bone marrow with hUCBDSCs significantly decreased the expression of VLA-4 compared with control mice (p b 0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in haploidentical mice cotransplanted with hUCBDSCs. Our study shows that hUCBDSCs may protect mouse recipients of haploidentical stem cell transplantation from aGVHD via downregulating the expression of VLA-4. © 2011 Elsevier Inc. All rights reserved.
Abbreviations: hUCBDSCs, Human umbilical cord blood derived stromal cells; VLA-4, Very late activation antigen-4; GVHD, Graft-versus-host disease; hBMSCs, Human bone marrow stromal cells; HSCT, Hematopoietic stem cell transplantation; MNCs, Mononuclear cells. ⁎ Corresponding author. Fax: + 86 23 6520 0687. E-mail addresses: [email protected] (C. Zhang), [email protected] (X.-H. Chen), [email protected] (X. Zhang), [email protected] (L. Gao), [email protected] (L. Gao), [email protected] (P.-Y. Kong), [email protected] (X.-G. Peng), [email protected] (A.-H. Sun), [email protected] (Q.-Y. Wang). 1 Fax: +86 23 6520 0687. 1521-6616/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2011.01.004
aGVHD regulated by hUCBDSCs through VLA-4
1. Introduction Acute graft-versus-host disease (aGVHD) is still a major hurdle for successful HLA-haploidentical/mismatched hematopoietic stem cell transplantation (HSCT), a proven therapeutic modality for the treatment of various benign and malignant hematopoietic diseases. The use of HLA-haploidentical/mismatched HSCT has been disappointing due to the high incidence of GVHD and infectious complications resulting in high transplant-related mortality, and the fact that there were only a few survivors as a result of this therapy led to the closure of many of these trials [1,2]. Although many immunosuppressive drugs are available, none of them alone or in combination is able to completely abolish aGVHD [3]. The development of new strategies aimed at preventing and/or decreasing GVHD has become critical research areas in HLA-haploidentical/mismatched HSCT. Adhesion molecules are involved in GVHD [4,5], but data on the expression of adhesion molecules in GVHD are limited. The a4β1 integrin very late activation antigen-4 (VLA-4), expressed mainly on lymphocytes and monocytes, is an a4 (CD49d)/β1(CD29) heterodimer that plays a key role in the adhesion of hematopoietic progenitor cells to bone marrow stromal cells. VLA-4 has been associated with GVHD, and anti–VLA-4 treatment could significantly delay the occurrence of aGVHD [6,7]. Cellular therapy for GVHD has attracted much attention. We have previously isolated a novel population of adherent fibroblast-like cells from human umbilical cord blood CD34+ cells, called hUCBDSCs, and confirmed that hUCBDSCs exert identical immunomodulatory effects on T cells in vitro across MHC species barriers as do MSCs [8,9]. Therefore, in this study, the effect of hUCBDSCs on VLA-4 expression was investigated by co-culturing hUCBDSCs with spleen lymphocytes of donor mice and in haploidentical HSCT mice with aGVHD. Our findings could provide indirect insights as to whether and how hUCBDSCs affect human transplant recipients.
2. Materials and methods 2.1. Cell separation of hUCB Cell separation was performed according to our previous report [10]. Briefly, the majority of RBCs in collected hUCB were depleted by a 6% gelatin sedimentation method. The leukocyte-rich fraction was washed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) to remove the gelatin and then loaded onto Percoll density-gradient fractionation columns (density= 1.077 g/l) (Pharmacia Biotech, Uppsala, Sweden). Cells were centrifuged at 400 ×g for 20 min at 4 °C. The mononuclear cells (MNCs) at the interface were washed with PBS and resuspended in PBS. Blood collection was approved by the institutional ethics committee in compliance with national guidelines regarding the use of fetal tissue for research purposes. Informed consent was obtained in all cases.
2.2. CD34+ cell purification from hUCB CD34+ cells were separated using a magnetic cell-sorting (MACS) system (Miltenyi Biotec; Bergisch Gladbach, Germany)
95 according to our previous report [8]. Briefly, 1 × 108 MNCs were mixed with anti-CD34 monoclonal antibody for 20 min at 4 °C. Then, anti-mouse immunomagnetic beads were added and incubated with the MNCs for 15 min at 8 °C. The positive fraction was isolated, and the percent purity of the positive fraction was determined using anti-CD34 fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugate-tagged antibodies (Santa Cruz, CA, USA) by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
2.3. Establishment of hUCBDSCs The hUCB CD34+ cells were cultured in a Dexter system to obtain hUCBDSCs. CD34+ cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 12.5% fetal bovine serum (Hyclone, USA), 12.5% horse serum (HS, Gibco, USA), 10− 6 M hydrocortisone, 10 ng/ml recombinant human stem cell factor (rhSCF) (Sigma, USA), 10 ng/ml recombinant human basic fibroblast growth factor (rhbFGF) (Sigma, USA), 100 U/ml penicillin and 100 ng/ml streptomycin. Fresh medium was replaced after 48 h and then demidepopulated weekly accompanied by the addition of fresh medium. After the cells reached 80% confluence, the hUCBDSCs were subcultured at a 1:2 ratio using the same culture medium and culturing conditions.
2.4. Isolation and cultivation of human bone marrow stroma cells (hBMSCs) The bone marrow samples obtained from healthy donors were diluted with RPMI1640 medium containing 2% newborn calf serum, then loaded on Percoll density-gradient fractionation columns (density= 1.077 g/l). Cells were centrifuged at 400 ×g for 15 min at 4 °C. The MNCs at the interface were washed with PBS and resuspended in PBS. Subsequently, 2–3 ml culture fluid was mixed fully with the sedimented cell clump, and the cell numbers were counted. The cells were cultured at 2–4 × 106 MNCs/ml in a 100-ml plastic culture flask with 5 ml bulk volume in RPMI1640 with 12.5% horse serum, 12.5% calf serum, 100 U/ml penicillin, 100 ng/ml streptomycin, and 10−6 mol/ml hydrocortisone (all from Gibco, USA). The medium was replaced after 48 h. Then the cells were demi-depopulated weekly with fresh medium. After the cells had reached 80% confluence, hBMSCs were subcultured at a 1:2 ratio under the same culture conditions.
2.5. In vitro co-culture The spleen cells of C57BL/6 (H-2b) (termed B6) mice were obtained by passing minced spleen through a 60-mm sterile nylon mesh. Erythrocytes were removed from spleen cell suspensions by density-gradient centrifugation on FicollPaque. The MNCs were harvested from the interface and cultured with DMEM containing 10% fetal bovine serum for 24 h, and then the non-adhesive cells were collected. Some non-adhesive cells were co-cultured with hUCBDSCs or hBMSCs for 7 days, while others were cultured alone for another 7 days as control group.
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2.6. Mice The B6 and F1 (H-2b/d) (C57BL/6 × BALB/c) male mice were purchased from the animal center of the Third Military Medical University, China. Mice were used at 4 to 12 weeks of age and housed in a specific pathogen–free facility in microisolator cages. Experiments adhered to the principles and guidelines for scientific experiments on animals of the Third Military Medical University animal committee.
2.7. Haploidentical bone marrow transplantation and induction of aGVHD Four- to 8-week-old inbred male B6 mice were used as donors. Eight- to 12-week-old male F1 mice were used as the bone marrow transplantation (BMT) recipients. They were given on water containing 250 mg/l of erythromycin and 320 mg/l of gentamicin to prevent infection with Grampositive and Gram-negative bacteria, as well as an ad libitum diet after total body irradiation (TBI). The F1 mice were housed in ventilated cage racks after BMT. The mouse BMT model was established according to previous reports by Li et al. [3]. Recipient F1 mice were anesthetized and subsequently irradiated with 6 MV X-rays from a medical linear accelerator. The total dosage given was 7.0 Gy at a dose rate of 0.5 Gy/min, the day before transplantation. On the day of transplantation, donor mice were killed by cervical dislocation. Their femurs and tibias were removed aseptically and kept on ice in DMEM supplemented with 0.5% fetal calf serum and 1% penicillin–streptomycin. Bones were cut open at both ends, and bone marrow (BM) cells were subsequently flushed out from the shaft by forcing cold DMEM through a 21gauge needle inserted at the proximal end of the bones. Large fragments were removed by filtering the cell suspension through a 60-mm sterile nylon mesh. BM cells were washed twice with PBS and resuspended in DMEM. Donor spleen cells were obtained by passing minced spleen through a 60-mm sterile nylon mesh. Erythrocytes were removed from spleen cell suspensions by density-gradient centrifugation on FicollPaque. The MNCs were harvested from the interface. The BM and spleen cells were counted, and viability was determined by Trypan blue dye exclusion. To induce aGVHD after BMT, 300 ml of medium containing 1 × 107 BM cells and 1 × 107 splenocytes (as the main source of T cells) from B6 donors were injected into each of the irradiated F1 recipients through the tail vein. hUCBDSCs (1 × 106) were injected into the F1 mice of the experimental group. Mice that died within 1 week post-BMT were considered to have succumbed to radiation poisoning and were excluded from the data analysis. In total, 13 recipient mice in the control group and 16 recipient mice in the experimental group were evaluated. Acute GVHD was evident by rapid and sustained weight loss after recovery from irradiation, as well as from features such as hunchback, diarrhea, hair loss and skin thickening.
C. Zhang et al. USA) as isotype control for 20 min and then washed with PBS three times. The stained cells were resuspended in PBS. Ten thousand cells were counted for each preparation on a Coulter flow cytometer (Epics Esp, Coulter Corp., Hialeah, FL, USA), and data analysis was performed with FlowJo software (Treestar, San Carlos, CA, USA). The cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, USA).The co-cultured cells in control group and hUCBDSCs group were incubated on ice with FITC-VLA-4PE-CD4 (PE-CD4 from eBioscience, USA) and FITC-VLA-4PE-CD8 (PE-CD8 from eBioscience, USA) as double labeling and FITC-IgGPE-IgG as isotype control, the following steps were performed as the aforementioned details. Percentages of cells in each group were estimated. Spleen MNCs were obtained from mice 0, 7, 14 and 21 days post-transplantation, and then the level of VLA-4 was analyzed by flow cytometry according to the aforementioned methods.
2.9. Analysis of chimerism Four weeks after transplantation, peripheral WBCs were analyzed to determine the number of recipient- or donortype cells by flow cytometry. Briefly, peripheral blood was incubated with PE-labeled anti-mouse H-2d and FITCconjugated anti-2b mAb, followed by hemolysis using BD PharM Lyse (BD Biosciences Pharmingen, San Jose, CA). The stained cells were analyzed using FACScan (Becton Dickinson, Mountain View, CA). The percent donor chimerism is defined as donor/(donor + host) × 100%.
2.10. Fluorescence immunocytochemistry Immunocytochemical labeling was performed. Briefly, cells cocultured with hUCBDSCs or hBMSCs or the control group were slided and incubated with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience, USA) for 20 min in a humid chamber at room temperature. The slides were washed with PBS and photographed immediately. Sets of slides were processed in duplicate. The extent of positive staining was calculated from the average optical density using Image pro-plus 5.0 software.
2.11. Fluorescence immunohistochemistry Immunohistochemical labeling was performed as previously described [11]. Briefly, Cryostat sections of the liver, intestine and skin obtained from the F1 recipients posttransplantation were incubated with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience, USA) for 20 min in a humid chamber at room temperature. The slides were washed with PBS and photographed immediately. Sets of slides were processed in duplicate. The extent of positive staining was calculated from the average optical density using Image pro-plus 5.0 software.
2.12. Statistical analysis 2.8. Flow cytometry The lymphocytes in co-culture were collected and washed with PBS three times. The cells were incubated on ice with FITC-VLA-4 (Southern Biotec USA) and FITC-IgG (eBioscience,
Data are shown as mean ± standard deviation. Comparisons of two means were determined by t-test, and multiple comparisons of means were determined by the Tukey honestly significant difference test and Tamhane T2 of
aGVHD regulated by hUCBDSCs through VLA-4 post-hoc comparisons. The statistical significance level was set at P-values lower than 0.05. SPSS, version 13.0 (SPSS Inc., Chicago, IL, USA) was used to process the data.
3. Results 3.1. Expression of VLA-4 in lymphocytes in co-culture by flow cytometry To investigate the regulation of lymphocyte VLA-4 by hUCBDSCs in co-culture, the expression of VLA-4 was
97 detected by flow cytometry. Co-culture with either hUCBDSCs or hBMSCs decreased the expression of VLA-4 on the spleen lymphocytes. There was no statistical significance between VLA-4 expression in the hBMSC and control groups (p N 0.05). However, there was a significant difference between VLA-4 in the hUCBDSC and control groups (p b 0.05). The expression of VLA-4 was lower in the presence of hUCBDSCs compared with hBMSCs (p b 0.05) (Fig. 1). We further observed the expression of VLA-4 on CD4 and CD8 T cell subtype after co-cultured with hUCBDSCs , the data showed that the level of VLA-4 decreased on both T cell subtype (p b 0.05), however, the decrease was greater on
Figure 1 Expression of VLA-4 in splenic lymphocytes of donor mice in co-culture by flow cytometry analysis. Both hUCBDSCs and hBMSCs decreased the expression of VLA-4. hUCBDSCs decreased the expression of VLA-4 significantly more than control conditions (p b 0.05). The expression of VLA-4 was lower in the hUCBDSC group than in the hBMSCs group (p b 0.05). The expression of VLA-4 on CD4 and CD8 T cell subtype decreased on both T cell subtype (p b 0.05), however, the decrease was greater on CD8 T cell subtype than that on CD4 T cell subtype. (A) Isotype control, (B) control group, (C) hBMSC group, (D) hUCBDSC group, (E) the expression of VLA-4 in splenic lymphocytes of donor mice in co-culture, (F) the expression of VLA-4 on CD4 and CD8 T cell subtype of donor mice in co-culture.
98 CD8 T cell subtype than that on CD4 T cell subtype (35% versus 28%) (Fig. 1 F).
3.2. Expression of VLA-4 in lymphocytes in co-culture by fluorescence immunocytochemistry To further observe the effect of hUCBDSCs on VLA-4, VLA-4 was detected by fluorescence immunocytochemistry, and the AOD was calculated. The AOD was decreased in both groups, but the decrease was greater in the hUCBDSC group than in the hBMSC group (p b 0.05) (Fig. 2).
3.3. Expression of VLA-4 in spleen cells post-transplantation Our in vitro experiments showed that hUCBDSCs decreased the expression of VLA-4 on spleen lymphocytes, so we further investigated the effect of hUCBDSCs on VLA-4 in haploidentical transplantation mice by flow cytometry. VLA-4 decreased in spleen MNCs of mouse recipients post-transplantation and reached the lowest level on day 14, then increased in both hUCBDSC and control transplant groups. However, the degree of decrease was much sharper in the BMT + hUCBDSC group than in the BMT-only group, and the subsequent rate of increase was slower in the former (p b 0.05) (Fig. 3).
3.4. Expression of VLA-4 in target organs of GVHD post-transplantation Immunohistochemical VLA-4 labeling increased in the target organs of GVHD (skin, liver and intestine) after irradiation, but VLA-4 decreased post-transplantation in both BMT + hUCBDSC and BMT groups. A significant reduction of VLA-4 in the target organs of GVHD occurred in the BMT + hUCBDSC group compared with the BMT group (p b 0.05) (Fig. 4).
3.5. GVHD observation The body weight of all the recipient mice decreased by about 10–40% in the first week after TBI. In the BMT + hUCBDSC group, the body weight of the mice began to increase
Figure 2 Expression of VLA-4 in splenic lymphocytes of donor mice in co-culture by fluorescence immunocytochemistry. The AOD was decreased in the presence of both hUCBDSCs and hBMSCs, but the hUCBDSCs decreased VLA-4 more than the hBMSCs did (pb 0.05).
C. Zhang et al. approximately 2 weeks post-transplantation and continued to increase gradually. In the BMT group, the changes in body weight followed the same general pattern. However, the body weight of mice in the BMT + hUCBDSC group decreased significantly more slowly and increased faster than in the BMT group (P b 0.05).
3.6. Donor chimerism At 28 days post-transplantation, the proportion of chimerism was 75.95 ± 4.20% in the BMT + hUCBDSC group, which was higher than that of the BMT group (62.49 ± 5.48%) (P b 0.05).
4. Discussion Acute GVHD, a complex pathological process involving numerous cell types and target tissues, remains a major hurdle for successful BMT, a proven therapeutic modality for the treatment of various benign and malignant hematopoietic diseases. Although many immunosuppressive drugs are available, none of them alone or in combination is able to completely abolish aGVHD. Most current immunosuppressive protocols in organ/cell transplantation rely on calcineurin inhibitors, such as cyclosporine or FK506, which are nephrotoxic and diabetogenic and which may promote the establishment and growth of virally induced tumors. Furthermore, patients need to take these drugs continuously post-BMT to prevent the occurrence or reoccurrence of aGVHD. This extended immunosuppression profoundly reduces the quality of life of BMT recipients. Therefore, new therapeutic approaches are needed to overcome these side-effects. Adhesion molecules have been shown to play roles in effector cell migration in GVHD after experimental and clinical allo-HSCT. The integrins are a family of heterodimeric transmembrane proteins functioning in cell–cell and cell-extracellular matrix (ECM) adhesion. There are currently 18 known α subunits and β subunits in mammals [12]. The integrins relevant to the immune system include those in the β1, β2, and β7 families.VLA-4 belongs to the β1 family and LFA-1 belongs to the β2 family. Adhesion molecule–integrin interactions are critical in target organ inflammation in multiple specific transplantation settings. For example, targeting receptors important in migration after reducedintensity allo-BMT may exacerbate GVHD after a fully myeloablative transplantation. Thus, the tools with which to prevent target organ inflammation through inhibition of adhesion molecules may already be in place, but they currently await a more thorough understanding of the function of this complex system during GVHD [13]. Both ICAM-1 and VCAM-1 belong to the receptors of β2integrins, which are members of the immunoglobulin superfamily. ICAM-1 is expressed on the cell surface of cytokine-stimulated cells (e.g., leukocytes, endothelial cells, melanocytes, and dermal fibroblasts) and certain types of carcinomas. It binds to LFA-1 and Mac-1 on T cells, neutrophils, and macropages and provides a mechanism for selective recruitment of leukeocytes in different pathologic situations. Many cytokines, such as INF-γ, IL-1 and TNF-α, induce expression of ICAM-1 on the surface of endothelial cells. VCAM-1 is expressed on activated endothelial cells and
aGVHD regulated by hUCBDSCs through VLA-4 contains six or seven Ig domains of the H-type. VCAM-1 regulates adhension of monocytes, lymphocytes, eosinophils, and basophils to activated endothelial cells; the activation is induced by lipopolysaccharide, TNF, or IL-1. VCAM-1 binds to VLA-4 and has a role in the localization of B and T cells in the humoral immune response, which possibly providing a mechanism for selective recruitment of granulocytes in different pathologic conditions, such as rheumatoid arthritis, transplant-rejection, and allergy [14]. The expression of ICAM-1 and VCAM-1 plays an important role in GVHD. It has become a fundamental basis to understand the mechanism of LFA-1 and VLA-4 binding to their ligands in the developing therapeutic agents for immune diseases [14].
99 Data on expression of adhesion molecules in human GVHD are limited. In murine models of GVHD, however, enhanced expression of ICAM-1 on biliary and portal vein endothelial cells and sinusoidal cells is commonly observed [4]. Also, the number of T cells expressing the VCAM-1 ligand, VLA-4, is increased [15]. The expression of VLA-4 is significantly increased after allogenic transplantation, and all mice develop chronic GVHD. VLA-4 on donor CD4+ and CD8+ T cells is strongly up-regulated in GVHD mice [16], and antiLFA-1 mAb and anti-VLA-4 mAb have been shown to reduce the severity and incidence of murine GVHD [17,18]. In an aGVHD model using SCID mice as recipients, incubating donor spleen cells with mAb directed at CD49d and CD62L
Figure 3 Expression of VLA-4 in splenic MNCs of recipients at different times post-transplantation by flow cytometry analysis. VLA-4 decreased post-transplantation and reached its nadir on day 14, then increased in both groups. However, the degree of decrease was sharper in the BMT + hUCBDSC group than in the BMT group, and the rate of increase was slower in the former (p b 0.05). (A) Isotype control, (B) expression of VLA-4 in normal mice, (C) expression of VLA-4 on day 7 in the BMT group, (D) expression of VLA-4 on day 7 in the BMT + hUCBDSC group, (E) expression of VLA-4 on day 14 in the BMT group, (F) expression of VLA-4 on day 14 in the BMT + hUCBDSC group, (G) expression of VLA-4 on day 21 in the BMT group, (H) expression of VLA-4 on day 21 in the BMT + hUCBDSC group.
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Figure 3 (continued).
significantly delays the occurrence of aGVHD. We have found that hUCBDSCs play a role in immune regulation [9]. Here, we detected the effect of hUCBDSCs on VLA-4 in mice haploidentical transplantation. We found that in vitro hUCBDSCs significantly downregulated the expression of VLA-4 on the spleen lymphocytes of donor mice. It has been reported that Anti-VLA-4 antibodies inhibited CD8+ mediated primed CTL response, a critical parameter in
Figure 4 Expression of VLA-4 in the target organs of GVHD (skin, liver and intestine) on day 21 post-transplantation. Immunohistochemical VLA-4 labeling increased in skin, liver and intestine after irradiation, but VLA-4 decreased posttransplantation in both BMT + hUCBDSC and BMT groups. The post-transplantation VLA-4 level was lower in all three tissues in the BMT + hUCBDSC than in the BMT group (p b 0.05).
GVHD. We further detected that the level of VLA-4 decreased on both CD4 and CD8 T cell subtype and the decrease was greater on CD8 T cell subtype than that on CD4 T cell subtype. Also, we observed that hUCBDSCs more strongly downregulated lymphocyte VLA-4 than did hBMSCs, which shows that umbilical cord blood, which is easily obtained and contains many hematopoietic progenitor cells, is a potential source of stromal cells that regulate GVHD. As anticipated, treatment of mice during GVHD induction with anti-ICAM-1 mAb was ineffective. Others have reported successful inhibition of hepatic lesions using anti-ICAM-1 mAb, but not anti–LFA-1 mAb. Inhibition is optimal using a combination of the two mAbs. Blocking the interaction between VCAM and VLA-4 with anti-VLA-4 antibody leads to a significant reduction in the incidence and severity of hepatic lesions. We found that in vivo, hUCBDSCs significantly downregulated the expression of VLA-4 in spleen and target organs of haploidentical transplantation mice with aGVHD. Further, the lesions of GVHD organs were much less severe in the BMT + hUCBDSC group than in the BMT group (data not shown), suggesting that hUCBDSCs alleviate GVHD in part through the downregulation of VLA-4. However, There was no blocking of VCAM-1 in this study, this is only a speculative conclusion, further study should be done with the anti-VCAM1 to understand this phenomenon. It has been reported that antibodies against VLA-4 have been shown to mobilize HSPCs to the peripheral circulation [19,20]. In this study, hUCBDSCs significantly decreased the expression of VLA-4, which may be correlated with increased proportion of chimerism for BMT + hUCBDSCs transplantation. Altogether, our study shows that hUCBDSCs may protect haploidentical transplantation mice from aGVHD through
aGVHD regulated by hUCBDSCs through VLA-4 downregulating the expression of VLA-4, which could provide indirect insight as to whether and how hUCBDSCs protect human transplant recipients from GVHD.
Acknowledgments This work was funded by grants from the National Natural Science Foundation (No. 30971109) and Natural Science Foundation Project of CQ “CSTC” (CSTC, 2009BA5011), and by the Innovation Foundation for Young Scientists of the Third Military Medical University (No. 2009D226).
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