- Email: [email protected]
Umbilical cord mesenchymal stem cells suppress B-cell proliferation and differentiation
Cellular Immunology 274 (2012) 46–53
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Umbilical cord mesenchymal stem cells suppress B-cell proliferation and differentiation Nan Che a,1, Xia Li a,1, Shiliang Zhou b, Rui Liu a, Dongyan Shi b, Liwei Lu c,⇑, Lingyun Sun a,⇑ a
Department of Immunology and Rheumatology, Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing, Jiangsu, PR China Department of Immunology and Rheumatology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, PR China c Department of Pathology and Center of Infection and Immunology, The University of Hong Kong, Hong Kong, PR China b
a r t i c l e
i n f o
Article history: Received 11 November 2011 Accepted 7 February 2012 Available online 23 February 2012 Keywords: Umbilical cord mesenchymal stem cells B cells B-cell proliferation and differentiation
a b s t r a c t Mesenchymal stem cells (MSCs) may be obtained from umbilical cord as an abundant and noninvasive source. However, the immunomodulatory properties of umbilical cord–MSCs (UC–MSCs) were poorly studied. In this study, we aimed to investigate the effects of UC–MSCs on B-cell proliferation and differentiation. UC–MSCs were found to suppress the proliferation of B cells isolated from murine spleen. Moreover, UC–MSCs markedly suppressed B-cell differentiation as shown by the decreased number of CD138 + cells and reduced levels of IgM and IgG production in coculture. As revealed by transwell experiments, soluble factors produced by UC–MSCs might be involved in mediating B-cell suppression. The Blimp-1 mRNA expression was suppressed whereas the PAX-5 mRNA expression was induced in coculture. Finally, UC–MSCs modified the phosphorylation pattern of Akt and p38 pathways, which were involved in B-cell proliferation and differentiation. These results may further support the potential therapeutic use of UC–MSCs in treating autoimmune disorders. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bone marrow–mesenchymal stem cells (BM–MSCs) are multipotent adult stem cells capable of differentiating into multiple cell lineages including osteoblasts, chondrocytes and adipocytes [1]. In addition to the differentiation potential, BM–MSCs constitutively express low levels of major histocompatibility complex-I molecules and do not express costimulatory molecules such as CD80, CD86 and CD40, thus lacking immunogenicity. Recent studies have shown that BM–MSCs can efficiently inhibit the maturation, cytokine production and T-cell stimulatory capacity of dendritic cells (DCs) [2–4]. BM–MSCs can also markedly inhibit the proliferation, cytokine secretion and cytotoxic potential of natural killer cells and T cells [5–7]. Furthermore, many studies have focused on the effect of ex vivo-expanded BM–MSCs on B cells, which have shown that BM–MSCs could suppress the activation, proliferation, and differentiation of B cells [8–13]. Due to their
Abbreviations: MSCs, mesenchymal stem cells; BM–MSCs, bone marrow– mesenchymal stem cells; UC–MSCs, umbilical cord-mesenchymal stem cells; Blimp-1, B-lymphocyte-induced maturation protein-1; PAX-5, paired box gene-5; Xbp-1, X-box binding protein-1; Bcl-6, B-cell lymphoma-6. ⇑ Corresponding authors. E-mail addresses: [email protected] (L. Lu), [email protected] (L. Sun). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2012.02.004
immunomodulatory capability and their low immunogenicity, MSCs have been used as promising candidates for the prevention and treatment of immune-mediated diseases. Although BM–MSCs currently represent the main source of multipotent MSCs, bone marrow harvesting is invasive. In addition, the proliferation efficiency, multipotent differentiation potential, and maximal lifespan of BM–MSCs show distinguished individual differences and decrease significantly with aging [14–17]. However, MSCs can also be collected from other tissues including umbilical cord, umbilical cord blood, peripheral blood and adipose tissue [18–20]. In particular, umbilical cord-MSCs (UC–MSCs) may be acquired without invasive approach to the donors, which are more primitive MSCs than those isolated from bone marrow [21]. UC–MSCs share the unique features with BM–MSCs, such as their differentiation potential, low immunogenicity, and the immunomodulatory effects [19,22].Therefore, UC–MSCs should be considered as a promising alternative source of MSCs for allogeneic cell therapy [23]. Despite some functional similarities shared between UC–MSCs and BM–MSCs, there is a controversy on whether these cells are different apart from their tissue origin. Li et al. showed that the migration capacity and migration-related proteins were varying in these cells [24]. Furthermore, recent studies comparing both cell types have reported the diversities in adipogenic and osteogenic differential capabilities, suggesting that UC–MSCs and BM–MSCs, while sharing many similarities, may be actually different in
N. Che et al. / Cellular Immunology 274 (2012) 46–53
several aspects [25,26]. Therefore, the mechanisms underlying the immunomodulatory properties of UC–MSCs, which have so far been poorly studied, may be distinct from those of BM–MSCs. Recently, it has been reported that UC–MSCs could inhibit T cells activation [27–29]. However, the effects of UC–MSCs on B cells remain unclear. Here, we demonstrated that UC–MSCs significantly suppressed the proliferation, differentiation, and immunoglobulin secretion of B cells in vitro. These results provide further support to the notion that administration of UC–MSCs may represent a promising therapeutic strategy for immune-mediated disorders. 2. Materials and methods 2.1. UC–MSCs purification and identification Samples of UC–MSCs were prepared by the Stem Cell Center of Jiangsu Province (Beike Bio-Technology) as previously described [30]. All of the UC–MSCs used in the experiment were derived from passage 4. Flow cytometric analysis showed CD29, CD44, and CD105 expression of >95%, in parallel with CD45, CD34, CD14, and HLA-DR expression of <2% (Supplemental Fig. S1). 2.2. B-cell isolation and culture To avoid inadvertent activation of B cells, B cells were isolated from the spleen of the 6–8-week-old C57BL/6 female mice (Chinese Academy of Military Medical Experimental Animal Center, Beijing, China) by negative selection (CD43 depletion) with the use of CD43 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Following staining for B220 (eBioscience), B cells were sorted with a FACSCalibur (Becton Dickinson). The purity of the sorted cells was >95% (Supplemental Fig. S2). To monitor the effector functions of activated B cells, B cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 units/ml of penicillin (Gibco), 100 lg/ml of streptomycin (Gibco) in the absence or presence of the following stimuli: the CpG 2395 synthetic oligonucleotide
47
(CpG 2395; 2.5 lg/mL; Hycult Biotech), soluble CD40L (sCD40L; 100 ng/mL; R&D Systems), F(ab0 )2 anti-mouse IgM (anti-IgM; 2 lg/mL; Jackson ImmunoResearch), IL-4 (10 ng/mL, R&D Systems). 2.3. Proliferation assay Purified B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 3 days, 60 lM BrdU (Sigma) was introduced to cultures for another 3 h. Following the incorporation of BrdU, non-adherent cells were washed with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) (Sigma) and fixed overnight with 1% paraformaldehyde in PBS 0.1% Tween-20. After fixation, cells were washed and resuspended in Ca2+ and Mg2+ containing PBS with 100 Kunitz units/ ml of bovine pancreatic DNase-I (Sigma). Following digestion, the cells were washed and resuspended in PBS supplemented with BSA and Tween-20. Then, 5 ll of the FITC-coupled anti-BrdU (eBioscience) were added. After an hour of incubation, cells were washed and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) using the Cell Quest software program (Becton Dickinson). A minimum of 10,000 events per tube were collected for analysis. 2.4. Differentiation assay Purified B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 4 days, non-adherent cells were washed and then resuspended in PBS. Then, 5 ll of the PE-coupled antiCD138 mAb (Becton Dickinson) were added. After half an hour of incubation, cells were washed and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) using the Cell Quest software program (Becton Dickinson). A minimum of 10,000 events per tube were collected for analysis.
Fig. 1. UC–MSCs inhibited B-cell proliferation. B cells purified from spleen of 6–8-week old C57BL/6 female mice incubated either alone or in combination with UC–MSCs at different ratios. Cultures were simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) for 3 days. Cell proliferation was assessed by fluoresceinactivated cell sorting (FACS) after BrdU incorporation. Results were shown as percentage of positive cells. One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
48
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Fig. 2. UC–MSCs inhibited B-cell differentiation and immunoglobulin production. Purified B cells were incubated either alone or in combination with UC–MSCs at different ratios. Cultures were simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4). B-cell differentiation was evaluated using two assays, namely, the enumeration of CD138 positive cells by FACS on day 4 of culture (A) and the quantitation of IgM and IgG in culture supernatants by ELISA on day 6 of culture (B). One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
2.5. Detection of immunoglobulin production Purified mouse B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 6 days, supernatants were collected and IgM, IgG levels were tested by enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to instructions. 2.6. Transwell experiments Transwell chambers with 0.4-lm pore membranes (Costar) were used to physically separate the stimulated B cells from the UC–MSCs. B cells were cocultured in the lower Transwell chamber, and UC–MSCs were placed in the upper chamber at the same ratios as described above (1:1, 10:1, and 100:1 ratio of B cells to UC– MSCs). 2.7. RNA isolation and real-time PCR Total RNA was extracted from the cultured B cells using Trizol reagent (Invitrogen) according to the manufacturer’s recommendations. RNA was reverse-transcribed and quantified by real-time polymerase chain reaction (PCR) using the PrimeScript RT-PCR and SYBRÒ Premix Ex Taq™ kit (TaKaRa Biotechnology). The relative expressions of B-lymphocyte-induced maturation protein-1 (Blimp-1), paired box gene-5 (PAX-5), X-box binding protein-1 (Xbp-1) and B-cell lymphoma-6 (Bcl-6) mRNA were determined and normalized to the expression of the internal housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). Primer sequences were described as follows: Blimp-1: forward, 50 -ACA GTTCCCAAGAATGCCAACAG-30 , reverse, 50 -GGATTCACGTAGCGCA TCCA-30 ; PAX-5: forward, 50 -CAGCCATGGTTGTGTCAGCA-30 , reverse, 50 -GCAACCTTTGGTTTGGATCCTC-30 ; Xbp-1: forward, 50 -AGTTAAGAACACG CTTGGGAATGG-3’, reverse, 5’-CTGCTGCAGA GGTGCACATAGTC-30 ; Bcl-6: forward, 50 -GTGGTGAGCCGTGAGCA
GTTTA-30 , reverse, 50 -CCTCAGGGCTGA TTTCAGGATCTA-30 ; GAPDH: forward, 50 -AAATGGTGAAGGTCGGTGTGAAC -30 , reverse, 50 -CAA CAATCTCCACTTTGCCAC TG-30 . Relative quantification was calculated using the comparative Ct method. 2.8. Western blot analysis For Western blot analysis, B cells were cultured in the lower chamber of a 0.4 lm pore transwell insert, and UC–MSCs were cultured separately in the upper chamber. Accordingly, purified B cells were obtained for the analyses. The B cells were pelleted, washed twice with PBS (Gibco) and lysed in ice-cold lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet P-40, 20 mM Tris pH 7.0, 1 mM pepstatin, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mM sodium orthovanadate and 1 mM phenylmethylsulphonyl fluoride). Samples were centrifuged at 13,000 rpm at 4 °C for 10 min and the supernatants were collected into new tubes. Cell extracts were resuspended in sample buffer boiled and separated using sodium dodecyl sulfate gel electrophoresis. After transfer to PVDF membranes (Millipore), filters were blocked for 1 h in 10 mM Tris pH 7, 150 mM NaCl, 0, 1% Tween 20 (TBST) plus 1% bovine serum albumin (BSA) and then incubated with the corresponding antibody. Membranes were incubated with rabbit antip-p38 (1:1000), mouse anti-p38 (1:1000), anti-pAkt (1:1000), anti-Akt (1:1000) and anti-Gapdh (1:1000) (Cell Signaling Technology). After washing with TBST, filters were incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min and bands were visualized in a luminol-based detection system with p-iodophenol enhancement. 2.9. Statistical analysis Data are expressed as means ± standard deviation (SD) of the mean. SPSS 11.0 software was used for statistical analysis. For comparison between groups, the Student’s t-test was performed. The p-values < 0.05 were considered to be significant.
N. Che et al. / Cellular Immunology 274 (2012) 46–53
3. Results 3.1. Effects of UC–MSCs on B-cell proliferation We first investigated the effects of UC–MSCs on the proliferation of B cells simultaneously activated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4). The maximum inhibition of Bcell proliferation was observed at the UC–MSCs/B-cell ratio of 1:1 (p < 0.01; Fig. 1). This inhibitory effect was still detectable at a 1:10 ratio of UC–MSCs/B-cell (p < 0.05; Fig. 1), and diminished at 1:100 ratios (p > 0.05; Fig. 1). 3.2. Effects of UC–MSCs on B-cell differentiation and immunoglobulin production Next, purified B cells were incubated with or without UC–MSCs at 1:1, 1:10, and 1:100 ratios in the presence of four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) for the assessment of B-cell differentiation. As shown in Fig. 2A, the presence of UC–MSCs in the culture significantly decreased the percentage of CD138+ cells at the UC–MSCs/B-cell ratios of 1:1 and 1:10, an effect diminished at 1:100 ratios.
49
Then we examined whether the decreased percentage of CD138+ cells was paralleled by reduced amounts of immunoglobulin assessed by an ELISA assay. UC–MSCs significantly suppressed the production of IgM and IgG at the UC–MSCs/B-cell ratios of 1:1 and 1:10 (p < 0.01 for IgM, p < 0.01 for IgG) (Fig. 2B). No inhibition of immunoglobulin production was observed at the UC–MSCs/Bcell ratio of 1:100. 3.3. Soluble factors mediated UC–MSCs-dependent inhibition of B-cell proliferation and differentiation Because previous reports suggested that inhibition of B-cell proliferation by BM–MSCs was mainly mediated by soluble factors [8], we next evaluated B-cell proliferation and differentiation in a transwell system in which B cells, plated in the lower chamber, were physically separated from UC–MSCs dispensed in the upper chamber at a 1:1 ratio. Paired cultures in which B cells were admixed with UC–MSCs at the same ratio were also set up. Statistically significant inhibition of B cells proliferation and differentiation was observed irrespective of the presence or absence of the filter separating B cells from UC–MSCs (p < 0.01; Fig. 3A–C). These results demonstrate that soluble factors, released
Fig. 3. UC–MSCs inhibited B-cell proliferation and differentiation through the release of soluble factors. B cells, simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM and IL-4), and UC–MSCs at a 1: 1 ratio were seeded in 24-well plates on the opposite sides of a 0.4-lm pore-size polycarbonate membrane (transwell) and cultured for 3 days to detect B-cell proliferation (A), for 4 days to test B-cell differentiation (B), and for 6 days to measure immunoglobulin production (C). B-cell proliferation and differentiation were assessed by FACS, and immunoglobulin production was assessed by ELISA. One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄p < 0.05).
50
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Fig. 4. UC–MSCs suppressed B-lymphocyte-induced maturation protein-1 (Blimp-1) expression and induced paired box gene-5 (PAX-5) expression in cocultured B cells. Messenger RNA (mRNA) was extracted from B cells cultured alone or with UC–MSCs in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 together in transwells for 3 days. Expression levels of Blimp-1 (A), PAX-5 (B), X-box binding protein-1 (XBP-1) (C), and B-cell lymphoma 6 (Bcl-6) (D) mRNA were analyzed by realtime polymerase chain reaction (PCR). Results were calculated by the comparative threshold cycle (Ct) method, with the Ct for the glyceraldehyde phosphate dehydrogenase (GAPDH) used to normalize the results. Expression of each gene was calculated with the endogenous level of the corresponding gene in activated B cells defined as 1. Three independent tests per group showed similar results. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
Fig. 5. Phosphorylation of p38 and Akt was suppressed in B cells cocultured with UC–MSCs. Phosphorylation of p38 and Akt in B cells measured by western blot assays on day 3 of culture after simultaneously treatment with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) alone or in combination with UC–MSCs. One representative experiment of three independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
by UC–MSCs cultured together with B cells but physically separated from them, are sufficient to provide maximal inhibitory effects on B-cell proliferation and differentiation. 3.4. Effects of UC–MSCs on the expressions of Blimp-1 and PAX-5 mRNA during B-cell proliferation and differentiation To examine the genes involved in suppression of B-cell proliferation and differentiation by UC–MSCs, the expressions of Blimp-1, PAX-5, Xbp-1 and Bcl-6 mRNA by B cells were assayed using realtime-PCR analysis. Blimp-1 expression was significantly lower in B cells cocultured with UC–MSCs and the difference was highest at the UC–MSCs/B-cell ratio of 1:1, and also decreased at the UC– MSCs/B-cell ratio of 1:10. In contrast, the PAX-5 expression on activated B cells, was significantly increased when cocultured with
UC–MSCs as compared to without UC–MSCs, which also showed UC–MSCs/B-cell ratio dependent fashion. There were no significant differences in the Xbp-1 or Bcl-6 expressions on B cells in both groups (Fig. 4). In summary, the expressions of Blimp-1 mRNA were suppressed throughout the culture period in B cells cocultured with UC–MSCs. Conversely, PAX-5 expression was increased. These results suggest that UC–MSCs may prevent the proliferation and differentiation of B cells by the downregulation of Blimp-1 and upregulation of PAX-5. 3.5. Pathways involved in the effect of UC–MSCs on B-cell proliferation and differentiation In order to define the pathways involved in the effects of UC–MSCs on B cells, we examined the signal transduction pathways
N. Che et al. / Cellular Immunology 274 (2012) 46–53
involved in cell proliferation and differentiation by Western blot analysis. We evaluated each pathway in three different experiments. For this purpose, we used B cell cultured in transwells for 3 days in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4, with or without UC–MSCs at different ratios. Regarding the pathways related to cell proliferation and differentiation, we observed that Akt and p38 phosphorylation occurred upon stimulation with CpG 2395, sCD40L, anti-IgM, and IL-4. The addition of UC–MSCs suppressed phosphorylation of p-p38 and p-Akt in B-cells (Fig. 5). This inhibitory effect was enhanced along with the increased ratio of UC–MSCs to B cells. 4. Discussion In this study, we showed for the first time that UC–MSCs inhibited B-cell proliferation and differentiation to antibody-secreting cells in vitro. When UC–MSCs were cocultured with B cells stimulated with CpG 2395, sCD40L, anti-IgM, and IL-4, B-cell proliferation and differentiation to plasma cells were significantly suppressed. There are some contradictory studies with regard to BM–MSCs mediated immunomodulation of B cells. On the one hand, Corcione et al. showed that BM–MSCs suppressed B cells proliferation and differentiation, which was concluded as a result of the decrease of immunoglobulin production in the presence of CpG, CD40L, anti-immunoglobulin, and IL-2, IL-4 and IL-10 [8]. On the other hand, Traggiai et al. recently reported that BM–MSCs could induce B-cell expansion and differentiation treated with an agonist of Tolllike receptor 9 in the absence of the B-cell antigen receptors (BCRs) triggering [31]. In addition, Rasmusson et al. revealed that BM– MSCs increased B cells immunoglobulin secretion stimulated with lipopolysaccharide, cytomegalovirus or varicellazoster virus although this effect varied depending on the stimulus used to trigger B cells. Therefore, in cases in which lipopolysaccharide, cytomegalovirus or varicellazoster virus induce a weak response, BM–MSCs can stimulate immunoglobulin secretion, but the contrary occurs when a strong primary stimulus is used [32]. UC–MSCs share many similar characteristics with BM–MSCs, including (i) the pluripotent to differentiate into bone, cartilage, and adipose cells, (ii) the low immunogenic potential, and (iii) the capability to inhibit immune responses. However, recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that UC–MSCs and BM–MSCs, although sharing many similarities, may possess different biological functional characteristics, including the immunomodulatory properties. Compared to BM–MSCs, the specific mechanisms underlying UC–MSCs mediated immunoregulation of B cells have been not fully explored. The effects of UC–MSCs on B cells appeared to be strongly influenced by the relative in vitro concentrations, as suggested by a gradual loss of inhibition observed at lower UC–MSCs/B-cell ratios. Because T-cell activation is strongly suppressed by MSCs even at low concentrations of MSCs [33], we cannot exclude the possibility that their effect on B cells in vivo may be stronger when T-cell help is required. The similar inhibition of B-cell proliferation and differentiation was found in this study when UC–MSCs and B cells were cocultured together or physically separated by a filter in the same transwell system, which indicates that the inhibitory effect may be mainly mediated by the soluble factors. Further studies are warranted to investigate the key cytokines or molecules involved in mediating the suppressive effect of MSCs on B-cell differentiation and function. Since the contradicting results of pro-inflammatory or immune suppressive effects of BM–MSCs have been observed possibly due to different conditions both in vitro and in vivo, it remains to be confirmed whether transplanted UC- MSCs will maintain their immune suppressive effects in vivo.
51
It has been reported that transcription factors Blimp-1 [34], Xbp1 [35], PAX-5 [36], and Bcl-6 [37] are the master regulators of B-cell differentiation to immunoglobulin-secreting cells. Blimp-1 suppresses the expressions of both PAX-5 [38,39] and Bcl-6 [37,40,41], which are needed for preservation of B-cell phenotypes and germinal center reactions. Plasma cells generation also requires repression of PAX-5 and Bcl-6 expressions [42]. Among these genes, only the expression of Blimp-1 mRNA was suppressed in the UC– MSCs/B-cell cocultures, while the expression of PAX-5 mRNA increased. BCR signaling activates the MAPK signaling pathway and enhances transcriptional activity mediated by the transcription factor activator protein–1 (AP-1) [43], which subsequently induce Blimp-1 expression [44,45]. The Blimp-1 promoter is directly regulated by Bcl-6 [46] or AP-1 [44,45]. Thus, the humoral factor(s) released by UC–MSCs may influence this signaling pathway, leading to the suppression of Blimp-1 and induction of PAX-5. Regarding the activated signal transduction pathways underlying these effects, we focused our analysis on those which are involved in B cells proliferation and differentiation. Although we found that UC–MSCs inhibited Akt and p38 MAPK phosphorylation in a UC–MSCs dose-dependent fashion, further studies are needed to determine whether and which soluble factor(s) secreted by UC– MSCs mediate these inhibitory effects on B cells. Interestingly, both pathways have emerged as central regulators of cell proliferation and differentiation. Upon antigen binding to BCRs, B cells can rapidly proliferate and differentiate into antibody-secreting plasma cells. The Akt and p38 mitogen-activated protein kinase (MAPK) pathway functions as the downstream of the BCRs to control cell proliferation and differentiation. Interestingly, Yuichi et al. showed that BANK, a recently described adaptor molecule, attenuated CD40-mediated Akt activation, thereby preventing hyperactive B cell responses [47]. Similar findings were reported by Ju et al., who revealed that LPS-induced proliferation and differentiation of resting B cells were mainly mediated through a PTK-associated PKC/PKA-dependent p38 MAPK pathway [48]. Moreover, studies by Aase et al. also indicated that retinoic acid could strengthen the humoral immunity by promoting CpG-mediated stimulation of B cells via activation of p38 MAPK, resulting in increased proliferation and differentiation to Ig-secreting plasma cells [49]. Since MSCs are known to express multiple TLRs including TLR9, whether CpG-mediated TLR9 ligation affects the immunomodulatory functions of MSCs remain to be addressed. In conclusion, we show that UC–MSCs inhibit the proliferation and differentiation of B cells, which are accompanied with the downregulation of Blimp-1 and upregulation of PAX-5. Although current evidence indicates a non-specifically immune suppressive function of MSCs, cautions of the long-term side effects and possible alteration of vaccine responses following MSCs transplantation in clinical application need to be further investigated. Nevertheless, our data support the clinical application of UC–MSCs for the treatment of immune-mediated disorders and may facilitate the possible use of UC–MSCs as cell therapy for autoimmune diseases, including those in which B cells may play a major role.
Author disclosure statement No competing financial interests exist.
Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 81072473, 81172847, 30972736); Jiangsu Province Natural Science Foundation (BK2009034); Major International (Regional) Joint Research Project (81120108021), Jiangsu Province
52
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Kejiao Xingwei Program and National Basic Research Program of China (Grant No. 2010 CB 529100).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2012.02.004.
References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] W. Zhang, W. Ge, C. Li, S. You, L. Liao, Q. Han, W. Deng, R.C. Zhao, Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells, Stem Cells Dev. 13 (2004) 263–271. [3] X.X. Jiang, Y. Zhang, B. Liu, S.X. Zhang, Y. Wu, X.D. Yu, N. Mao, Human mesenchymal stem cells inhibit differentiation and function of monocytederived dendritic cells, Blood 105 (2005) 4120–4126. [4] A.J. Nauta, A.B. Kruisselbrink, E. Lurvink, R. Willemze, W.E. Fibbe, Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells, J. Immunol. 177 (2006) 2080–2087. [5] P.A. Sotiropoulou, S.A. Perez, A.D. Gritzapis, C.N. Baxevanis, M. Papamichail, Interactions between human mesenchymal stem cells and natural killer cells, Stem Cells 24 (2006) 74–85. [6] G.M. Spaggiari, A. Capobianco, S. Becchetti, M.C. Mingari, L. Moretta, Mesenchymal stem cell–natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL2-induced NK-cell proliferation, Blood 107 (2006) 1484–1490. [7] S.H. Yang, M.J. Park, I.H. Yoon, S.Y. Kim, S.H. Hong, J.Y. Shin, H.Y. Nam, Y.H. Kim, B. Kim, C.G. Park, Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10, Exp. Mol. Med. 41 (2009) 315–324. [8] A. Corcione, F. Benvenuto, E. Ferretti, D. Giunti, V. Cappiello, F. Cazzanti, M. Risso, F. Gualandi, G.L. Mancardi, V. Pistoia, Human mesenchymal stem cells modulate B cell functions, Blood 107 (2006) 367–372. [9] M. Rafei, J. Hsieh, S. Fortier, M.Y. Li, S. Yuan, E. Birman, K. Forner, M.N. Boivin, K. Doody, M. Tremblay, B. Annabi, J. Galipeau, Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction, Blood 112 (2008) 4991–4998. [10] P. Comoli, F. Ginevri, R. Maccario, M.A. Avanzini, M. Marconi, A. Groff, A. Cometa, M. Cioni, L. Porretti, W. Barberi, F. Frassoni, F. Locatelli, Human mesenchymal stem cells inhibit antibody production induced in vitro by allostimulation, Nephrol. Dial. Transplant. 23 (2008) 1196–1202. [11] S. Tabera, J.A.P. Simón, M.D. Campelo, L.I.S. Abarca, B. Blanco, A. López, A. Benito, E. Ocio, F.M.S. Guijo, C. Cañizo, J.F.S. Miguel, The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes, Haematologica 93 (2008) 1301–1309. [12] S. Asaria, S. Itakuraa, K. Ferreria, C. Liub, Y. Kurodaa, F. Kandeela, Y. Mullen, Mesenchymal stem cells suppress B-cell terminal differentiation, Exp. Hematol. 37 (2009) 604–615. [13] F. Schena, C. Gambini, A. Gregorio, M. Mosconi, D. Reverberi, M. Gattorno, S. Casazza, A. Uccelli, L. Moretta, A. Martini, E. Traggiai, Interferon-c-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of Systemic Lupus Erythematosus, Arthritis. Rheum. 62 (2010) 2776–2786. [14] K. Stenderup, J. Justesen, C. Clausen, M. Kassem, Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells, Bone 33 (2003) 919–926. [15] S. Nishida, N. Endo, H. Yamagiwa, T. Tanizawa, H.E. Takahashi, Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation, J. Bone. Miner. Metab. 17 (1999) 171–177. [16] S.M. Mueller, J. Glowacki, Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges, J. Cell. Biochem. 82 (2001) 583–590. [17] H. Zhang, S. Fazel, H. Tian, D.A. Mickle, R.D. Weisel, T. Fujii, R.K. Li, Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy, Am. J. Physiol. Heart Circ. Physiol. 289 (2005) H2089–H2096. [18] A.J. Friedenstein, K.V. Petrakova, A.I. Kurolesova, G.P. Frolova, Heterotopic of bone marrow: Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation 6 (1968) 230–247. [19] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R.O. Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41–49. [20] A.J. Nauta, W.E. Fibbe, Immunomodulatory properties of mesenchymal stromal cells, Blood 110 (2007) 3499–3506. [21] M.L. Weiss, S. Medicetty, A.R. Bledsoe, R.S. Rachakatla, M. Choi, S. Merchav, Y. Luo, M.S. Rao, G. Velagaleti, D. Troyer, Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson0 s disease, Stem Cells 24 (2006) 781–792.
[22] S. Medicetty, A.R. Bledsoe, C.B. Fahrenholtz, D. Troyer, M.L. Weiss, Transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks, Exp. Neurol. 190 (2004) 32–41. [23] L.L. Lu, Y.J. Liu, S.G. Yang, Q.J. Zhao, X. Wang, W. Gong, Z.B. Han, Z.S. Xu, Y.X. Lu, D. Liu, Z.Z. Chen, Z.C. Han, Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials, Haematologica 91 (2006) 1017–1026. [24] G. Li, X.A. Zhang, H. Wang, X. Wang, C.L. Meng, C.Y. Chan, D.T.W. Yew, K.S. Tsang, K. Li, S.N. Tsai, S.M. Ngai, Z.C. Han, M.C.M. Lin, M.L. He, H.F. Kung, Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: implication in the migration, Proteomics 9 (2009) 20–30. [25] H. Cheng, L. Qiu, J. Ma, H. Zhang, M. Cheng, W. Li, X. Zhao, K. Liu, Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts, Mol. Biol. Rep. Epub ahead of print (2010). [26] M.Y. Chen, P.C. Lie, Z.L. Li, X. Wei, Endothelial differentiation of Wharton’s jelly-derived mesenchymal stem cells in comparison with bone marrowderived mesenchymal stem cells, Exp. Hematol. 37 (2009) 629–640. [27] K. Chen, D. Wang, W.T. Du, Z.B. Han, H. Ren, Y. Chi, S.G. Yang, D. Zhu, F. Bayard, Z.C. Han, Human umbilical cord mesenchymal stem cells hUC–MSCs exert immunosuppressive activities through a PGE2-dependent mechanism, Clin. Immunol. 135 (2010) 448–458. [28] A.J. Cutler, V. Limbani, J. Girdlestone, C.V. Navarrete, Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation, J. Immunol. 185 (2010) 6617–6623. [29] Y. Liu, R. Mu, S. Wang, L. Long, X. Liu, R. Li, J. Sun, J. Guo, X. Zhang, J. Guo, P. Yu, C. Li, X. Liu, Z. Huang, D. Wang, H. Li, Z. Gu, B. Liu, Z. Li, Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis, Arthritis. Res. Ther. 12 (2010) R210. [30] L. Sun, D. Wang, J. Liang, H. Zhang, X. Feng, H. Wang, B. Hua, B. Liu, S. Ye, X. Hu, W. Xu, X. Zeng, Y. Hou, G.S. Gilkeson, R.M. Silver, L. Lu, S. Shi, Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus, Arthritis. Rheum. 62 (2010) 2467–2475. [31] E. Traggiai, S. Volpi, F. Schena, M. Gattorno, F. Ferlito, L. Moretta, A. Martini, Bone marrow-derived mesenchymal stem cells induce both polyclonal expansion and differentiation of B cells isolated from healthy donors and systemic lupus erythematosus patients, Stem cells 26 (2008) 562–569. [32] I. Rasmusson, K.L. Blanc, B. Sundberg, O. Ringdén, Mesenchymal stem cells stimulate antibody secretion in human B cells, Scand. J. Immunol. 65 (2007) 336–343. [33] E. Zappia, S. Casazza, E. Pedemonte, F. Benvenuto, I. Bonanni, E. Gerdoni, D. Giunti, A. Ceravolo, F. Cazzanti, F. Frassoni, G. Mancardi, A. Uccelli, Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy, Blood 106 (2005) 1755–1761. [34] Y. Lin, K. Wong, K. Calame, Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation, Science 276 (1997) 596–599. [35] A.M. Reimold, N.N. Iwakoshi, J. Manis, P. Vallabhajosyula, E.S. Tsuda, E.M. Gravallese, D. Friend, M.J. Grusby, F. Alt, L.H. Glimcher, Plasma cell differentiation requires the transcription factor XBP-1, Nature 412 (2001) 300–307. [36] M. Horcher, A. Souabni, M. Busslinger, Pax5/BSAP maintains the identity of B cells in late B lymphopoiesis, Immunity 14 (2001) 779–790. [37] A.L. Dent, A.L. Shaffer, X. Yu, D. Allman, L.M. Staudt, Control of inflammation, cytokine expression, and germinal center formation by BCL-6, Science 276 (1997) 589–592. [38] K.P. Nera, P. Kohonen, E. Narvi, A. Peippo, L. Mustonen, P. Terho, K. Koskela, J.M. Buerstedde, O. Lassila, Loss of Pax5 promotes plasma cell differentiation, Immunity 24 (2006) 283–293. [39] A. Delogu, A. Schebesta, Q. Sun, K. Aschenbrenner, T. Perlot, M. Busslinger, Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells, Immunity 24 (2006) 269–281. [40] B.H. Ye, G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. Waard, C. Leung, M.N. Shirazi, A. Orazi, R.S. Chaganti, P. Rothman, A.M. Stall, P.P. Pandolfi, R.D. Favera, The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation, Nat. Genet. 16 (1997) 161–170. [41] T. Fukuda, T. Yoshida, S. Okada, M. Hatano, T. Miki, K. Ishibashi, S. Okabe, H. Koseki, S. Hirosawa, M. Taniguchi, N. Miyasaka, T. Tokuhisa, Disruption of the Bcl6 gene results in an impaired germinal center formation, J. Exp. Med. 186 (1997) 439–448. [42] M.S. Shelef, K. Calame, Regulation of plasma-cell development, Nat. Rev. Immunol. 5 (2005) 230–242. [43] P. Hess, G. Pihan, C.L. Sawyers, R.A. Flavell, R.J. Davis, Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts, Nat. Genet. 32 (2002) 201–205. [44] F.H. Vasanwala, S. Kusam, L.M. Toney, A.L. Dent, Repression of AP-1 function: a mechanism for the regulation of Blimp-1 expression and B lymphocyte differentiation by the B cell lymphoma-6 protooncogene, J. Immunol. 169 (2002) 1922–1929. [45] Y. Ohkubo, M. Arima, E. Arguni, S. Okada, K. Yamashita, S. Asari, S. Obata, A. Sakamoto, M. Hatano, J.O. Wang, M. Ebara, H. Saisho, T. Tokuhisa, A role for cfos/activator protein 1 in B lymphocyte terminal differentiation, J. Immunol. 174 (2005) 7703–7710. [46] C. Tunyaplin, A.L. Shaffer, C.D. Angelin-Duclos, X. Yu, L.M. Staudt, K.L. Calame, Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation, J. Immunol. 173 (2004) 1158–1165.
N. Che et al. / Cellular Immunology 274 (2012) 46–53 [47] Y. Aiba, T. Yamazaki, T. Okada, K. Gotoh, H. Sanjo, M. Ogata, T. Kurosaki, BANK negatively regulates Akt activation and subsequent B cell responses, Immunity 24 (2006) 259–268. [48] J. Kim, H.Y. Yang, Y.S. Jang, A G-protein-associated ERK pathway is involved in LPS-induced proliferation and a PTK-associated p38 MAPK pathway is
53
involved in LPS-induced differentiation in resting mB cells, Mol. Immunol. 43 (2006) 1232–1242. [49] A. Ertesvag, H.C. Aasheim, S. Naderi, H.K. Blomhoff, Vitamin A potentiates CpGmediated memory B-cell proliferation and differentiation: involvement of early activation of p38 MAPK, Blood 109 (2007) 3865–3872.
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Umbilical cord mesenchymal stem cells suppress B-cell proliferation and differentiation Nan Che a,1, Xia Li a,1, Shiliang Zhou b, Rui Liu a, Dongyan Shi b, Liwei Lu c,⇑, Lingyun Sun a,⇑ a
Department of Immunology and Rheumatology, Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing, Jiangsu, PR China Department of Immunology and Rheumatology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, PR China c Department of Pathology and Center of Infection and Immunology, The University of Hong Kong, Hong Kong, PR China b
a r t i c l e
i n f o
Article history: Received 11 November 2011 Accepted 7 February 2012 Available online 23 February 2012 Keywords: Umbilical cord mesenchymal stem cells B cells B-cell proliferation and differentiation
a b s t r a c t Mesenchymal stem cells (MSCs) may be obtained from umbilical cord as an abundant and noninvasive source. However, the immunomodulatory properties of umbilical cord–MSCs (UC–MSCs) were poorly studied. In this study, we aimed to investigate the effects of UC–MSCs on B-cell proliferation and differentiation. UC–MSCs were found to suppress the proliferation of B cells isolated from murine spleen. Moreover, UC–MSCs markedly suppressed B-cell differentiation as shown by the decreased number of CD138 + cells and reduced levels of IgM and IgG production in coculture. As revealed by transwell experiments, soluble factors produced by UC–MSCs might be involved in mediating B-cell suppression. The Blimp-1 mRNA expression was suppressed whereas the PAX-5 mRNA expression was induced in coculture. Finally, UC–MSCs modified the phosphorylation pattern of Akt and p38 pathways, which were involved in B-cell proliferation and differentiation. These results may further support the potential therapeutic use of UC–MSCs in treating autoimmune disorders. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bone marrow–mesenchymal stem cells (BM–MSCs) are multipotent adult stem cells capable of differentiating into multiple cell lineages including osteoblasts, chondrocytes and adipocytes [1]. In addition to the differentiation potential, BM–MSCs constitutively express low levels of major histocompatibility complex-I molecules and do not express costimulatory molecules such as CD80, CD86 and CD40, thus lacking immunogenicity. Recent studies have shown that BM–MSCs can efficiently inhibit the maturation, cytokine production and T-cell stimulatory capacity of dendritic cells (DCs) [2–4]. BM–MSCs can also markedly inhibit the proliferation, cytokine secretion and cytotoxic potential of natural killer cells and T cells [5–7]. Furthermore, many studies have focused on the effect of ex vivo-expanded BM–MSCs on B cells, which have shown that BM–MSCs could suppress the activation, proliferation, and differentiation of B cells [8–13]. Due to their
Abbreviations: MSCs, mesenchymal stem cells; BM–MSCs, bone marrow– mesenchymal stem cells; UC–MSCs, umbilical cord-mesenchymal stem cells; Blimp-1, B-lymphocyte-induced maturation protein-1; PAX-5, paired box gene-5; Xbp-1, X-box binding protein-1; Bcl-6, B-cell lymphoma-6. ⇑ Corresponding authors. E-mail addresses: [email protected] (L. Lu), [email protected] (L. Sun). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2012.02.004
immunomodulatory capability and their low immunogenicity, MSCs have been used as promising candidates for the prevention and treatment of immune-mediated diseases. Although BM–MSCs currently represent the main source of multipotent MSCs, bone marrow harvesting is invasive. In addition, the proliferation efficiency, multipotent differentiation potential, and maximal lifespan of BM–MSCs show distinguished individual differences and decrease significantly with aging [14–17]. However, MSCs can also be collected from other tissues including umbilical cord, umbilical cord blood, peripheral blood and adipose tissue [18–20]. In particular, umbilical cord-MSCs (UC–MSCs) may be acquired without invasive approach to the donors, which are more primitive MSCs than those isolated from bone marrow [21]. UC–MSCs share the unique features with BM–MSCs, such as their differentiation potential, low immunogenicity, and the immunomodulatory effects [19,22].Therefore, UC–MSCs should be considered as a promising alternative source of MSCs for allogeneic cell therapy [23]. Despite some functional similarities shared between UC–MSCs and BM–MSCs, there is a controversy on whether these cells are different apart from their tissue origin. Li et al. showed that the migration capacity and migration-related proteins were varying in these cells [24]. Furthermore, recent studies comparing both cell types have reported the diversities in adipogenic and osteogenic differential capabilities, suggesting that UC–MSCs and BM–MSCs, while sharing many similarities, may be actually different in
N. Che et al. / Cellular Immunology 274 (2012) 46–53
several aspects [25,26]. Therefore, the mechanisms underlying the immunomodulatory properties of UC–MSCs, which have so far been poorly studied, may be distinct from those of BM–MSCs. Recently, it has been reported that UC–MSCs could inhibit T cells activation [27–29]. However, the effects of UC–MSCs on B cells remain unclear. Here, we demonstrated that UC–MSCs significantly suppressed the proliferation, differentiation, and immunoglobulin secretion of B cells in vitro. These results provide further support to the notion that administration of UC–MSCs may represent a promising therapeutic strategy for immune-mediated disorders. 2. Materials and methods 2.1. UC–MSCs purification and identification Samples of UC–MSCs were prepared by the Stem Cell Center of Jiangsu Province (Beike Bio-Technology) as previously described [30]. All of the UC–MSCs used in the experiment were derived from passage 4. Flow cytometric analysis showed CD29, CD44, and CD105 expression of >95%, in parallel with CD45, CD34, CD14, and HLA-DR expression of <2% (Supplemental Fig. S1). 2.2. B-cell isolation and culture To avoid inadvertent activation of B cells, B cells were isolated from the spleen of the 6–8-week-old C57BL/6 female mice (Chinese Academy of Military Medical Experimental Animal Center, Beijing, China) by negative selection (CD43 depletion) with the use of CD43 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Following staining for B220 (eBioscience), B cells were sorted with a FACSCalibur (Becton Dickinson). The purity of the sorted cells was >95% (Supplemental Fig. S2). To monitor the effector functions of activated B cells, B cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 units/ml of penicillin (Gibco), 100 lg/ml of streptomycin (Gibco) in the absence or presence of the following stimuli: the CpG 2395 synthetic oligonucleotide
47
(CpG 2395; 2.5 lg/mL; Hycult Biotech), soluble CD40L (sCD40L; 100 ng/mL; R&D Systems), F(ab0 )2 anti-mouse IgM (anti-IgM; 2 lg/mL; Jackson ImmunoResearch), IL-4 (10 ng/mL, R&D Systems). 2.3. Proliferation assay Purified B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 3 days, 60 lM BrdU (Sigma) was introduced to cultures for another 3 h. Following the incorporation of BrdU, non-adherent cells were washed with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) (Sigma) and fixed overnight with 1% paraformaldehyde in PBS 0.1% Tween-20. After fixation, cells were washed and resuspended in Ca2+ and Mg2+ containing PBS with 100 Kunitz units/ ml of bovine pancreatic DNase-I (Sigma). Following digestion, the cells were washed and resuspended in PBS supplemented with BSA and Tween-20. Then, 5 ll of the FITC-coupled anti-BrdU (eBioscience) were added. After an hour of incubation, cells were washed and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) using the Cell Quest software program (Becton Dickinson). A minimum of 10,000 events per tube were collected for analysis. 2.4. Differentiation assay Purified B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 4 days, non-adherent cells were washed and then resuspended in PBS. Then, 5 ll of the PE-coupled antiCD138 mAb (Becton Dickinson) were added. After half an hour of incubation, cells were washed and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) using the Cell Quest software program (Becton Dickinson). A minimum of 10,000 events per tube were collected for analysis.
Fig. 1. UC–MSCs inhibited B-cell proliferation. B cells purified from spleen of 6–8-week old C57BL/6 female mice incubated either alone or in combination with UC–MSCs at different ratios. Cultures were simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) for 3 days. Cell proliferation was assessed by fluoresceinactivated cell sorting (FACS) after BrdU incorporation. Results were shown as percentage of positive cells. One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
48
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Fig. 2. UC–MSCs inhibited B-cell differentiation and immunoglobulin production. Purified B cells were incubated either alone or in combination with UC–MSCs at different ratios. Cultures were simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4). B-cell differentiation was evaluated using two assays, namely, the enumeration of CD138 positive cells by FACS on day 4 of culture (A) and the quantitation of IgM and IgG in culture supernatants by ELISA on day 6 of culture (B). One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
2.5. Detection of immunoglobulin production Purified mouse B cells were cocultured in 96-well flat-bottom plates (Costar) with UC–MSCs at different ratios (1:1, 10:1, and 100:1 ratio of B cells to UC–MSCs) in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 in a total volume of 0.2 mL RPMI 1640 medium per well in triplicate. After 6 days, supernatants were collected and IgM, IgG levels were tested by enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to instructions. 2.6. Transwell experiments Transwell chambers with 0.4-lm pore membranes (Costar) were used to physically separate the stimulated B cells from the UC–MSCs. B cells were cocultured in the lower Transwell chamber, and UC–MSCs were placed in the upper chamber at the same ratios as described above (1:1, 10:1, and 100:1 ratio of B cells to UC– MSCs). 2.7. RNA isolation and real-time PCR Total RNA was extracted from the cultured B cells using Trizol reagent (Invitrogen) according to the manufacturer’s recommendations. RNA was reverse-transcribed and quantified by real-time polymerase chain reaction (PCR) using the PrimeScript RT-PCR and SYBRÒ Premix Ex Taq™ kit (TaKaRa Biotechnology). The relative expressions of B-lymphocyte-induced maturation protein-1 (Blimp-1), paired box gene-5 (PAX-5), X-box binding protein-1 (Xbp-1) and B-cell lymphoma-6 (Bcl-6) mRNA were determined and normalized to the expression of the internal housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). Primer sequences were described as follows: Blimp-1: forward, 50 -ACA GTTCCCAAGAATGCCAACAG-30 , reverse, 50 -GGATTCACGTAGCGCA TCCA-30 ; PAX-5: forward, 50 -CAGCCATGGTTGTGTCAGCA-30 , reverse, 50 -GCAACCTTTGGTTTGGATCCTC-30 ; Xbp-1: forward, 50 -AGTTAAGAACACG CTTGGGAATGG-3’, reverse, 5’-CTGCTGCAGA GGTGCACATAGTC-30 ; Bcl-6: forward, 50 -GTGGTGAGCCGTGAGCA
GTTTA-30 , reverse, 50 -CCTCAGGGCTGA TTTCAGGATCTA-30 ; GAPDH: forward, 50 -AAATGGTGAAGGTCGGTGTGAAC -30 , reverse, 50 -CAA CAATCTCCACTTTGCCAC TG-30 . Relative quantification was calculated using the comparative Ct method. 2.8. Western blot analysis For Western blot analysis, B cells were cultured in the lower chamber of a 0.4 lm pore transwell insert, and UC–MSCs were cultured separately in the upper chamber. Accordingly, purified B cells were obtained for the analyses. The B cells were pelleted, washed twice with PBS (Gibco) and lysed in ice-cold lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet P-40, 20 mM Tris pH 7.0, 1 mM pepstatin, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mM sodium orthovanadate and 1 mM phenylmethylsulphonyl fluoride). Samples were centrifuged at 13,000 rpm at 4 °C for 10 min and the supernatants were collected into new tubes. Cell extracts were resuspended in sample buffer boiled and separated using sodium dodecyl sulfate gel electrophoresis. After transfer to PVDF membranes (Millipore), filters were blocked for 1 h in 10 mM Tris pH 7, 150 mM NaCl, 0, 1% Tween 20 (TBST) plus 1% bovine serum albumin (BSA) and then incubated with the corresponding antibody. Membranes were incubated with rabbit antip-p38 (1:1000), mouse anti-p38 (1:1000), anti-pAkt (1:1000), anti-Akt (1:1000) and anti-Gapdh (1:1000) (Cell Signaling Technology). After washing with TBST, filters were incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min and bands were visualized in a luminol-based detection system with p-iodophenol enhancement. 2.9. Statistical analysis Data are expressed as means ± standard deviation (SD) of the mean. SPSS 11.0 software was used for statistical analysis. For comparison between groups, the Student’s t-test was performed. The p-values < 0.05 were considered to be significant.
N. Che et al. / Cellular Immunology 274 (2012) 46–53
3. Results 3.1. Effects of UC–MSCs on B-cell proliferation We first investigated the effects of UC–MSCs on the proliferation of B cells simultaneously activated with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4). The maximum inhibition of Bcell proliferation was observed at the UC–MSCs/B-cell ratio of 1:1 (p < 0.01; Fig. 1). This inhibitory effect was still detectable at a 1:10 ratio of UC–MSCs/B-cell (p < 0.05; Fig. 1), and diminished at 1:100 ratios (p > 0.05; Fig. 1). 3.2. Effects of UC–MSCs on B-cell differentiation and immunoglobulin production Next, purified B cells were incubated with or without UC–MSCs at 1:1, 1:10, and 1:100 ratios in the presence of four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) for the assessment of B-cell differentiation. As shown in Fig. 2A, the presence of UC–MSCs in the culture significantly decreased the percentage of CD138+ cells at the UC–MSCs/B-cell ratios of 1:1 and 1:10, an effect diminished at 1:100 ratios.
49
Then we examined whether the decreased percentage of CD138+ cells was paralleled by reduced amounts of immunoglobulin assessed by an ELISA assay. UC–MSCs significantly suppressed the production of IgM and IgG at the UC–MSCs/B-cell ratios of 1:1 and 1:10 (p < 0.01 for IgM, p < 0.01 for IgG) (Fig. 2B). No inhibition of immunoglobulin production was observed at the UC–MSCs/Bcell ratio of 1:100. 3.3. Soluble factors mediated UC–MSCs-dependent inhibition of B-cell proliferation and differentiation Because previous reports suggested that inhibition of B-cell proliferation by BM–MSCs was mainly mediated by soluble factors [8], we next evaluated B-cell proliferation and differentiation in a transwell system in which B cells, plated in the lower chamber, were physically separated from UC–MSCs dispensed in the upper chamber at a 1:1 ratio. Paired cultures in which B cells were admixed with UC–MSCs at the same ratio were also set up. Statistically significant inhibition of B cells proliferation and differentiation was observed irrespective of the presence or absence of the filter separating B cells from UC–MSCs (p < 0.01; Fig. 3A–C). These results demonstrate that soluble factors, released
Fig. 3. UC–MSCs inhibited B-cell proliferation and differentiation through the release of soluble factors. B cells, simultaneously treated with four stimuli (CpG 2395, sCD40L, anti-IgM and IL-4), and UC–MSCs at a 1: 1 ratio were seeded in 24-well plates on the opposite sides of a 0.4-lm pore-size polycarbonate membrane (transwell) and cultured for 3 days to detect B-cell proliferation (A), for 4 days to test B-cell differentiation (B), and for 6 days to measure immunoglobulin production (C). B-cell proliferation and differentiation were assessed by FACS, and immunoglobulin production was assessed by ELISA. One representative experiment of 4 independent experiments performed was shown. Data were expressed as mean ± SD (⁄p < 0.05).
50
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Fig. 4. UC–MSCs suppressed B-lymphocyte-induced maturation protein-1 (Blimp-1) expression and induced paired box gene-5 (PAX-5) expression in cocultured B cells. Messenger RNA (mRNA) was extracted from B cells cultured alone or with UC–MSCs in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 together in transwells for 3 days. Expression levels of Blimp-1 (A), PAX-5 (B), X-box binding protein-1 (XBP-1) (C), and B-cell lymphoma 6 (Bcl-6) (D) mRNA were analyzed by realtime polymerase chain reaction (PCR). Results were calculated by the comparative threshold cycle (Ct) method, with the Ct for the glyceraldehyde phosphate dehydrogenase (GAPDH) used to normalize the results. Expression of each gene was calculated with the endogenous level of the corresponding gene in activated B cells defined as 1. Three independent tests per group showed similar results. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
Fig. 5. Phosphorylation of p38 and Akt was suppressed in B cells cocultured with UC–MSCs. Phosphorylation of p38 and Akt in B cells measured by western blot assays on day 3 of culture after simultaneously treatment with four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) alone or in combination with UC–MSCs. One representative experiment of three independent experiments performed was shown. Data were expressed as mean ± SD (⁄⁄p < 0.01; ⁄p < 0.05).
by UC–MSCs cultured together with B cells but physically separated from them, are sufficient to provide maximal inhibitory effects on B-cell proliferation and differentiation. 3.4. Effects of UC–MSCs on the expressions of Blimp-1 and PAX-5 mRNA during B-cell proliferation and differentiation To examine the genes involved in suppression of B-cell proliferation and differentiation by UC–MSCs, the expressions of Blimp-1, PAX-5, Xbp-1 and Bcl-6 mRNA by B cells were assayed using realtime-PCR analysis. Blimp-1 expression was significantly lower in B cells cocultured with UC–MSCs and the difference was highest at the UC–MSCs/B-cell ratio of 1:1, and also decreased at the UC– MSCs/B-cell ratio of 1:10. In contrast, the PAX-5 expression on activated B cells, was significantly increased when cocultured with
UC–MSCs as compared to without UC–MSCs, which also showed UC–MSCs/B-cell ratio dependent fashion. There were no significant differences in the Xbp-1 or Bcl-6 expressions on B cells in both groups (Fig. 4). In summary, the expressions of Blimp-1 mRNA were suppressed throughout the culture period in B cells cocultured with UC–MSCs. Conversely, PAX-5 expression was increased. These results suggest that UC–MSCs may prevent the proliferation and differentiation of B cells by the downregulation of Blimp-1 and upregulation of PAX-5. 3.5. Pathways involved in the effect of UC–MSCs on B-cell proliferation and differentiation In order to define the pathways involved in the effects of UC–MSCs on B cells, we examined the signal transduction pathways
N. Che et al. / Cellular Immunology 274 (2012) 46–53
involved in cell proliferation and differentiation by Western blot analysis. We evaluated each pathway in three different experiments. For this purpose, we used B cell cultured in transwells for 3 days in the presence of CpG 2395, sCD40L, anti-IgM, and IL-4, with or without UC–MSCs at different ratios. Regarding the pathways related to cell proliferation and differentiation, we observed that Akt and p38 phosphorylation occurred upon stimulation with CpG 2395, sCD40L, anti-IgM, and IL-4. The addition of UC–MSCs suppressed phosphorylation of p-p38 and p-Akt in B-cells (Fig. 5). This inhibitory effect was enhanced along with the increased ratio of UC–MSCs to B cells. 4. Discussion In this study, we showed for the first time that UC–MSCs inhibited B-cell proliferation and differentiation to antibody-secreting cells in vitro. When UC–MSCs were cocultured with B cells stimulated with CpG 2395, sCD40L, anti-IgM, and IL-4, B-cell proliferation and differentiation to plasma cells were significantly suppressed. There are some contradictory studies with regard to BM–MSCs mediated immunomodulation of B cells. On the one hand, Corcione et al. showed that BM–MSCs suppressed B cells proliferation and differentiation, which was concluded as a result of the decrease of immunoglobulin production in the presence of CpG, CD40L, anti-immunoglobulin, and IL-2, IL-4 and IL-10 [8]. On the other hand, Traggiai et al. recently reported that BM–MSCs could induce B-cell expansion and differentiation treated with an agonist of Tolllike receptor 9 in the absence of the B-cell antigen receptors (BCRs) triggering [31]. In addition, Rasmusson et al. revealed that BM– MSCs increased B cells immunoglobulin secretion stimulated with lipopolysaccharide, cytomegalovirus or varicellazoster virus although this effect varied depending on the stimulus used to trigger B cells. Therefore, in cases in which lipopolysaccharide, cytomegalovirus or varicellazoster virus induce a weak response, BM–MSCs can stimulate immunoglobulin secretion, but the contrary occurs when a strong primary stimulus is used [32]. UC–MSCs share many similar characteristics with BM–MSCs, including (i) the pluripotent to differentiate into bone, cartilage, and adipose cells, (ii) the low immunogenic potential, and (iii) the capability to inhibit immune responses. However, recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that UC–MSCs and BM–MSCs, although sharing many similarities, may possess different biological functional characteristics, including the immunomodulatory properties. Compared to BM–MSCs, the specific mechanisms underlying UC–MSCs mediated immunoregulation of B cells have been not fully explored. The effects of UC–MSCs on B cells appeared to be strongly influenced by the relative in vitro concentrations, as suggested by a gradual loss of inhibition observed at lower UC–MSCs/B-cell ratios. Because T-cell activation is strongly suppressed by MSCs even at low concentrations of MSCs [33], we cannot exclude the possibility that their effect on B cells in vivo may be stronger when T-cell help is required. The similar inhibition of B-cell proliferation and differentiation was found in this study when UC–MSCs and B cells were cocultured together or physically separated by a filter in the same transwell system, which indicates that the inhibitory effect may be mainly mediated by the soluble factors. Further studies are warranted to investigate the key cytokines or molecules involved in mediating the suppressive effect of MSCs on B-cell differentiation and function. Since the contradicting results of pro-inflammatory or immune suppressive effects of BM–MSCs have been observed possibly due to different conditions both in vitro and in vivo, it remains to be confirmed whether transplanted UC- MSCs will maintain their immune suppressive effects in vivo.
51
It has been reported that transcription factors Blimp-1 [34], Xbp1 [35], PAX-5 [36], and Bcl-6 [37] are the master regulators of B-cell differentiation to immunoglobulin-secreting cells. Blimp-1 suppresses the expressions of both PAX-5 [38,39] and Bcl-6 [37,40,41], which are needed for preservation of B-cell phenotypes and germinal center reactions. Plasma cells generation also requires repression of PAX-5 and Bcl-6 expressions [42]. Among these genes, only the expression of Blimp-1 mRNA was suppressed in the UC– MSCs/B-cell cocultures, while the expression of PAX-5 mRNA increased. BCR signaling activates the MAPK signaling pathway and enhances transcriptional activity mediated by the transcription factor activator protein–1 (AP-1) [43], which subsequently induce Blimp-1 expression [44,45]. The Blimp-1 promoter is directly regulated by Bcl-6 [46] or AP-1 [44,45]. Thus, the humoral factor(s) released by UC–MSCs may influence this signaling pathway, leading to the suppression of Blimp-1 and induction of PAX-5. Regarding the activated signal transduction pathways underlying these effects, we focused our analysis on those which are involved in B cells proliferation and differentiation. Although we found that UC–MSCs inhibited Akt and p38 MAPK phosphorylation in a UC–MSCs dose-dependent fashion, further studies are needed to determine whether and which soluble factor(s) secreted by UC– MSCs mediate these inhibitory effects on B cells. Interestingly, both pathways have emerged as central regulators of cell proliferation and differentiation. Upon antigen binding to BCRs, B cells can rapidly proliferate and differentiate into antibody-secreting plasma cells. The Akt and p38 mitogen-activated protein kinase (MAPK) pathway functions as the downstream of the BCRs to control cell proliferation and differentiation. Interestingly, Yuichi et al. showed that BANK, a recently described adaptor molecule, attenuated CD40-mediated Akt activation, thereby preventing hyperactive B cell responses [47]. Similar findings were reported by Ju et al., who revealed that LPS-induced proliferation and differentiation of resting B cells were mainly mediated through a PTK-associated PKC/PKA-dependent p38 MAPK pathway [48]. Moreover, studies by Aase et al. also indicated that retinoic acid could strengthen the humoral immunity by promoting CpG-mediated stimulation of B cells via activation of p38 MAPK, resulting in increased proliferation and differentiation to Ig-secreting plasma cells [49]. Since MSCs are known to express multiple TLRs including TLR9, whether CpG-mediated TLR9 ligation affects the immunomodulatory functions of MSCs remain to be addressed. In conclusion, we show that UC–MSCs inhibit the proliferation and differentiation of B cells, which are accompanied with the downregulation of Blimp-1 and upregulation of PAX-5. Although current evidence indicates a non-specifically immune suppressive function of MSCs, cautions of the long-term side effects and possible alteration of vaccine responses following MSCs transplantation in clinical application need to be further investigated. Nevertheless, our data support the clinical application of UC–MSCs for the treatment of immune-mediated disorders and may facilitate the possible use of UC–MSCs as cell therapy for autoimmune diseases, including those in which B cells may play a major role.
Author disclosure statement No competing financial interests exist.
Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 81072473, 81172847, 30972736); Jiangsu Province Natural Science Foundation (BK2009034); Major International (Regional) Joint Research Project (81120108021), Jiangsu Province
52
N. Che et al. / Cellular Immunology 274 (2012) 46–53
Kejiao Xingwei Program and National Basic Research Program of China (Grant No. 2010 CB 529100).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2012.02.004.
References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] W. Zhang, W. Ge, C. Li, S. You, L. Liao, Q. Han, W. Deng, R.C. Zhao, Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells, Stem Cells Dev. 13 (2004) 263–271. [3] X.X. Jiang, Y. Zhang, B. Liu, S.X. Zhang, Y. Wu, X.D. Yu, N. Mao, Human mesenchymal stem cells inhibit differentiation and function of monocytederived dendritic cells, Blood 105 (2005) 4120–4126. [4] A.J. Nauta, A.B. Kruisselbrink, E. Lurvink, R. Willemze, W.E. Fibbe, Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells, J. Immunol. 177 (2006) 2080–2087. [5] P.A. Sotiropoulou, S.A. Perez, A.D. Gritzapis, C.N. Baxevanis, M. Papamichail, Interactions between human mesenchymal stem cells and natural killer cells, Stem Cells 24 (2006) 74–85. [6] G.M. Spaggiari, A. Capobianco, S. Becchetti, M.C. Mingari, L. Moretta, Mesenchymal stem cell–natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL2-induced NK-cell proliferation, Blood 107 (2006) 1484–1490. [7] S.H. Yang, M.J. Park, I.H. Yoon, S.Y. Kim, S.H. Hong, J.Y. Shin, H.Y. Nam, Y.H. Kim, B. Kim, C.G. Park, Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10, Exp. Mol. Med. 41 (2009) 315–324. [8] A. Corcione, F. Benvenuto, E. Ferretti, D. Giunti, V. Cappiello, F. Cazzanti, M. Risso, F. Gualandi, G.L. Mancardi, V. Pistoia, Human mesenchymal stem cells modulate B cell functions, Blood 107 (2006) 367–372. [9] M. Rafei, J. Hsieh, S. Fortier, M.Y. Li, S. Yuan, E. Birman, K. Forner, M.N. Boivin, K. Doody, M. Tremblay, B. Annabi, J. Galipeau, Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction, Blood 112 (2008) 4991–4998. [10] P. Comoli, F. Ginevri, R. Maccario, M.A. Avanzini, M. Marconi, A. Groff, A. Cometa, M. Cioni, L. Porretti, W. Barberi, F. Frassoni, F. Locatelli, Human mesenchymal stem cells inhibit antibody production induced in vitro by allostimulation, Nephrol. Dial. Transplant. 23 (2008) 1196–1202. [11] S. Tabera, J.A.P. Simón, M.D. Campelo, L.I.S. Abarca, B. Blanco, A. López, A. Benito, E. Ocio, F.M.S. Guijo, C. Cañizo, J.F.S. Miguel, The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes, Haematologica 93 (2008) 1301–1309. [12] S. Asaria, S. Itakuraa, K. Ferreria, C. Liub, Y. Kurodaa, F. Kandeela, Y. Mullen, Mesenchymal stem cells suppress B-cell terminal differentiation, Exp. Hematol. 37 (2009) 604–615. [13] F. Schena, C. Gambini, A. Gregorio, M. Mosconi, D. Reverberi, M. Gattorno, S. Casazza, A. Uccelli, L. Moretta, A. Martini, E. Traggiai, Interferon-c-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of Systemic Lupus Erythematosus, Arthritis. Rheum. 62 (2010) 2776–2786. [14] K. Stenderup, J. Justesen, C. Clausen, M. Kassem, Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells, Bone 33 (2003) 919–926. [15] S. Nishida, N. Endo, H. Yamagiwa, T. Tanizawa, H.E. Takahashi, Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation, J. Bone. Miner. Metab. 17 (1999) 171–177. [16] S.M. Mueller, J. Glowacki, Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges, J. Cell. Biochem. 82 (2001) 583–590. [17] H. Zhang, S. Fazel, H. Tian, D.A. Mickle, R.D. Weisel, T. Fujii, R.K. Li, Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy, Am. J. Physiol. Heart Circ. Physiol. 289 (2005) H2089–H2096. [18] A.J. Friedenstein, K.V. Petrakova, A.I. Kurolesova, G.P. Frolova, Heterotopic of bone marrow: Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation 6 (1968) 230–247. [19] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R.O. Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41–49. [20] A.J. Nauta, W.E. Fibbe, Immunomodulatory properties of mesenchymal stromal cells, Blood 110 (2007) 3499–3506. [21] M.L. Weiss, S. Medicetty, A.R. Bledsoe, R.S. Rachakatla, M. Choi, S. Merchav, Y. Luo, M.S. Rao, G. Velagaleti, D. Troyer, Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson0 s disease, Stem Cells 24 (2006) 781–792.
[22] S. Medicetty, A.R. Bledsoe, C.B. Fahrenholtz, D. Troyer, M.L. Weiss, Transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks, Exp. Neurol. 190 (2004) 32–41. [23] L.L. Lu, Y.J. Liu, S.G. Yang, Q.J. Zhao, X. Wang, W. Gong, Z.B. Han, Z.S. Xu, Y.X. Lu, D. Liu, Z.Z. Chen, Z.C. Han, Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials, Haematologica 91 (2006) 1017–1026. [24] G. Li, X.A. Zhang, H. Wang, X. Wang, C.L. Meng, C.Y. Chan, D.T.W. Yew, K.S. Tsang, K. Li, S.N. Tsai, S.M. Ngai, Z.C. Han, M.C.M. Lin, M.L. He, H.F. Kung, Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: implication in the migration, Proteomics 9 (2009) 20–30. [25] H. Cheng, L. Qiu, J. Ma, H. Zhang, M. Cheng, W. Li, X. Zhao, K. Liu, Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts, Mol. Biol. Rep. Epub ahead of print (2010). [26] M.Y. Chen, P.C. Lie, Z.L. Li, X. Wei, Endothelial differentiation of Wharton’s jelly-derived mesenchymal stem cells in comparison with bone marrowderived mesenchymal stem cells, Exp. Hematol. 37 (2009) 629–640. [27] K. Chen, D. Wang, W.T. Du, Z.B. Han, H. Ren, Y. Chi, S.G. Yang, D. Zhu, F. Bayard, Z.C. Han, Human umbilical cord mesenchymal stem cells hUC–MSCs exert immunosuppressive activities through a PGE2-dependent mechanism, Clin. Immunol. 135 (2010) 448–458. [28] A.J. Cutler, V. Limbani, J. Girdlestone, C.V. Navarrete, Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation, J. Immunol. 185 (2010) 6617–6623. [29] Y. Liu, R. Mu, S. Wang, L. Long, X. Liu, R. Li, J. Sun, J. Guo, X. Zhang, J. Guo, P. Yu, C. Li, X. Liu, Z. Huang, D. Wang, H. Li, Z. Gu, B. Liu, Z. Li, Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis, Arthritis. Res. Ther. 12 (2010) R210. [30] L. Sun, D. Wang, J. Liang, H. Zhang, X. Feng, H. Wang, B. Hua, B. Liu, S. Ye, X. Hu, W. Xu, X. Zeng, Y. Hou, G.S. Gilkeson, R.M. Silver, L. Lu, S. Shi, Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus, Arthritis. Rheum. 62 (2010) 2467–2475. [31] E. Traggiai, S. Volpi, F. Schena, M. Gattorno, F. Ferlito, L. Moretta, A. Martini, Bone marrow-derived mesenchymal stem cells induce both polyclonal expansion and differentiation of B cells isolated from healthy donors and systemic lupus erythematosus patients, Stem cells 26 (2008) 562–569. [32] I. Rasmusson, K.L. Blanc, B. Sundberg, O. Ringdén, Mesenchymal stem cells stimulate antibody secretion in human B cells, Scand. J. Immunol. 65 (2007) 336–343. [33] E. Zappia, S. Casazza, E. Pedemonte, F. Benvenuto, I. Bonanni, E. Gerdoni, D. Giunti, A. Ceravolo, F. Cazzanti, F. Frassoni, G. Mancardi, A. Uccelli, Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy, Blood 106 (2005) 1755–1761. [34] Y. Lin, K. Wong, K. Calame, Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation, Science 276 (1997) 596–599. [35] A.M. Reimold, N.N. Iwakoshi, J. Manis, P. Vallabhajosyula, E.S. Tsuda, E.M. Gravallese, D. Friend, M.J. Grusby, F. Alt, L.H. Glimcher, Plasma cell differentiation requires the transcription factor XBP-1, Nature 412 (2001) 300–307. [36] M. Horcher, A. Souabni, M. Busslinger, Pax5/BSAP maintains the identity of B cells in late B lymphopoiesis, Immunity 14 (2001) 779–790. [37] A.L. Dent, A.L. Shaffer, X. Yu, D. Allman, L.M. Staudt, Control of inflammation, cytokine expression, and germinal center formation by BCL-6, Science 276 (1997) 589–592. [38] K.P. Nera, P. Kohonen, E. Narvi, A. Peippo, L. Mustonen, P. Terho, K. Koskela, J.M. Buerstedde, O. Lassila, Loss of Pax5 promotes plasma cell differentiation, Immunity 24 (2006) 283–293. [39] A. Delogu, A. Schebesta, Q. Sun, K. Aschenbrenner, T. Perlot, M. Busslinger, Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells, Immunity 24 (2006) 269–281. [40] B.H. Ye, G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. Waard, C. Leung, M.N. Shirazi, A. Orazi, R.S. Chaganti, P. Rothman, A.M. Stall, P.P. Pandolfi, R.D. Favera, The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation, Nat. Genet. 16 (1997) 161–170. [41] T. Fukuda, T. Yoshida, S. Okada, M. Hatano, T. Miki, K. Ishibashi, S. Okabe, H. Koseki, S. Hirosawa, M. Taniguchi, N. Miyasaka, T. Tokuhisa, Disruption of the Bcl6 gene results in an impaired germinal center formation, J. Exp. Med. 186 (1997) 439–448. [42] M.S. Shelef, K. Calame, Regulation of plasma-cell development, Nat. Rev. Immunol. 5 (2005) 230–242. [43] P. Hess, G. Pihan, C.L. Sawyers, R.A. Flavell, R.J. Davis, Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts, Nat. Genet. 32 (2002) 201–205. [44] F.H. Vasanwala, S. Kusam, L.M. Toney, A.L. Dent, Repression of AP-1 function: a mechanism for the regulation of Blimp-1 expression and B lymphocyte differentiation by the B cell lymphoma-6 protooncogene, J. Immunol. 169 (2002) 1922–1929. [45] Y. Ohkubo, M. Arima, E. Arguni, S. Okada, K. Yamashita, S. Asari, S. Obata, A. Sakamoto, M. Hatano, J.O. Wang, M. Ebara, H. Saisho, T. Tokuhisa, A role for cfos/activator protein 1 in B lymphocyte terminal differentiation, J. Immunol. 174 (2005) 7703–7710. [46] C. Tunyaplin, A.L. Shaffer, C.D. Angelin-Duclos, X. Yu, L.M. Staudt, K.L. Calame, Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation, J. Immunol. 173 (2004) 1158–1165.
N. Che et al. / Cellular Immunology 274 (2012) 46–53 [47] Y. Aiba, T. Yamazaki, T. Okada, K. Gotoh, H. Sanjo, M. Ogata, T. Kurosaki, BANK negatively regulates Akt activation and subsequent B cell responses, Immunity 24 (2006) 259–268. [48] J. Kim, H.Y. Yang, Y.S. Jang, A G-protein-associated ERK pathway is involved in LPS-induced proliferation and a PTK-associated p38 MAPK pathway is
53
involved in LPS-induced differentiation in resting mB cells, Mol. Immunol. 43 (2006) 1232–1242. [49] A. Ertesvag, H.C. Aasheim, S. Naderi, H.K. Blomhoff, Vitamin A potentiates CpGmediated memory B-cell proliferation and differentiation: involvement of early activation of p38 MAPK, Blood 109 (2007) 3865–3872.