- Email: [email protected]
Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors
Cytokine xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors☆ ⁎
Wojciech Fidyk , Iwona Mitrus, Agnieszka Ciomber, Andrzej Smagur, Agata Chwieduk, Magdalena Głowala-Kosińska, Sebastian Giebel Department of Bone Marrow Transplantation and Onco-Hematology, Maria Sklodowska-Curie Institute Oncology Center, Gliwice Branch, 44-101 Gliwice, Wybrzeże Armii Krajowej 15 Street, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Immune privilege CD47 CD274 Cytokine levels Bone marrow Stem cells
Introduction: Cytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. Methodology: Study consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. Results: The results has shown different composition of analyzed cytokines (IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF-β1 which possibly creates a more immunosuppressive environment. The IL-10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G-CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors.
1. Introduction The hematopoietic stem cells (HSCs) transplantation is an important tool in the treatment of hematological diseases often being the sole curative option for patients with leukemia, lymphoma or myeloma [1]. HSCs reside in bone marrow, but they are capable of migrating out of their niche and coming back [2]. HSCs for transplantation may be collected either directly from bone marrow cavities or from peripheral blood with the use of leukapheresis. The latter requires preceding stimulation with granulocyte - colony stimulating factor (G-CSF) which causes the release of HSCs from bone marrow to peripheral blood. Those two types of material are successfully used for transplantation. It is known that the source of stem cells may have an impact on outcome [3]. The reconstitution of hematopoiesis is usually more rapid after peripheral blood stem cells (PBSCs) transplantation. On the other hand
the PBSCs transplantation may lead to a higher risk of chronic graft versus host disease (cGVHD) [4]. It can be explained with different cellular composition (both in percentage and absolute volume) of transplantation material or with different properties of cells being transplanted. HSCs properties can be regulated by a variety of factors including the surrounding microenvironment, cell to cell signaling and interaction, cytokine and chemokine influence and many others. The microenvironment of the HSCs, including cytokine composition can have an impact on the state and properties of the cells and therefore on the properties of the transplantation material. It is believed, that HSCs in bone marrow are actively protected from toxic or infectious factors. This protective role of HSCs niche was studied by Fujisaki et al. They observed prolonged survival of allogeneic HSCs in the bone marrow of mice after HSC transplantation without immunosuppression [5]. Fujisaki et al. hypothesized that the HSC niche
Abbreviations: allo-HSCT, allogeneic hematopoietic stem cell transplantation; G-CSF, granulocyte - colony stimulating factor; PBSCs, peripheral blood stem cells; GVHD, graft-versushost disease; cGVHD, chronic graft-versus-host disease; IL, interleukin; Tregs, T regulatory lymphocytes; PD-L1, Programmed death ligand 1; TGF-β1, transforming growth factor β1; TNFα, tumor necrosis factor α; IFN-γ, interferon γ ☆ The project was supported by the Grant No. 2011/03/B/NZ6/04917 (National Science Centre, Poland). ⁎ Corresponding author. E-mail address: wojciech.fi[email protected] (W. Fidyk). http://dx.doi.org/10.1016/j.cyto.2017.09.001 Received 10 May 2017; Received in revised form 17 August 2017; Accepted 2 September 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Fidyk, W., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.09.001
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
within the bone marrow is an immune-privileged site [5,6]. Currently it is considered, that immune privileged places include the eyes, brain, placenta and testis [7,8]. These sites are able to tolerate the presence of antigen (like allogeneic graft), without the initiation of immune response. This phenomenon is an active process, related to the higher frequency of Tregs, increased concentration of immunosuppressive cytokines like TGF-β1, IL-4, IL-10, IL-12 and decreased level of proinflammatory cytokines (IL-1β, IL-6) [9]. It provides protection for the more valuable cells like HSCs. Despite of protection offered by microenvironment, HSCs themselves are able to inhibit attack of the immune system. Fujisaki et al. showed, that HSCs secrete IL-10, which is critical for Treg-mediated immunosuppression [5]. HSCs are also capable of direct interaction with the immune system cells, by altered expression of surface molecules [10]. CD47 is a protein, known as a “don't-eat-me” signal, because it inhibits host cell phagocytosis by binding the signal regulatory protein on macrophage surface [11]. CD274 (also called B7-H1or PD-L1 - programmed death ligand 1) downregulates several T lymphocytes functions such as proliferation and production of effector cytokines [12]. It promotes T-cell apoptosis and plays a potential role in inhibiting T cell immune response [13]. Probably the elevated expression of CD47 and CD274 on the surface of HSCs could be another evolutionary adaptation, which prevents potential damage of HSCs by immune system. Protection mechanisms of HSCs in bone marrow are still under investigation and we believe it could be an important aspect of HSCs transplantation. According to our best knowledge, this is first study of differences in protection mechanisms between HSCs collected by two different ways: peripheral blood stem cells (PBSCs) collected by leukapheresis procedure and HSCs collected directly from bone marrow cavities. The main aim of this study was to compare concentration of selected pro- and anti-inflammatory cytokines and also the expression of immunosuppressive CD47 and CD274 proteins on HCSs from PBSCs and bone marrow of healthy donors.
Table 1 Samples collected for cytokine concentration analysis. Type of Donor
PBSCs donors
Bone marrow donors
Material
Whole blood sample taken before G-CSF stimulation and on the day of leukapheresis 28
Bone marrow sample taken at the start of the donation 23
28 (20–47)
23 (19–40)
0.33
22/6
12/11
0.11
Number of samples Median age of donors (range) Sex male/female
p
Table 2 Samples collected for flow cytometry analysis. Type of Donor
PBSCs donors
Bone marrow donors
p
Material
Whole blood sample taken on the day of leukapheresis
Number of samples Median age of donors (range) Sex Male/Female
16
Bone marrow sample taken at the start of the donation 18
27.5 (20–37)
25 (20–45)
0.34
9/7
13/5
0.29
manufacturer’s manual (to increase assay sensitivity, the samples were incubated with capture antibody overnight – 16–18 h). After collection samples of blood and bone marrow were centrifuged at 500 ×g for 20 min. Next, aliquots of plasma samples, were divided into portions and immediately stored in liquid nitrogen until the time of analysis. Synergy 2 Multi-Mode Microplate Readers (BioTek, Winooski, USA) was used for the analysis.
2. Material and methods 2.3. Flow cytometry analyses
2.1. Sampling methodology
The flow cytometry analysis of CD47 and CD274 expression on HSCs was performed immediately after PBSCs or bone marrow collection. Whole blood cells were incubated for 20 min., at room temperature, with appropriate antibodies. To remove erythrocytes, the cells were incubated with BD Pharm Lyse™ lysing buffer (BD Biosciences, San Jose, CA, USA). After washing in Cell Wash Buffer (BD Biosciences, San Jose, CA, USA), the cells were suspended in Cell Wash Buffer and analyzed using fluorocytometer FACS Canto (BD Biosciences, San Jose, CA, USA). The subpopulations of HSCs (CD45+CD34+ phenotype) showing high expression of CD274 or CD47 markers were identified using CD34PE, CD45-PerCP, CD47-Alexa Fluor 647, CD274-FITC antibodies (all antibodies obtained from BD Biosciences, San Jose, CA, USA).
All samples were obtained from healthy donors of HSCs. We have collected samples from two groups of donors: PBSCs donors and bone marrow donors. All of the donors are thoroughly examined for any past and present medical conditions and also for any form of taken medication. All of the donors were in full health and were not taking any form of medication. PBSCs donors prior to leukapheresis were stimulated with G-CSF (granulocyte colony stimulating factor) 10 µg/kg/day for 4 consecutive days. Whole blood samples were collected two times from each donor. First sample was taken before the G-CSF stimulation, the second sample was taken on the day of leukapheresis (before the procedure). Bone marrow samples were collected at the start of the bone marrow donation. The first syringe (approx. 2 ml) was taken for the analysis to minimize the possible dilution caused by peripheral blood. All samples were collected into EDTA vacutainer tubes (BectonDickinson, New Jersey, USA). They were used to evaluate cytokine concentration by ELISA test and immunosuppressive protein expression on HSCs by FACS analysis. A characteristic of groups of donors is shown in Tables 1 and 2. Permission for studies was obtained from the local Bioethical Committee (Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland).
2.4. Statistical analysis U Mann–Whitney test was used to evaluate differences between concentration of cytokines in the whole blood and bone marrow samples and also to compare expression of CD47 and CD274 on HSCs. Wilcoxon test was used to evaluate differences between concentration of cytokines in peripheral blood of donors prior to and past the mobilization. Patients’ characteristics were compared using the U Mann–Whitney test.
2.2. Determination of cytokines concentration The levels of IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α were analyzed. All cytokines concentrations were measured using ELISA kits (eBioscience, San Diego, USA) according to the 2
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
state condition (p = 0.023). The level of IL-10 prior to mobilization was significantly higher in blood compared to bone marrow: 12.98 pg/ ml (0.31–82.32) vs 3.15 pg/ml (0–75.03) respectively (p = 0.025). Other cytokines analyzed were very often non-detectable or present at very low concentrations (IL-1β, IL-2, IL-4, IL17A, TNF-α). IFN-γ and TGF-β1 were detected in all samples but their level has not changed after the G-CSF mobilization.
Table 3 Cytokine concentrations in the bone marrow and peripheral blood after mobilization [Values in pg/ml, median and range]. Cytokine
Peripheral blood
Bone marrow
p
IL-10 IFN-γ IL-2 TGF-β1 IL-1β TNF-α IL-17 a IL-4 IL-6
27.86 (10.13–110.19) 38.01 (9.76–79.15) 3.39 (0.84–4.72) 30.91 (0–164.13) 3.34 (0–7.5) 0.35 (0–7.86) 0 (0–7.03) 1.14 (0–2.08) 7.14 (2.22–37.94)
3.15 (0–75.03) 9.86 (4.57–462.86) 0 (0–12.21) 1697.76 (642.51–24 000) 0 (0–4.6) 0 (0–6.48) 1.54 (0–6.48) 0.64 (0–9.44) 0 (0–72.16)
0.0009* 0.011* 0.038* 0.0002* 0.025* 0.066 0.349 0.943 0.446
3.3. The expression of CD47 and CD274 proteins on HSCs in bone marrow and peripheral blood Approximately 1x105 cells were analyzed for each sample. The average levels of CD34+ cells was 1.0% vs 0.22% in bone marrow and peripheral blood after mobilization respectively. The percentage of CD47+ HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors – (median 88.15% vs 50.35% respectively, p = 0.0001) (Fig. 1). The expression of CD274 was not detectable in the majority of HSCs (median CD274+ HSCs 0% both for bone marrow and peripheral blood).
Bone marrow samples were taken from healthy donors at the start of donation. Peripheral blood samples were collected on the day of leukapheresis from PBSC donors. Concentrations of cytokines were evaluated with an ELISA test. Statistically significant differences obtained with the U-Mann Whitney Test are marked with asterisks (*p < 0.05).
3. Results
4. Discussion
3.1. Cytokine concentrations in bone marrow and peripheral blood after mobilization
HSCs reside in bone marrow, but they are capable of migrating out of their niche and coming back. Migration of HSCs from the bone marrow into circulation following a stimulation is termed mobilization. In clinical practice, this phenomenon is used to obtain cells for transplantation. Administration of the G-CSF is a standard way of HSCs mobilization in healthy donors [14]. This allows for relatively simple harvest of HSCs from peripheral blood by leukapheresis. The other possibility utilizes direct bone marrow harvest from bones (specifically iliac crest) performed in an operating room. Those two totally different ways of collecting transplantation material provide a unique opportunity to compare them and investigate the environment in which the HSCs reside during collection. In our study we have decided to compare the concentration of selected cytokines and expression of immunosuppressive proteins on HSCs between blood from PBSCs donors and bone marrow of donors. In the first part of our research, we have determined the levels of selected cytokines, in peripheral blood (after the mobilization with GCSF) and bone marrow of healthy donors. The concentrations of cytokines in bone marrow of healthy people are poorly researched. We assumed that the cytokine levels in our samples will provide some insight into the state of HSCs present in different localizations. In our research, nearly half of the analyzed cytokines concentrations (IL-4, IL6, IL-17a, TNF-α) were comparable in both localizations, providing no additional information on their possible influence on HSCs. However we have observed significantly higher concentrations of three proinflammatory cytokines (IFN-γ, IL-2 and IL-1β) and one immunosuppressive cytokine (IL-10) in peripheral blood of mobilized donors relative to bone marrow. In contrast, the concentration of TGFβ1 was higher in bone marrow compared to peripheral blood. TGF-β1 is secreted mainly by regulatory T cells to inhibit the hostile activity of other T cells and also to prevent activation of T helper cells and cytotoxic T cells [15]. Previous studies has shown that the bone marrow can harbor high number of Treg cells [16] to prevent possible inflammation of this crucial organ [17]. TGF-β1 is also known to block the secretion of several proinflammatory cytokines. High concentrations of TGF-β1 in bone marrow (over 50-fold higher than in peripheral blood) confirms the immunosuppressive properties of HSCs microenvironment. We theorize that the elevated level of TGF-β1 can act as a protective factor for the HSCs residing in bone marrow, ensuring their survival from possible lymphocyte attack and thus contributing towards the immune privileged state. The increased level of TGF-β1 may be also the results of its potential to maintain stemness and quiescence of HSCs [18]. IFN-γ is generally perceived as a strongly proinflammatory
We have analyzed concentration of nine cytokines in both bone marrow and peripheral blood samples after the administration of GCSF. The results of analysis are presented in the Table 3. The levels of IL-10, IFN-gamma, IL-2 and IL-1β were higher in peripheral blood of mobilized donors than in the bone marrow. The concentration of TGF-β1 was significantly higher in bone marrow than in peripheral blood after the mobilization. There were no differences in the concentration of IL-17A, IL-4, IL-6 and TNF-α between bone marrow and peripheral blood samples. The levels of these cytokines were very low or close to the detection limit. We have checked the possible correlations between cytokine levels, total white blood cells count and CD3+ or CD34+ cells percentages. We have not found any statistically significant associations (data not shown). 3.2. Cytokine concentration in peripheral blood prior to and after the mobilization with G-CSF We have analyzed concentration of the same nine cytokines in peripheral blood samples before and after the mobilization procedure. The results of the analysis are presented in Table 4. We have observed increased level of IL-10 in donors’ blood in the samples collected after G-CSF was administered compared to steady Table 4 Cytokine concentrations in peripheral blood of HSCs donors before and after the mobilization with G-CSF. [Values in pg/ml, median and range]. Peripheral blood Cytokine
Before mobilization
After mobilization
p
IL-10 IFN-γ IL-2 TGF-β1 IL-1β TNF-α IL-17a IL-4 IL-6
12.98 (0.31–82.32) 50.48 (21.4–187.81) 2.40 (1.92–3.74) 36.01 (0–1693.62) 2.04 (0.27–20.32) 0.62 (0–11.62) 1.34 (0–4.53) 0.87 (0–1.8) 6.36 (2.1–30.08)
27.86 (10.12–110.18) 38.01 (9.76–79.15) 3.40 (0.85–4.72) 30.91 (0–164.13) 3.34 (0–7.5) 0.35 (0–7.86) 0 (0–7.04) 1.14 (0–2.08) 7.14 (2.22–37.95)
0.023* 0.6 0.224 0.286 0.779 0.6 0.236 0.44 0.789
Peripheral blood samples were collected from healthy donors of PBSC before and after mobilization with G-CSF. Concentrations of cytokines were evaluated with an ELISA test. Statistically significant differences obtained with the Wilcoxon Test are marked with asterisks (*p < 0.05).
3
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
Fig. 1. (A) Expression of CD47 protein on HSCs in bone marrow and blood. CD47 and CD274 proteins dot plot on CD34 + cells in bone marrow (B) and peripheral blood (C) evaluated by flow cytometry. Bone marrow samples were taken from healthy donors at the start of donation. Peripheral blood samples were collected on the day of leukapheresis from PBSC donors. Expression of CD47 on HSCs was evaluated by flow cytometry. Statistically significant differences were determined with the Mann-Whitney U test (p = 0.0001).
produce IL-1β why is it nearly non detectable in bone marrow. It can be explained by the lack of a proper stimuli or by the fact of very high level of TGF-β1 as described above. It is possible that the levels of proinflammatory cytokines in bone marrow are generally lower than in peripheral blood but the levels of these cytokines may be also derivative of the TGF-β1 concentration, as it may directly inhibit the secretion of IFN-γ, IL-2 and IL-1β specifically when present in such high concentrations. We have shown that the level of IL-10 in peripheral blood (prior to mobilization) is significantly higher than in bone marrow. It may be associated with the higher levels of Treg cells present in circulation of donors mobilized with G-CSF as described by Chevallier et al. [20].The presence of activated Treg cells is strongly associated with the level of IL-10 [23]. In our results we have shown that the administration of GCSF further increases the level of IL-10. It is highly probable that the increased level of IL-10 in peripheral blood after the mobilization is directly associated to the presence of large amount of injected G-CSF and acts as an counter-measure to inhibit the production of additional G-CSF as it was previously described by Raychaudhuri et al. [24]. To
cytokine. It is essential for the anti-microbial and anti-tumor response, stimulates the development of cytotoxic T lymphocytes, improves antigen presentation and many more [19]. IL-2 is produced by activated CD4+ cells after the appropriate antigen stimulation but its elevated level can also be explained by the nearly 2-fold higher amount of dendritic cells in PBSCs [20]. Dendritic cells subsets 1 and 2 can initiate the differentiation of T cells into Th1 and Th2 subsets respectively. Th1 cells are known to secrete a number of proinflammatory cytokines. Their primary product after the differentiation is IL-2 [21]. IL-1β is a member of IL-1 superfamily of cytokines responsible for immediate inflammatory response. Its most known functions are to enhance inflammatory state by promoting the production of other inflammatory cytokines. There are studies describing HSCs collected from the cord blood to be able to secrete IL-1β [22]. The reason for this is not entirely clear. It is a fact that the IL-1β can induce differentiation of HSCs into myeloid lineage. It is possible that the elevated level of IL-1β in peripheral blood can be associated with the higher levels of other proinflammatory cytokines. The question remains that if the HSCs can 4
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
HSCs both, collected directly from bone marrow and harvested by the means of leukapheresis are widely used for transplantation. Both materials are utilized in treatment of various hematological malignancies and are used uniformly as a source of hematopoietic stem cells. PBSCs harvest is the most frequent choice due to the simpler procedure and smaller amount of stress the donor undergoes. So far, there were no papers describing and comparing the cytokine composition regarding these two transplantation materials. Results of our study indicate that the cytokine composition of PBSCs vs bone marrow is different. White blood cells present in G-CSF mobilized peripheral blood are exposed to higher concentration of proinflammatory cytokines in comparison to bone marrow cells, which may augment their potential alloreactivity. This mechanism may contribute to increased risk of GVHD reported after PBSCs transplantations [4]. On the other hand, we demonstrated that CD34+ cells derived from bone marrow are characterized by increased expression of CD47. It is possible it may favor their engraftment. Altogether, our findings give insight into the biology of hematopoietic stem cell transplantation with respect to the source of stem cells.
rule out that possibility the experiments should be repeated on donors mobilized with other mobilizing factor. Some are currently being developed like, GRO-βT and GRO-β. They are known to operate differently from the G-CSF (possibly holding different impact on cytokine levels) [25], Those mobilizing factors are not routinely used in healthy donors mobilization procedure, so we are unable to confirm, whether the effect of elevated IL-10 level is specific solely for G-CSF. There was evidence suggesting the presence of feedback loop between the donors injected with G-CSF and their IL-10 level in peripheral blood. We have checked the concentrations of selected cytokines in peripheral blood before and after the administration with G-CSF. The results showed no significant change in cytokine levels with an exception of IL-10 concentration. IL-10 level rose from a median of 12.98 pg/ ml up to 27.86 pg/ml, (p = 0.023). It could prove the connection of IL10 levels with administration of G-CSF in blood and therefore not to be connected with the presence of HSCs. On the other hand G-CSF not only mobilizes HSC into the periphery but also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. G-CSF also mobilizes mature DCs, pDC1 cells, and pDC2 cells [26]. The increased numbers of DC could there correlate to the increased levels of IL-10 expression in peripheral blood of G-CSF treated recipients. In the second part of our research, we analyzed the expression of CD274 and CD47 on HSCs. Both proteins have immunosuppressive properties. CD47 is a membrane protein that interacts with the receptor SIRPα on macrophage surface and inhibits phagocytosis. CD47 is expressed in various types of cells, including e.g. erythrocytes and tumor cells [27,28]. This protein is also constitutively upregulated on mouse and human leukemia cells – probably as a mechanism which allows cancer cells to avoid destruction by macrophages. [29,30]. CD274 prevents destruction of cells by cytotoxic lymphocytes, via inducing the apoptosis pathway in this cells. This protein is abnormally expressed on many tumor cells, and play a major role in suppressing the immune system [10]. In our study we measured expression of immunosuppressive proteins on HSCs in blood (after mobilization) and in bone marrow (from donors of bone marrow). The percentage of CD47 positive HSCs was significant higher in bone marrow than in blood. These results are different than those obtained by Jaiswal. In our study, the level of CD47 decreased, when HSCs migrated outside of the bone marrow, but Jaiswal reported, that the expression of CD47 on HSCs is transiently upregulated during mobilization. Possible explanation to these contradictory observations is that Jaiswal investigated the most primitive HSCs (CD90+ CD34+ CD38- Lin-) – in our research we decided to check expression on all CD34 positive cells (not only most primitive, but progenitors as well). The level of CD274 positive HSCs was low (< 1% of cells) and similar in both localizations. Expression of CD274 and CD47 on HSCs may be compared with result published by Zheng et al. [2]. This group tested multiple markers on mouse HSCs, including MHC-I, MHC-II, CD274, CD275, CD47, and CD86. The level of CD274 was low, but in vitro culturing led to an increase in its expression. Authors demonstrated, that upregulation of CD274 during culture supports HSC allograft in mice. Next, they measured level of this protein on human HSCs, obtained from human cord blood. About 10% of freshly isolated HSCs expressed CD274 on their surface, and – as in the case of mouse cells – in vitro conditions induced elevated expression of this protein [31]. We observed similar effect in HSC culturing (data not shown). It is possible that CD47 and CD274 proteins can provide additional protection to the HSCs, mainly from the hostile activity of the immune system. There is also the evidence that suggested the correlation between the expression of CD47 and concentration of TGF-B1 [32]. TGF-B1 expression is regulated by the binding of CD47. Moreover CD47 expression reduces the maturation of DC cells [33]. The decreased expression of CD47 in the periphery would therefore allow maturation of DC's and may explain the reduced concentration of TGF-B1 in the peripheral blood.
References [1] J.R. Passweg, J. Halter, C. Bucher, S. Gerull, D. Heim, A. Rovó, et al., Hematopoietic stem cell transplantation: a review and recommendations for follow-up care for the general practitioner, Swiss. Med. Wkly. 142 (2012) w13696, http://dx.doi.org/10. 4414/smw.2012.13696. [2] J. Zheng, M. Umikawa, S. Zhang, H. Huynh, R. Silvany, B.P.C. Chen, et al., Ex vivo expanded hematopoietic stem cells overcome the mhc barrier in allogeneic transplantation, Cell Stem Cell 9 (2011) 119–130, http://dx.doi.org/10.1016/j.stem. 2011.06.003. [3] I. Sirinoglu Demiriz, E. Tekgunduz, F. Altuntas, What is the most appropriate source for hematopoietic stem cell transplantation? peripheral stem cell/bone marrow/ cord blood, Bone Marrow Res. 2012 (2012) 1–5, http://dx.doi.org/10.1155/2012/ 834040. [4] C. Anasetti, B.R. Logan, S.J. Lee, E.K. Waller, D.J. Weisdorf, J.R. Wingard, et al., Peripheral-blood stem cells versus bone marrow from unrelated donors, N. Engl. J. Med. 367 (2012) 1487–1496, http://dx.doi.org/10.1056/NEJMoa1203517. [5] J. Fujisaki, J. Wu, A.L. Carlson, L. Silberstein, P. Putheti, R. Larocca, et al., In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche, Nature 474 (2011) 216–219, http://dx.doi.org/10.1038/nature10160. [6] T. Reya, Illuminating Immune privilege — a role for regulatory T cells in preventing rejection, N Engl J Med 365 (2011) 956–957, http://dx.doi.org/10.1056/ NEJMcibr1107781. [7] P. Arck, M.E. Solano, M. Walecki, A. Meinhardt, The immune privilege of testis and gravid uterus: same difference? Mol. Cell. Endocrinol. 382 (2014) 509–520, http:// dx.doi.org/10.1016/j.mce.2013.09.022. [8] A. Louveau, T.H. Harris, J. Kipnis, Revisiting the mechanisms of CNS immune privilege, Trends. Immunol. 36 (2015) 569–577, http://dx.doi.org/10.1016/j.it. 2015.08.006. [9] S. Zhao, W. Zhu, S. Xue, D. Han, Testicular defense systems: immune privilege and innate immunity, Cell. Mol. Immunol. 11 (2014) 428–437, http://dx.doi.org/10. 1038/cmi.2014.38. [10] C.C. Zhang, Hematopoietic stem cells: interplay with immunity, Am. J. Blood Res. 2 (2012) 219–227. [11] M.P. Chao, I.L. Weissman, R. Majeti, The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications, Curr. Opin. Immunol. 24 (2012) 225–232, http://dx.doi.org/10.1016/j.coi.2012.01.010. [12] W. Li, X. Wang, R. Chen, H. Zhu, G. Chen, X. Sun, Overexpression of programmed death ligand 1 in dendritic cells inhibits allogeneic lymphocyte activation in mice, J. Surg. Res. 176 (2012) e79–e87, http://dx.doi.org/10.1016/j.jss.2011.12.009. [13] P. Greaves, J.G. Gribben, The role of B7 family molecules in hematologic malignancy, Blood 121 (2013) 734–744, http://dx.doi.org/10.1182/blood-2012-10385591. [14] R.K. Hopman, J.F. DiPersio, Advances in stem cell mobilization, Blood Rev. 28 (2014) 31–40, http://dx.doi.org/10.1016/j.blre.2014.01.001. [15] K.M. Gilbert, M. Thoman, K. Bauche, T. Pham, W.O. Weigle, Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells, Immunol. Invest. 26 (1997) 459–472. [16] L. Zou, B. Barnett, H. Safah, V.F. LaRussa, M. Evdemon-Hogan, P. Mottram, et al., Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals, Cancer Res. 64 (2004) 8451, http://dx.doi.org/10.1158/ 0008-5472.CAN-04-1987. [17] E. Zhao, L. Wang, J. Dai, I. Kryczek, S. Wei, L. Vatan, et al., Regulatory T cells in the bone marrow microenvironment in patients with prostate cancer, OncoImmunology 1 (2012) 152–161, http://dx.doi.org/10.4161/onci.1.2.18480. [18] S. Yamazaki, A. Iwama, S -i. Takayanagi, K. Eto, H. Ema, H. Nakauchi, TGF- as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation, Blood (2009) 113 1250–1256. doi:10.1182/blood-2008-04-146480. [19] Y.-G. Yang, H. Wang, W. Asavaroengchai, B.R. Dey, Role of Interferon-gamma in
5
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al. GVHD and GVL, Cell. Mol. Immunol. 2 (2005) 323–329. [20] P. Chevallier, N. Robillard, M. Illiaquer, J. Esbelin, M. Mohty, C. Bodin-Bressollette, et al., Characterization of various blood and graft sources: a prospective series: characterization of various blood and graft sources, Transfusion (Paris) 53 (2013) 2020–2026, http://dx.doi.org/10.1111/trf.12072. [21] S.L. Gaffen, K.D. Liu, Overview of interleukin-2 function, production and clinical applications, Cytokine 28 (2004) 109–123, http://dx.doi.org/10.1016/j.cyto.2004. 06.010. [22] K. Watari, H. Mayani, F. Lee, W. Dragowska, P.M. Lansdorp, J.W. Schrader, Production of interleukin 1beta by human hematopoietic progenitor cells, J. Clin. Invest. 97 (1996) 1666–1674, http://dx.doi.org/10.1172/JCI118593. [23] C.A. Stewart, H. Metheny, N. Iida, L. Smith, M. Hanson, F. Steinhagen, et al., Interferon-dependent IL-10 production by Tregs limits tumor Th17 inflammation, J. Clin. Invest. 123 (2013) 4859–4874, http://dx.doi.org/10.1172/JCI65180. [24] B. Raychaudhuri, C.J. Fisher, C.F. Farver, A. Malur, J. Drazba, M.S. Kavuru, et al., Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages, Cytokine 12 (2000) 1348–1355, http://dx.doi.org/ 10.1006/cyto.2000.0721. [25] B . Suárez-Álvarez, A. López-Vázque, C. López-Larrea , Mobilization and Homing of Hematopoietic Stem Cells, In: C. López-Larrea, A. López-Vázque, B. Suárez-Álvarez, editors. Stem Cell Transplant., vol. 741, New York, NY: Springer US; 2012, p. 152–70. [26] P.J. Shaughnessy, C. Bachier, C.F. Lemaistre, C. Akay, B.H. Pollock, Y. Gazitt, Granulocyte colony-stimulating factor mobilizes more dendritic cell subsets than granulocyte-macrophage colony-stimulating factor with no polarization of dendritic
[27]
[28]
[29]
[30]
[31]
[32]
[33]
6
cell subsets in normal donors, Stem. Cells Dayt. Ohio. 24 (2006) 1789–1797, http:// dx.doi.org/10.1634/stemcells.2005-0492. S. Jaiswal, M.P. Chao, R. Majeti, I.L. Weissman, Macrophages as mediators of tumor immunosurveillance, Trends. Immunol. 31 (2010) 212–219, http://dx.doi.org/10. 1016/j.it.2010.04.001. N. Navarro-Alvarez, Y.-G. Yang, CD47: a new player in phagocytosis and xenograft rejection, Cell. Mol. Immunol. 8 (2011) 285–288, http://dx.doi.org/10.1038/cmi. 2010.83. S. Jaiswal, C.H.M. Jamieson, W.W. Pang, C.Y. Park, M.P. Chao, R. Majeti, et al., CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis, Cell 138 (2009) 271–285, http://dx.doi.org/10.1016/j.cell. 2009.05.046. H. Jiang, R. Fu, H. Wang, L. Li, H. Liu, Z. Shao, CD47 is expressed abnormally on hematopoietic cells in myelodysplastic syndrome, Leuk. Res. 37 (2013) 907–910, http://dx.doi.org/10.1016/j.leukres.2013.04.008. J. Zheng, C. Song, C.C. Zhang, A new chapter: hematopoietic stem cells are direct players in immunity, Cell Biosci. 1 (2011) 33, http://dx.doi.org/10.1186/20453701-1-33. K. Shimada, A. Nakajima, K. Ikeda, K. Ishibashi, N. Shimizu, K. Ito, CD47 regulates the TGF-β signaling pathway in osteoblasts and is distributed in Meckel’s cartilage, J. Oral Sci. 53 (2011) 169–175. V. Doyen, M. Rubio, D. Braun, T. Nakajima, J. Abe, H. Saito, et al., Thrombospondin 1 is an autocrine negative regulator of human dendritic cell activation, J. Exp. Med. 198 (2003) 1277–1283, http://dx.doi.org/10.1084/jem.20030705.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors☆ ⁎
Wojciech Fidyk , Iwona Mitrus, Agnieszka Ciomber, Andrzej Smagur, Agata Chwieduk, Magdalena Głowala-Kosińska, Sebastian Giebel Department of Bone Marrow Transplantation and Onco-Hematology, Maria Sklodowska-Curie Institute Oncology Center, Gliwice Branch, 44-101 Gliwice, Wybrzeże Armii Krajowej 15 Street, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Immune privilege CD47 CD274 Cytokine levels Bone marrow Stem cells
Introduction: Cytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. Methodology: Study consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. Results: The results has shown different composition of analyzed cytokines (IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF-β1 which possibly creates a more immunosuppressive environment. The IL-10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G-CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors.
1. Introduction The hematopoietic stem cells (HSCs) transplantation is an important tool in the treatment of hematological diseases often being the sole curative option for patients with leukemia, lymphoma or myeloma [1]. HSCs reside in bone marrow, but they are capable of migrating out of their niche and coming back [2]. HSCs for transplantation may be collected either directly from bone marrow cavities or from peripheral blood with the use of leukapheresis. The latter requires preceding stimulation with granulocyte - colony stimulating factor (G-CSF) which causes the release of HSCs from bone marrow to peripheral blood. Those two types of material are successfully used for transplantation. It is known that the source of stem cells may have an impact on outcome [3]. The reconstitution of hematopoiesis is usually more rapid after peripheral blood stem cells (PBSCs) transplantation. On the other hand
the PBSCs transplantation may lead to a higher risk of chronic graft versus host disease (cGVHD) [4]. It can be explained with different cellular composition (both in percentage and absolute volume) of transplantation material or with different properties of cells being transplanted. HSCs properties can be regulated by a variety of factors including the surrounding microenvironment, cell to cell signaling and interaction, cytokine and chemokine influence and many others. The microenvironment of the HSCs, including cytokine composition can have an impact on the state and properties of the cells and therefore on the properties of the transplantation material. It is believed, that HSCs in bone marrow are actively protected from toxic or infectious factors. This protective role of HSCs niche was studied by Fujisaki et al. They observed prolonged survival of allogeneic HSCs in the bone marrow of mice after HSC transplantation without immunosuppression [5]. Fujisaki et al. hypothesized that the HSC niche
Abbreviations: allo-HSCT, allogeneic hematopoietic stem cell transplantation; G-CSF, granulocyte - colony stimulating factor; PBSCs, peripheral blood stem cells; GVHD, graft-versushost disease; cGVHD, chronic graft-versus-host disease; IL, interleukin; Tregs, T regulatory lymphocytes; PD-L1, Programmed death ligand 1; TGF-β1, transforming growth factor β1; TNFα, tumor necrosis factor α; IFN-γ, interferon γ ☆ The project was supported by the Grant No. 2011/03/B/NZ6/04917 (National Science Centre, Poland). ⁎ Corresponding author. E-mail address: wojciech.fi[email protected] (W. Fidyk). http://dx.doi.org/10.1016/j.cyto.2017.09.001 Received 10 May 2017; Received in revised form 17 August 2017; Accepted 2 September 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Fidyk, W., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.09.001
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
within the bone marrow is an immune-privileged site [5,6]. Currently it is considered, that immune privileged places include the eyes, brain, placenta and testis [7,8]. These sites are able to tolerate the presence of antigen (like allogeneic graft), without the initiation of immune response. This phenomenon is an active process, related to the higher frequency of Tregs, increased concentration of immunosuppressive cytokines like TGF-β1, IL-4, IL-10, IL-12 and decreased level of proinflammatory cytokines (IL-1β, IL-6) [9]. It provides protection for the more valuable cells like HSCs. Despite of protection offered by microenvironment, HSCs themselves are able to inhibit attack of the immune system. Fujisaki et al. showed, that HSCs secrete IL-10, which is critical for Treg-mediated immunosuppression [5]. HSCs are also capable of direct interaction with the immune system cells, by altered expression of surface molecules [10]. CD47 is a protein, known as a “don't-eat-me” signal, because it inhibits host cell phagocytosis by binding the signal regulatory protein on macrophage surface [11]. CD274 (also called B7-H1or PD-L1 - programmed death ligand 1) downregulates several T lymphocytes functions such as proliferation and production of effector cytokines [12]. It promotes T-cell apoptosis and plays a potential role in inhibiting T cell immune response [13]. Probably the elevated expression of CD47 and CD274 on the surface of HSCs could be another evolutionary adaptation, which prevents potential damage of HSCs by immune system. Protection mechanisms of HSCs in bone marrow are still under investigation and we believe it could be an important aspect of HSCs transplantation. According to our best knowledge, this is first study of differences in protection mechanisms between HSCs collected by two different ways: peripheral blood stem cells (PBSCs) collected by leukapheresis procedure and HSCs collected directly from bone marrow cavities. The main aim of this study was to compare concentration of selected pro- and anti-inflammatory cytokines and also the expression of immunosuppressive CD47 and CD274 proteins on HCSs from PBSCs and bone marrow of healthy donors.
Table 1 Samples collected for cytokine concentration analysis. Type of Donor
PBSCs donors
Bone marrow donors
Material
Whole blood sample taken before G-CSF stimulation and on the day of leukapheresis 28
Bone marrow sample taken at the start of the donation 23
28 (20–47)
23 (19–40)
0.33
22/6
12/11
0.11
Number of samples Median age of donors (range) Sex male/female
p
Table 2 Samples collected for flow cytometry analysis. Type of Donor
PBSCs donors
Bone marrow donors
p
Material
Whole blood sample taken on the day of leukapheresis
Number of samples Median age of donors (range) Sex Male/Female
16
Bone marrow sample taken at the start of the donation 18
27.5 (20–37)
25 (20–45)
0.34
9/7
13/5
0.29
manufacturer’s manual (to increase assay sensitivity, the samples were incubated with capture antibody overnight – 16–18 h). After collection samples of blood and bone marrow were centrifuged at 500 ×g for 20 min. Next, aliquots of plasma samples, were divided into portions and immediately stored in liquid nitrogen until the time of analysis. Synergy 2 Multi-Mode Microplate Readers (BioTek, Winooski, USA) was used for the analysis.
2. Material and methods 2.3. Flow cytometry analyses
2.1. Sampling methodology
The flow cytometry analysis of CD47 and CD274 expression on HSCs was performed immediately after PBSCs or bone marrow collection. Whole blood cells were incubated for 20 min., at room temperature, with appropriate antibodies. To remove erythrocytes, the cells were incubated with BD Pharm Lyse™ lysing buffer (BD Biosciences, San Jose, CA, USA). After washing in Cell Wash Buffer (BD Biosciences, San Jose, CA, USA), the cells were suspended in Cell Wash Buffer and analyzed using fluorocytometer FACS Canto (BD Biosciences, San Jose, CA, USA). The subpopulations of HSCs (CD45+CD34+ phenotype) showing high expression of CD274 or CD47 markers were identified using CD34PE, CD45-PerCP, CD47-Alexa Fluor 647, CD274-FITC antibodies (all antibodies obtained from BD Biosciences, San Jose, CA, USA).
All samples were obtained from healthy donors of HSCs. We have collected samples from two groups of donors: PBSCs donors and bone marrow donors. All of the donors are thoroughly examined for any past and present medical conditions and also for any form of taken medication. All of the donors were in full health and were not taking any form of medication. PBSCs donors prior to leukapheresis were stimulated with G-CSF (granulocyte colony stimulating factor) 10 µg/kg/day for 4 consecutive days. Whole blood samples were collected two times from each donor. First sample was taken before the G-CSF stimulation, the second sample was taken on the day of leukapheresis (before the procedure). Bone marrow samples were collected at the start of the bone marrow donation. The first syringe (approx. 2 ml) was taken for the analysis to minimize the possible dilution caused by peripheral blood. All samples were collected into EDTA vacutainer tubes (BectonDickinson, New Jersey, USA). They were used to evaluate cytokine concentration by ELISA test and immunosuppressive protein expression on HSCs by FACS analysis. A characteristic of groups of donors is shown in Tables 1 and 2. Permission for studies was obtained from the local Bioethical Committee (Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland).
2.4. Statistical analysis U Mann–Whitney test was used to evaluate differences between concentration of cytokines in the whole blood and bone marrow samples and also to compare expression of CD47 and CD274 on HSCs. Wilcoxon test was used to evaluate differences between concentration of cytokines in peripheral blood of donors prior to and past the mobilization. Patients’ characteristics were compared using the U Mann–Whitney test.
2.2. Determination of cytokines concentration The levels of IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α were analyzed. All cytokines concentrations were measured using ELISA kits (eBioscience, San Diego, USA) according to the 2
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
state condition (p = 0.023). The level of IL-10 prior to mobilization was significantly higher in blood compared to bone marrow: 12.98 pg/ ml (0.31–82.32) vs 3.15 pg/ml (0–75.03) respectively (p = 0.025). Other cytokines analyzed were very often non-detectable or present at very low concentrations (IL-1β, IL-2, IL-4, IL17A, TNF-α). IFN-γ and TGF-β1 were detected in all samples but their level has not changed after the G-CSF mobilization.
Table 3 Cytokine concentrations in the bone marrow and peripheral blood after mobilization [Values in pg/ml, median and range]. Cytokine
Peripheral blood
Bone marrow
p
IL-10 IFN-γ IL-2 TGF-β1 IL-1β TNF-α IL-17 a IL-4 IL-6
27.86 (10.13–110.19) 38.01 (9.76–79.15) 3.39 (0.84–4.72) 30.91 (0–164.13) 3.34 (0–7.5) 0.35 (0–7.86) 0 (0–7.03) 1.14 (0–2.08) 7.14 (2.22–37.94)
3.15 (0–75.03) 9.86 (4.57–462.86) 0 (0–12.21) 1697.76 (642.51–24 000) 0 (0–4.6) 0 (0–6.48) 1.54 (0–6.48) 0.64 (0–9.44) 0 (0–72.16)
0.0009* 0.011* 0.038* 0.0002* 0.025* 0.066 0.349 0.943 0.446
3.3. The expression of CD47 and CD274 proteins on HSCs in bone marrow and peripheral blood Approximately 1x105 cells were analyzed for each sample. The average levels of CD34+ cells was 1.0% vs 0.22% in bone marrow and peripheral blood after mobilization respectively. The percentage of CD47+ HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors – (median 88.15% vs 50.35% respectively, p = 0.0001) (Fig. 1). The expression of CD274 was not detectable in the majority of HSCs (median CD274+ HSCs 0% both for bone marrow and peripheral blood).
Bone marrow samples were taken from healthy donors at the start of donation. Peripheral blood samples were collected on the day of leukapheresis from PBSC donors. Concentrations of cytokines were evaluated with an ELISA test. Statistically significant differences obtained with the U-Mann Whitney Test are marked with asterisks (*p < 0.05).
3. Results
4. Discussion
3.1. Cytokine concentrations in bone marrow and peripheral blood after mobilization
HSCs reside in bone marrow, but they are capable of migrating out of their niche and coming back. Migration of HSCs from the bone marrow into circulation following a stimulation is termed mobilization. In clinical practice, this phenomenon is used to obtain cells for transplantation. Administration of the G-CSF is a standard way of HSCs mobilization in healthy donors [14]. This allows for relatively simple harvest of HSCs from peripheral blood by leukapheresis. The other possibility utilizes direct bone marrow harvest from bones (specifically iliac crest) performed in an operating room. Those two totally different ways of collecting transplantation material provide a unique opportunity to compare them and investigate the environment in which the HSCs reside during collection. In our study we have decided to compare the concentration of selected cytokines and expression of immunosuppressive proteins on HSCs between blood from PBSCs donors and bone marrow of donors. In the first part of our research, we have determined the levels of selected cytokines, in peripheral blood (after the mobilization with GCSF) and bone marrow of healthy donors. The concentrations of cytokines in bone marrow of healthy people are poorly researched. We assumed that the cytokine levels in our samples will provide some insight into the state of HSCs present in different localizations. In our research, nearly half of the analyzed cytokines concentrations (IL-4, IL6, IL-17a, TNF-α) were comparable in both localizations, providing no additional information on their possible influence on HSCs. However we have observed significantly higher concentrations of three proinflammatory cytokines (IFN-γ, IL-2 and IL-1β) and one immunosuppressive cytokine (IL-10) in peripheral blood of mobilized donors relative to bone marrow. In contrast, the concentration of TGFβ1 was higher in bone marrow compared to peripheral blood. TGF-β1 is secreted mainly by regulatory T cells to inhibit the hostile activity of other T cells and also to prevent activation of T helper cells and cytotoxic T cells [15]. Previous studies has shown that the bone marrow can harbor high number of Treg cells [16] to prevent possible inflammation of this crucial organ [17]. TGF-β1 is also known to block the secretion of several proinflammatory cytokines. High concentrations of TGF-β1 in bone marrow (over 50-fold higher than in peripheral blood) confirms the immunosuppressive properties of HSCs microenvironment. We theorize that the elevated level of TGF-β1 can act as a protective factor for the HSCs residing in bone marrow, ensuring their survival from possible lymphocyte attack and thus contributing towards the immune privileged state. The increased level of TGF-β1 may be also the results of its potential to maintain stemness and quiescence of HSCs [18]. IFN-γ is generally perceived as a strongly proinflammatory
We have analyzed concentration of nine cytokines in both bone marrow and peripheral blood samples after the administration of GCSF. The results of analysis are presented in the Table 3. The levels of IL-10, IFN-gamma, IL-2 and IL-1β were higher in peripheral blood of mobilized donors than in the bone marrow. The concentration of TGF-β1 was significantly higher in bone marrow than in peripheral blood after the mobilization. There were no differences in the concentration of IL-17A, IL-4, IL-6 and TNF-α between bone marrow and peripheral blood samples. The levels of these cytokines were very low or close to the detection limit. We have checked the possible correlations between cytokine levels, total white blood cells count and CD3+ or CD34+ cells percentages. We have not found any statistically significant associations (data not shown). 3.2. Cytokine concentration in peripheral blood prior to and after the mobilization with G-CSF We have analyzed concentration of the same nine cytokines in peripheral blood samples before and after the mobilization procedure. The results of the analysis are presented in Table 4. We have observed increased level of IL-10 in donors’ blood in the samples collected after G-CSF was administered compared to steady Table 4 Cytokine concentrations in peripheral blood of HSCs donors before and after the mobilization with G-CSF. [Values in pg/ml, median and range]. Peripheral blood Cytokine
Before mobilization
After mobilization
p
IL-10 IFN-γ IL-2 TGF-β1 IL-1β TNF-α IL-17a IL-4 IL-6
12.98 (0.31–82.32) 50.48 (21.4–187.81) 2.40 (1.92–3.74) 36.01 (0–1693.62) 2.04 (0.27–20.32) 0.62 (0–11.62) 1.34 (0–4.53) 0.87 (0–1.8) 6.36 (2.1–30.08)
27.86 (10.12–110.18) 38.01 (9.76–79.15) 3.40 (0.85–4.72) 30.91 (0–164.13) 3.34 (0–7.5) 0.35 (0–7.86) 0 (0–7.04) 1.14 (0–2.08) 7.14 (2.22–37.95)
0.023* 0.6 0.224 0.286 0.779 0.6 0.236 0.44 0.789
Peripheral blood samples were collected from healthy donors of PBSC before and after mobilization with G-CSF. Concentrations of cytokines were evaluated with an ELISA test. Statistically significant differences obtained with the Wilcoxon Test are marked with asterisks (*p < 0.05).
3
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
Fig. 1. (A) Expression of CD47 protein on HSCs in bone marrow and blood. CD47 and CD274 proteins dot plot on CD34 + cells in bone marrow (B) and peripheral blood (C) evaluated by flow cytometry. Bone marrow samples were taken from healthy donors at the start of donation. Peripheral blood samples were collected on the day of leukapheresis from PBSC donors. Expression of CD47 on HSCs was evaluated by flow cytometry. Statistically significant differences were determined with the Mann-Whitney U test (p = 0.0001).
produce IL-1β why is it nearly non detectable in bone marrow. It can be explained by the lack of a proper stimuli or by the fact of very high level of TGF-β1 as described above. It is possible that the levels of proinflammatory cytokines in bone marrow are generally lower than in peripheral blood but the levels of these cytokines may be also derivative of the TGF-β1 concentration, as it may directly inhibit the secretion of IFN-γ, IL-2 and IL-1β specifically when present in such high concentrations. We have shown that the level of IL-10 in peripheral blood (prior to mobilization) is significantly higher than in bone marrow. It may be associated with the higher levels of Treg cells present in circulation of donors mobilized with G-CSF as described by Chevallier et al. [20].The presence of activated Treg cells is strongly associated with the level of IL-10 [23]. In our results we have shown that the administration of GCSF further increases the level of IL-10. It is highly probable that the increased level of IL-10 in peripheral blood after the mobilization is directly associated to the presence of large amount of injected G-CSF and acts as an counter-measure to inhibit the production of additional G-CSF as it was previously described by Raychaudhuri et al. [24]. To
cytokine. It is essential for the anti-microbial and anti-tumor response, stimulates the development of cytotoxic T lymphocytes, improves antigen presentation and many more [19]. IL-2 is produced by activated CD4+ cells after the appropriate antigen stimulation but its elevated level can also be explained by the nearly 2-fold higher amount of dendritic cells in PBSCs [20]. Dendritic cells subsets 1 and 2 can initiate the differentiation of T cells into Th1 and Th2 subsets respectively. Th1 cells are known to secrete a number of proinflammatory cytokines. Their primary product after the differentiation is IL-2 [21]. IL-1β is a member of IL-1 superfamily of cytokines responsible for immediate inflammatory response. Its most known functions are to enhance inflammatory state by promoting the production of other inflammatory cytokines. There are studies describing HSCs collected from the cord blood to be able to secrete IL-1β [22]. The reason for this is not entirely clear. It is a fact that the IL-1β can induce differentiation of HSCs into myeloid lineage. It is possible that the elevated level of IL-1β in peripheral blood can be associated with the higher levels of other proinflammatory cytokines. The question remains that if the HSCs can 4
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al.
HSCs both, collected directly from bone marrow and harvested by the means of leukapheresis are widely used for transplantation. Both materials are utilized in treatment of various hematological malignancies and are used uniformly as a source of hematopoietic stem cells. PBSCs harvest is the most frequent choice due to the simpler procedure and smaller amount of stress the donor undergoes. So far, there were no papers describing and comparing the cytokine composition regarding these two transplantation materials. Results of our study indicate that the cytokine composition of PBSCs vs bone marrow is different. White blood cells present in G-CSF mobilized peripheral blood are exposed to higher concentration of proinflammatory cytokines in comparison to bone marrow cells, which may augment their potential alloreactivity. This mechanism may contribute to increased risk of GVHD reported after PBSCs transplantations [4]. On the other hand, we demonstrated that CD34+ cells derived from bone marrow are characterized by increased expression of CD47. It is possible it may favor their engraftment. Altogether, our findings give insight into the biology of hematopoietic stem cell transplantation with respect to the source of stem cells.
rule out that possibility the experiments should be repeated on donors mobilized with other mobilizing factor. Some are currently being developed like, GRO-βT and GRO-β. They are known to operate differently from the G-CSF (possibly holding different impact on cytokine levels) [25], Those mobilizing factors are not routinely used in healthy donors mobilization procedure, so we are unable to confirm, whether the effect of elevated IL-10 level is specific solely for G-CSF. There was evidence suggesting the presence of feedback loop between the donors injected with G-CSF and their IL-10 level in peripheral blood. We have checked the concentrations of selected cytokines in peripheral blood before and after the administration with G-CSF. The results showed no significant change in cytokine levels with an exception of IL-10 concentration. IL-10 level rose from a median of 12.98 pg/ ml up to 27.86 pg/ml, (p = 0.023). It could prove the connection of IL10 levels with administration of G-CSF in blood and therefore not to be connected with the presence of HSCs. On the other hand G-CSF not only mobilizes HSC into the periphery but also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. G-CSF also mobilizes mature DCs, pDC1 cells, and pDC2 cells [26]. The increased numbers of DC could there correlate to the increased levels of IL-10 expression in peripheral blood of G-CSF treated recipients. In the second part of our research, we analyzed the expression of CD274 and CD47 on HSCs. Both proteins have immunosuppressive properties. CD47 is a membrane protein that interacts with the receptor SIRPα on macrophage surface and inhibits phagocytosis. CD47 is expressed in various types of cells, including e.g. erythrocytes and tumor cells [27,28]. This protein is also constitutively upregulated on mouse and human leukemia cells – probably as a mechanism which allows cancer cells to avoid destruction by macrophages. [29,30]. CD274 prevents destruction of cells by cytotoxic lymphocytes, via inducing the apoptosis pathway in this cells. This protein is abnormally expressed on many tumor cells, and play a major role in suppressing the immune system [10]. In our study we measured expression of immunosuppressive proteins on HSCs in blood (after mobilization) and in bone marrow (from donors of bone marrow). The percentage of CD47 positive HSCs was significant higher in bone marrow than in blood. These results are different than those obtained by Jaiswal. In our study, the level of CD47 decreased, when HSCs migrated outside of the bone marrow, but Jaiswal reported, that the expression of CD47 on HSCs is transiently upregulated during mobilization. Possible explanation to these contradictory observations is that Jaiswal investigated the most primitive HSCs (CD90+ CD34+ CD38- Lin-) – in our research we decided to check expression on all CD34 positive cells (not only most primitive, but progenitors as well). The level of CD274 positive HSCs was low (< 1% of cells) and similar in both localizations. Expression of CD274 and CD47 on HSCs may be compared with result published by Zheng et al. [2]. This group tested multiple markers on mouse HSCs, including MHC-I, MHC-II, CD274, CD275, CD47, and CD86. The level of CD274 was low, but in vitro culturing led to an increase in its expression. Authors demonstrated, that upregulation of CD274 during culture supports HSC allograft in mice. Next, they measured level of this protein on human HSCs, obtained from human cord blood. About 10% of freshly isolated HSCs expressed CD274 on their surface, and – as in the case of mouse cells – in vitro conditions induced elevated expression of this protein [31]. We observed similar effect in HSC culturing (data not shown). It is possible that CD47 and CD274 proteins can provide additional protection to the HSCs, mainly from the hostile activity of the immune system. There is also the evidence that suggested the correlation between the expression of CD47 and concentration of TGF-B1 [32]. TGF-B1 expression is regulated by the binding of CD47. Moreover CD47 expression reduces the maturation of DC cells [33]. The decreased expression of CD47 in the periphery would therefore allow maturation of DC's and may explain the reduced concentration of TGF-B1 in the peripheral blood.
References [1] J.R. Passweg, J. Halter, C. Bucher, S. Gerull, D. Heim, A. Rovó, et al., Hematopoietic stem cell transplantation: a review and recommendations for follow-up care for the general practitioner, Swiss. Med. Wkly. 142 (2012) w13696, http://dx.doi.org/10. 4414/smw.2012.13696. [2] J. Zheng, M. Umikawa, S. Zhang, H. Huynh, R. Silvany, B.P.C. Chen, et al., Ex vivo expanded hematopoietic stem cells overcome the mhc barrier in allogeneic transplantation, Cell Stem Cell 9 (2011) 119–130, http://dx.doi.org/10.1016/j.stem. 2011.06.003. [3] I. Sirinoglu Demiriz, E. Tekgunduz, F. Altuntas, What is the most appropriate source for hematopoietic stem cell transplantation? peripheral stem cell/bone marrow/ cord blood, Bone Marrow Res. 2012 (2012) 1–5, http://dx.doi.org/10.1155/2012/ 834040. [4] C. Anasetti, B.R. Logan, S.J. Lee, E.K. Waller, D.J. Weisdorf, J.R. Wingard, et al., Peripheral-blood stem cells versus bone marrow from unrelated donors, N. Engl. J. Med. 367 (2012) 1487–1496, http://dx.doi.org/10.1056/NEJMoa1203517. [5] J. Fujisaki, J. Wu, A.L. Carlson, L. Silberstein, P. Putheti, R. Larocca, et al., In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche, Nature 474 (2011) 216–219, http://dx.doi.org/10.1038/nature10160. [6] T. Reya, Illuminating Immune privilege — a role for regulatory T cells in preventing rejection, N Engl J Med 365 (2011) 956–957, http://dx.doi.org/10.1056/ NEJMcibr1107781. [7] P. Arck, M.E. Solano, M. Walecki, A. Meinhardt, The immune privilege of testis and gravid uterus: same difference? Mol. Cell. Endocrinol. 382 (2014) 509–520, http:// dx.doi.org/10.1016/j.mce.2013.09.022. [8] A. Louveau, T.H. Harris, J. Kipnis, Revisiting the mechanisms of CNS immune privilege, Trends. Immunol. 36 (2015) 569–577, http://dx.doi.org/10.1016/j.it. 2015.08.006. [9] S. Zhao, W. Zhu, S. Xue, D. Han, Testicular defense systems: immune privilege and innate immunity, Cell. Mol. Immunol. 11 (2014) 428–437, http://dx.doi.org/10. 1038/cmi.2014.38. [10] C.C. Zhang, Hematopoietic stem cells: interplay with immunity, Am. J. Blood Res. 2 (2012) 219–227. [11] M.P. Chao, I.L. Weissman, R. Majeti, The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications, Curr. Opin. Immunol. 24 (2012) 225–232, http://dx.doi.org/10.1016/j.coi.2012.01.010. [12] W. Li, X. Wang, R. Chen, H. Zhu, G. Chen, X. Sun, Overexpression of programmed death ligand 1 in dendritic cells inhibits allogeneic lymphocyte activation in mice, J. Surg. Res. 176 (2012) e79–e87, http://dx.doi.org/10.1016/j.jss.2011.12.009. [13] P. Greaves, J.G. Gribben, The role of B7 family molecules in hematologic malignancy, Blood 121 (2013) 734–744, http://dx.doi.org/10.1182/blood-2012-10385591. [14] R.K. Hopman, J.F. DiPersio, Advances in stem cell mobilization, Blood Rev. 28 (2014) 31–40, http://dx.doi.org/10.1016/j.blre.2014.01.001. [15] K.M. Gilbert, M. Thoman, K. Bauche, T. Pham, W.O. Weigle, Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells, Immunol. Invest. 26 (1997) 459–472. [16] L. Zou, B. Barnett, H. Safah, V.F. LaRussa, M. Evdemon-Hogan, P. Mottram, et al., Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals, Cancer Res. 64 (2004) 8451, http://dx.doi.org/10.1158/ 0008-5472.CAN-04-1987. [17] E. Zhao, L. Wang, J. Dai, I. Kryczek, S. Wei, L. Vatan, et al., Regulatory T cells in the bone marrow microenvironment in patients with prostate cancer, OncoImmunology 1 (2012) 152–161, http://dx.doi.org/10.4161/onci.1.2.18480. [18] S. Yamazaki, A. Iwama, S -i. Takayanagi, K. Eto, H. Ema, H. Nakauchi, TGF- as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation, Blood (2009) 113 1250–1256. doi:10.1182/blood-2008-04-146480. [19] Y.-G. Yang, H. Wang, W. Asavaroengchai, B.R. Dey, Role of Interferon-gamma in
5
Cytokine xxx (xxxx) xxx–xxx
W. Fidyk et al. GVHD and GVL, Cell. Mol. Immunol. 2 (2005) 323–329. [20] P. Chevallier, N. Robillard, M. Illiaquer, J. Esbelin, M. Mohty, C. Bodin-Bressollette, et al., Characterization of various blood and graft sources: a prospective series: characterization of various blood and graft sources, Transfusion (Paris) 53 (2013) 2020–2026, http://dx.doi.org/10.1111/trf.12072. [21] S.L. Gaffen, K.D. Liu, Overview of interleukin-2 function, production and clinical applications, Cytokine 28 (2004) 109–123, http://dx.doi.org/10.1016/j.cyto.2004. 06.010. [22] K. Watari, H. Mayani, F. Lee, W. Dragowska, P.M. Lansdorp, J.W. Schrader, Production of interleukin 1beta by human hematopoietic progenitor cells, J. Clin. Invest. 97 (1996) 1666–1674, http://dx.doi.org/10.1172/JCI118593. [23] C.A. Stewart, H. Metheny, N. Iida, L. Smith, M. Hanson, F. Steinhagen, et al., Interferon-dependent IL-10 production by Tregs limits tumor Th17 inflammation, J. Clin. Invest. 123 (2013) 4859–4874, http://dx.doi.org/10.1172/JCI65180. [24] B. Raychaudhuri, C.J. Fisher, C.F. Farver, A. Malur, J. Drazba, M.S. Kavuru, et al., Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages, Cytokine 12 (2000) 1348–1355, http://dx.doi.org/ 10.1006/cyto.2000.0721. [25] B . Suárez-Álvarez, A. López-Vázque, C. López-Larrea , Mobilization and Homing of Hematopoietic Stem Cells, In: C. López-Larrea, A. López-Vázque, B. Suárez-Álvarez, editors. Stem Cell Transplant., vol. 741, New York, NY: Springer US; 2012, p. 152–70. [26] P.J. Shaughnessy, C. Bachier, C.F. Lemaistre, C. Akay, B.H. Pollock, Y. Gazitt, Granulocyte colony-stimulating factor mobilizes more dendritic cell subsets than granulocyte-macrophage colony-stimulating factor with no polarization of dendritic
[27]
[28]
[29]
[30]
[31]
[32]
[33]
6
cell subsets in normal donors, Stem. Cells Dayt. Ohio. 24 (2006) 1789–1797, http:// dx.doi.org/10.1634/stemcells.2005-0492. S. Jaiswal, M.P. Chao, R. Majeti, I.L. Weissman, Macrophages as mediators of tumor immunosurveillance, Trends. Immunol. 31 (2010) 212–219, http://dx.doi.org/10. 1016/j.it.2010.04.001. N. Navarro-Alvarez, Y.-G. Yang, CD47: a new player in phagocytosis and xenograft rejection, Cell. Mol. Immunol. 8 (2011) 285–288, http://dx.doi.org/10.1038/cmi. 2010.83. S. Jaiswal, C.H.M. Jamieson, W.W. Pang, C.Y. Park, M.P. Chao, R. Majeti, et al., CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis, Cell 138 (2009) 271–285, http://dx.doi.org/10.1016/j.cell. 2009.05.046. H. Jiang, R. Fu, H. Wang, L. Li, H. Liu, Z. Shao, CD47 is expressed abnormally on hematopoietic cells in myelodysplastic syndrome, Leuk. Res. 37 (2013) 907–910, http://dx.doi.org/10.1016/j.leukres.2013.04.008. J. Zheng, C. Song, C.C. Zhang, A new chapter: hematopoietic stem cells are direct players in immunity, Cell Biosci. 1 (2011) 33, http://dx.doi.org/10.1186/20453701-1-33. K. Shimada, A. Nakajima, K. Ikeda, K. Ishibashi, N. Shimizu, K. Ito, CD47 regulates the TGF-β signaling pathway in osteoblasts and is distributed in Meckel’s cartilage, J. Oral Sci. 53 (2011) 169–175. V. Doyen, M. Rubio, D. Braun, T. Nakajima, J. Abe, H. Saito, et al., Thrombospondin 1 is an autocrine negative regulator of human dendritic cell activation, J. Exp. Med. 198 (2003) 1277–1283, http://dx.doi.org/10.1084/jem.20030705.