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22Citation: Cellular Therapy and Transplantation, Vol. 2, No. 6
Please cite this article as follows: Elena R. Chernykh, Ekaterina Ya. Shevela, Ludmila V. Sakhno, Marina A. Tikhonova, Yaroslav L. Petrovsky, Alexander A. Ostanin. The generation and properties of human M2-like macrophages: potential candidates for CNSrepair? Cell Ther Transplant. 2010;2:e.000080.01. doi:10.3205/ctt-2010-en-000080.01
© The Authors. This article is provided under the following license: Creative Commons Attribution 3.0 Unported, http://creativecommons.org/licenses/by/3.0/
Submitted: 12 March 2010, accepted: 6 December 2010, published: 21 December 2010
The generation and properties of human M2-like macrophages: potential candidates for CNS repair?
Elena R. Chernykh, Ekaterina Ya. Shevela, Ludmila V. Sakhno, Marina A. Tikhonova, Yaroslav L. Petrovsky, Alexander A. Ostanin
Laboratory of Cellular Immunotherapy, Institute of Clinical Immunology of Russian Academy of Medical Sciences, Siberian Branch, Novosibirsk, Russia
Correspondence: Elena Chernykh, Laboratory of Cellular Immunotherapy, Institute of Clinical Immunology RAMS SB, Yadrintsevskaya str., 14, Novosibirsk, 630099, Russia; Phone: +7(383)2360329; Fax: +7(383)2227028; E-mail: [email protected]
Abstract
Regulation of the immune response seems to be a promising strategy for a successful central nervous system (CNS) repair, and macrophages are considered to be prospective candidates for cell therapy. Using low serum conditions we generated human anti-inflammatory M2-like macrophages from peripheral blood monocytes and compared these cells (termed M^3) with "standard" pro-inflammatory M^l and anti-inflammatory M^2, generated in the presence of GM-CSF and M-CSF. We focused primarily on the differences in T-cell stimulatory activity and production of various cytokines, chemokines, and growth factors. Low serum conditions had no negative impact on macrophage yield, the largest of which was for M^3. We showed that M^3 more closely resembled M^2 than M9I. M^2 and particularly M^3, but not M9I expressed relatively low levels of CD86 and failed to stimulate T-cell proliferation. In contrast to pro-inflammatory M9I, unstimulated M^3 produced significantly lower levels of pro-inflammatory cytokines (IL-ip, TNF-a, IL-6, IL-18, IL-12) and Th1/Th2-cytokines (IFN-y, IL-2, IL-4) coupled with a higher IL-10 level. Moreover, concentrations of IL-1p and pro-inflammatory chemokines IL-8 and MCP-1 in M^-3 supernatants were lower not only when compared to M^1, but also to M^2 cultures. Like M^1 and M^2, M^3 was capable of producing neurotrophic- (BDNF, IGF-1), angiogenic- (VEGF), and other growth factors (EPO, G-CSF, FGF-basic, EGF) with neuroprotective and regenerative activity. In fact, IGF-1 production by M^-3 exceeds secretion of this factor by M9-1 and M^-2 by more than 25 fold. Thus, generated M^-3 represented M2-like macrophages with high regenerative potential.
Keywords: macrophage polarization, cytokines, chemokines, growth factors, CNS repair
Introduction
Following injury to the nervous system, the activation of the immune system profoundly affects the ability of neurons to survive and to regenerate damaged axons. The role of immune response is controversial. It has long been established that immune cells in the CNS can cause or augment tissue injury. However, recent investigations show that immune cells and their factors can contribute to neuroprotection and neurorege-neration. This dual role of the immune system is determined by the type and duration of the immune response and the balance between destructive and protective factors that ultimate-
ly define the net result of the neuro-immune interaction [5].
The immune system operates via innate (antigen-independent) and adaptive (antigen-specific) immunity. Inflammatory responses during traumatic injury or different CNS diseases are dominated by cells of the innate immune system, most importantly resident microglia and blood-borne macrophages. After phagocytosing cellular debris, microglia/macrophages present antigens to lymphocytes, thereby activating the antigen-specific immune response [33].
Unlike most other systems, the central nervous system has a limited capacity for regeneration. While the inhibitory effects of proteoglycans and myelin on axonal growth have been well established, the role of neuroinflammation in regeneration failure remains highly controversial [6]. Several studies have demonstrated the beneficial effects of macrophages (M9) following injury [23,25,27,37]; however, others revealed that macrophages promoted injury [9,19].
One of the possible explanations of these diverse macrophage effects could be connected with the differences between the macrophages used. Certainly, M9 are remarkable for the heterogeneity and diverse biological activities [11]. There are at least two distinct functional M9 subsets that are triggered in response to different stimuli: classical pro-inflammatory and nonclassical anti-inflammatory macrophages, also termed type 1 (M1) and type 2 (M2) macrophages. Ml are induced by IFN-y, either alone or in concert with a microbial stimulus, possess high antigen-presented activity, and support Th1 response. These cells are involved in pro-inflammatory responses, mediate resistance to intracellular pathogens and anti-tumor resistance and are tissue destructive. In contrast, various forms of M2, generated in the presence IL-4 or IL-13, immune complexes, IL-10, etc., are not efficient at antigen presentation, suppress Th1 and/or favor Th2 response, and produce high levels of matrix-associated proteins. These cells are tolerogenic and generally oriented toward resistance to parasites, immunoregulation, tissue remodeling and repair, and tumor promotion [20,10,18]. It is important to note that macrophages can reversibly shift their functional phenotype in response to changes in their microenvironment. Sequential treatment of macrophages with multiple cytokines results in a progression through various functional phenotypes. That is, macrophages may progress from one functional phenotype to another [32,21].
Recently, Kigerl et al has shown that in CNS injury rapidly induced M1 response than shift to M2 response. M1 were neurotoxic, whereas M2 promoted a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominated sites of CNS injury (e.g., proteo-glycans and myelin). The authors concluded that switching macrophages toward an M2 phenotype could promote CNS repair while limiting secondary inflammatory-mediated injury [14]. Thus, boosting or modulating the immune response seems to be a promising strategy for successful CNS repair.
Since macrophages may be prospective candidates for cell therapy, the development of simple and reproducible technologies of M2-like macrophage generation seems to be a necessary step for the clinical application of this approach. For human monocytes GM-CSF treatment leads to the formation of M^1 macrophages with features of pro-inflammatory M1 cells, while the equivalent population following culture in M-CSF has been termed M^2 macrophages with features of M2 anti-inflammatory cells [34,35]. In addition, macrophages that ingest apoptotic cells are shown to decrease pro-inflammatory and acquire anti-inflammatory properties [8]. Utilizing of M2-like macrophages in experimental models and clinical trail was successfully demonstrated by the Michel Schwartz group [27,16]. Recently we developed a simple approach for
generation of non-classical type2-like macrophages (M^3) in the presence of GM-CSF in serum-deficient conditions. The purpose of the current study was to compare the phenotype and functions of these M^3 with "standard" pro-inflammatory M^1 and anti-inflammatory M^2 subsets, generated in the presence of GM-CSF and M-CSF.
Materials and Methods
Isolation and generation of macrophages
Human blood samples were obtained from healthy donors with informed consent according to the policy approved by the local Ethical Committee. Human peripheral blood mononuclear cells (PBMCs) were obtained through density gradient centrifugation (Ficoll-Paque, Sigma-Aldrich) of heparini-zed whole blood samples. For monocyte separation PBMCs were plated at 3-5 x106/ml in tissue culture dishes (TPP, Switzerland) in RPMI-1640 (Sigma-Aldrich) with 5% FCS (Biolot, Russia) for 18 h and then washed to remove non-adherent residual lymphocytes. The percentage of CD14-positive cells was demonstrated by flow cytometry analysis to be greater than 90-93% of the total cells recovered.
Classical type-1 macrophages (M^1) were generated by culturing adherent cells in six-well tissue plates (Nunclon, Denmark) in RPMI-1640 supplemented with 5% autologous plasma, 2% FCS, 0.05 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.3 mg/ml L-glutamine (all reagents of Sigma-Aldrich), 1% nonessential amino acids, 100 ^g/ml genta-mycin and 50 ng/ml recombinant human GM-CSF (R&D Systems) at 37°C with 5% CO2 for 7 days. Non-classical type 2 macrophages (M^2) were obtained in identical culture conditions in complete RPMI-1640 supplemented with rhM-CSF (50 ng/ml; R&D Systems). Non-classical type 3 macrophages (M^3) were generated by incubation of monocytes in serum growth factors deficiency conditions. Specifically, adherent cells were cultured for 7 days in complete RPMI-1640 supplemented with 2% autologous plasma (without FCS) and 50 ng/ml rhGM-CSF. Polarized M9 (M^1, -2, -3) were harvested by using EDTA in Hanks' balanced salt solution, washed and counted.
Flow cytometry analysis
For evaluation of the M9 phenotype, cell suspensions were incubated for 20 min at 4°C with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies specific for human CD14, CD86, CD90, and HLA-DR or isotype controls. All monoclonal antibodies were obtained from BD Biosciences (USA). After incubation with antibodies, cells were washed with PBS containing 0.1% sodium azide (Sigma-Aldrich) and 0.1% bovine serum albumin, and were then analyzed with a FACSCalibur using CellQuest software (BD Biosciences).
T-cell proliferation assays
The antigen-presenting and allostimulatory activity of M9 was determined by measuring T-cell proliferation in the mixed lymphocyte culture (MLC). Different types of M9
were collected after generation and 1x105 cells were then plated in RPMI-1640 supplemented with 0.3 mg/ml L-glut-amine, 5 mM HEPES buffer, 100 ^g/ml gentamycin and 10% inactivated donor serum (AB (IV) group), and added to 1x106 allogeneic responder PBMCs. All cultures were carried out in triplicate in round-bottom 96-well tissue culture plates, in a final volume of 150 ^l of RPMI complete medium. T-cell proliferation was assessed after 5 days by adding [3H]thymidine (1 ^Ci/well) for 18 h. Cells were then harvested and thymidine incorporation was measured in a liquid scintillation counter SL-30 (Intertechnic, France). The stimulatory capacity of M9 in MLC was expressed by the stimulation index (SI) = cpm in MLC (PBMCs+M^) / cpm in control culture (PBMCs alone).
Cytokines, chemokines, and growth factor measurements
Culture supernatants of generated M9 (M^1, -2, -3) were collected and stored at -80°C prior to measurement. The concentration of secreted cytokines/chemokines was determined by using the Bio-Plex Protein Array System (kits and equipment of Bio-Rad, USA based on Luminex xMAP technology; sensitivity 2 pg/ml) in the case of TNF-a, IL-1p, IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, IFN-y, IL-8, MCP-1, and MIP-1P, and by using ELISAs from Diagnostic System Laboratories for insulin-like growth factor-I (IGF-I, sensitivity 0.01 ng/ml); from BioSource for basic fibroblast growth factor (FGF-basic, sensitivity 7 pg/ml); from R&D Systems for brain-derived neurotrophic factor (BDNF, sensitivity 20 pg/ml); from Invitrogen Corp. for vascular endothelial growth factor (VEGF, sensitivity 5 pg/ml); from Protein Contour (St-Petersburg, Russia) for erythropoietin (EPO, sensitivity 4 pg/ml) and epidermal growth factor (EGF, sensitivity 2 pg/ml); and from Vector-Best (Novosibirsk, Russia) for IL-18 (sensitivity 5 pg/ml).
Statistical analysis
Statistical analysis was performed using the STATISTICA software version 6.0 (StatSoft. Inc., USA). The Mann-Whitney non-parametric two-tailed U test was used to determine the significance of data, which are presented as median and inter-quartile range (IQR). Values of p < 0.05 were considered statistically significant.
Results
Characterization of generated M9
We generated three distinct M9 subsets in vitro from peripheral blood monocytes and performed a series of parallel comparisons between them. As a first step, we measured cell yield and their phenotype. The number of M^1 and M^2 obtained from 1x106 PBMCs was 3.35x104 (IQR 2.2-7.4x104) and 2.50x104 (IQR 1.4-4.5x104), whereas M^3 yield was significantly higher — 5.0x104 (IQR 3.3- 0.4x104, pU<0.01), indicating that a low serum condition increased the quantity of macrophages generated in the presence of GM-CSF.
After 7 days of culture, the majority of M^1, M^2, and M^3 were adherent cells with a classical "fried egg" morphology (data not shown) that expressed CD14 on their cell surface (Table 1). A small number of adherent cells had a stretched, spindle-like morphology (fibroblast-like cells). The average number of these cells in M^1 (n=8) and M^2 (n=8) populations was similar and constituted 25% (IQR 22-45 and 16.5-33.5%, respectively), and was slightly higher (Median 32.5%, IQR 17-43%, n=6) in the M^3 subset. However, the expression of CD90 antigen (a typical marker for a fibroblasts and mesenchymal stem cells) in all M9 populations was low and the percentage of CD90+ cells did not exceed 2-3%.
All three M9 populations strongly expressed the HLA-DR antigen, though the percentage of HLA-DR positive cells in the M^3 cultures was lower than in the M^1 and M^2. All types of monocyte-derived macrophages also expressed the CD86 antigen. The mean number of CD86+ cells in M^2 and M^3 was lower than in M^1, though not significantly.
Percentage of positive cells
Marker M9I M^2 M93
Median (IQR) N Median (IQR) N Median (IQR) N
CD14 78 (70-84) 17 87 (78-91) 9 82 (67-92) 25
HLA-DR 97 (91-98) 21 96 (96-98) 9 87 (73-97) 17
CD86 37 (23-53) 18 27 (15-39) 13 23 (11-58) 17
CD90 2.5 (0-5.0) 10 2.0 (0-5.0) 13 3 (0.6-5.0) 8
Table 1: Phenotype M9I, M92 and M93 subsets
Culture M9I (n=24) M^2 (n=24) M93 (n=24)
PBMCs alone Median 330 140 370
IQR 105-720 105-410 70-1300
PBMCs + M9 (10:1) Median 7380 3130 ** 2070 ** #
HLA-DR IQR 3500-13220 1600-3680 330-3230
Stimulation index Median 19.6 14.8 3.4 ** ##
CD90 IQR 14.9-74.5 6.2-35.3 1.4-13.7
Mqi (1x105 cells) were cultured with 1x106 allogeneic PBMCs over 5 days. 3[H]-thymidine (1 yCi/well) was added 18 h before harvesting to measure T-cell proliferation (cpm). The stimulation index is expressed in calculated units (cpm in MLC (PBMCs+My) / cpm in control culture (PBMCs alone). **pU < 0.01 vs Mq>1; # pU < 0.05 and ## pU < 0.01 vs Mq>2. Table 2: The stimulatory effect of M9I, M92 and M93 subsets on allogeneic T-cell proliferation
The ability of M9 to induce T-cell proliferation
The revealed differences of HLA-DR and CD86 expression in distinct M9 populations could influence their antigen-presenting function. To determine whether M^1, M^2, and M^3 differed quantitatively in their capacity to present antigen, we tested and compared their ability to induce an allogeneic T-cell response. For this purpose distinct M9 subsets derived from the same donor were cocultured with allogeneic PBMCs over a period of 5 days, and the T-cell proliferation was determined (Table 2).
Analysis of [3H]thymidine incorporation revealed a strong proliferative response in PBMCs cocultured with M9I, whereas weak proliferation could be observed in PBMCs cocultured with M^2 or M^3. Remarkably, the T-cell stimulatory capacity of M^3 expressed by the stimulation index (SI) was significantly lower than that of M9I and M^2.
Generated M9 differ in cytokine and chemokine production
To further characterize the secretory profile of generated M9 subsets, we measured the production of Thi/pro-inflamm-atory (IFN-y, IL-2, IL-ip, TNF-a, IL-12, IL-17, IL-18, IL-6) and Th2/anti-inflammatory cytokines (IL-4, IL-10, IL-13). Cytokine levels were measured in supernatants of 7-day cultures of M9I, M^2 and M^3. M9I spontaneously produced considerable levels of IL-1p, IL-6, TNF-a, IFN- y, IL-4, and IL-17 (Table 3). This finding confirms the pro-inflammatory nature of M9I and their capacity for T-cell activation. M^2 were characterized by lower secretory activity for some of these cytokines, though the differences were significant only for IL-4 and IL-18. In contrast, M^3 displayed remarkably decreased basal levels of pro-inflammatory cytokines (IL-ip, TNF-a, IL-6, IL-18), Thi-cytokines (IFN-y, IL-2), and IL-4.
M^3 also differed from M9I by a 2-fold lower IL-12 production and more pronounced production of IL-10, though not significantly. In addition to cytokines, we measured the levels of various inflammatory chemokines in the supernatants of unstimulated macrophages. Generated M9 constitutively produced high levels of IL-8, MCP-1, and MIP-1p. M9I and M^2 demonstrated similar levels in their production. In contrast, secretion of neutrophil-attracting IL-8 and monocyte-attracting MCP-1 by M^3 was significantly lower than by M9I and M^2. However, the production of T-cell attracting MIP-1P by M^3 did not differ from that by M9I and M^2. Together, these data confirm the pro-inflammatory nature of M9I and significantly less pro-inflammatory activity of M^3.
Production of growth factors by generated M9
All three types of unstimulated macrophages secreted detectable concentrations of erythropoietin, G-CSF, FGF-basic, BDNF, and IGF-1 (Table 4). M9I and M^2 produced analogous levels of these growth factors, although there was a strong tendency to higher production of EPO by M^2. Despite the decreased production of pro-inflammatory cytokines, M^3 secreted concentrations of G-CSF, EPO, FGF-basic and
Cytokines& chemokines (pg/ml) M9I (n=10) Median IQR M^2(n=10) Median IQR M93 (n=24) Median IQR
IFN-y 872 734-995 839 539-1010 626 * 4 440-830
IL-2 154 115-154 115 70-155 72 *4 47-115
IL-ip 405 246-670 313 150-790 195 * # 4 68-290
TNF-a 175 124-282 148 55-224 99 * 4 51-156
IL-12 28 20-29 19 7-25 14 3-33
IL-17 308 245-483 257 177-448 214 112-427
IL-18 33 29-51 27 * 4 16.5-31.2 19 * 4 15.7-35.8
IL-6 21340 13430-27340 20350 8380-25060 10900 * 4 4110-21770
IL-4 215 198-246 119 ** 4 79-141 106 ** 4 53-190
IL-10 5 2-10 2 2-2 15 2-60
IL-13 78 37-113 48 37-78 78 42-112
IL-8 90380 74280-93340 67400 57940-94430 44320 ** ## 4 29150-59000
MCP-1 11140 5680-14000 11910 4160-17660 3345 ** ## 4 1100-4460
MIP-1P 1 960 1250-5590 1 560 930-2700 2220 790-7620
* pv < 0.05 and ** pv < 0.01 vsMç>1; # pv< 0.05 and##pv < 0.01 vsMq2. Table 3: Cytokine/chemokine concentrations secreted by M9I, Mç2, and Mç3
Growth factors (pg/ml) M9I (n=10) Median IQR M^2(n=10) Median IQR M93 (n=24) Median IQR
G-CSF 670 505-1610 730 315-2310 430 180-1050
EPO 19.2 1.7-36.9 46.5 33.8-81.1 34.9 21.5-56.5
FGF-basic 104 57-124 150 87-180 109 45-126
EGF 207 148-331 283 245-420 138 38-310
BDNF 392 187-705 438 215-739 131 * # 4 78-235
IGF-1 322 170-8560 152 116-459 8310 *##Î 520-9500
VEGF (n=6) 5.0 5.0-97 92.8 * Î 59.2-298 422.4 * # î 107.7-524.7
* pv < 0.05 and ** pv < 0.01 vsMq>1; # pv < 0.05 and## pv< 0.01 vsMq2. Wilcoxon matched non-parametric paris test was used to determine the significance of VEGF.. Table 4: Growth factors production by M9I, Mç2 and Mç3
EGF comparable with M^2, though significantly lower concentration of BDNF. But the most prominent difference was revealed for the production of IGF-1, which was much higher in M^3 in comparison with M9I and M^2 cultures. Concerning VEGF, its detectable concentrations in 7-day cultures were determined only in a quarter of tested donors. Among these cultures VEGF was predominantly produced by M^2, and especially by M^-3, but not M9I.
Discussion
Over the last decade, there has been an increasing interest in the role of the inflammatory reaction in CNS injury. Moreover, this interest has focused on the dominant cell type observed during inflammation, the macrophage. However, in the CNS the contribution of these cells to the healing process remains questionable [6].
The contradictory data regarding the contribution of M9 to CNS recovery could be explained by diverse macrophage activities, many of which appear to be oppositional in nature. The destructive potential of macrophages in CNS pathology may be caused by pro-inflammatory activity, whereas their regenerative capacity may be linked with anti-inflammatory features [12].
In the search for macrophages with potential regenerative activity we developed a simple method for the generation of macrophages in growth factor deficient conditions and analyzed the phenotype and functional activity of these macrophages, termed M^3, with pro-inflammatory M9I and anti-inflammatory M^2. We speculated that the deficiency of growth factors in low serum conditions may be one of the key factors capable of activating regenerative properties of macrophages. Particularly, low serum conditions during macrophage cultivation could stimulate deprivation-induced apoptosis of culturing cells (including admixture of non-adherent cells), and the ingestion of apoptotic cells may change the functional activity of macrophages toward an anti-inflammatory pheno-type.
The received data demonstrated that low serum conditions did not influence the efficacy of M^3 generation. Moreover, the yield of M^3 significantly exceeded the number of M9I and M^2. These data are correspondent with Plesner's study, who showed an enhanced yield of M-CSF treated macrophages in cultures with 1% fetal calf serum [22].
According to study of Verreck et al, anti-inflammatory M^2 have a lower expression of HLA-DR and CD86 molecules after LPS stimulation, though unstimulated macrophages expressed similar levels of these molecules [34]. We have shown that as compared to M9I and M^2, M^3 cultures contained lower numbers of HLA-DR and CD86-positive cells. These differences, though not statistically significant, were important for the association with the decreased capacity of M^3 to stimulate allogeneic T cell proliferation. Type-2 anti-inflammatory macrophages are known to have a lower ability to stimulate T-cell proliferation in MLC [11]. This is in agreement with our data, and pointed to the lower allosti-mulatory activity of M^2 in comparison with M^1. Notably,
M^3 virtually failed to stimulate lymphocyte proliferation in MLC. The medium value of the M^3 stimulation index was more than 6-fold lower than that of M^1. This fact strongly suggests that generated M^3 are not immunogenic and in this respect resemble anti-inflammatory M2 macrophages.
To further evaluate the pro- and anti-inflammatory activity of generated macrophages we compared their capacity to spontaneous production of Th1/pro- and Th2/anti-inflammatory cytokines. In contrast to M^1, M^3 produced significantly (2-fold) lower concentrations of pro-inflammatory (IL-1p, TNF-a, IL-6, IL-18) and Th1/Th2-cytokines (IFN-y, IL-2, IL-4). M^3 supernatants also contained 2-fold lower concentrations of IL-12 and higher levels of IL-10, though these differences were not statistically significant.
Gordon and coworkers [11] have described alternatively activated macrophages after treatment with IL-4 or IL-13, which produce IL-10 without microbial stimulation. At the same time the study of Verreck demonstrated that unlike alternatively activated M9, M-CSF polarized M^2 failed to release IL-10 without activation, but effectively secreted IL-10 after mycobacterial activation. However, activated M^-2 produced no or relatively low levels of IL-12, IL-1p, IL-6, TNF-a [34]. We also did not reveal any significant concentrations of IL-10 in the supernatants of unstimulated M^2. In contrast to M9-2, M9-3 spontaneously produced IL-10 and displayed significantly less pro-inflammatory phenotype (as compare with M^1) without any additional stimulation.
Our results are also in agreement with findings suggesting a high ability of M-CSF polarized M^2 to secrete pro-inflammatory chemokines [35]. M^3 were also shown to secrete MIP-1p levels comparable with M^1 and M^2, but lower levels of IL-8 and MCP-1. This indicated that unlike M^1 and M^2 subsets, M^3 has less capacity to attract neutrophils and monocytes and therefore is less effective in supporting inflammation, whereas they could recruit effector Th1 cells and modify their functions.
One possible mechanism underlying the beneficial role of macrophages in CNS repair is connected with their capacity to produce a wide range of growth factors that can promote neuroprotection and regeneration [30,17,6]. The comparative analysis of some growth factors in the supernatants of generated macrophages revealed that all three M9 subsets spontaneously produced detectable levels of EPO, G-CSF, IGF-1, FGF-basic, EGF, and BDNF. M^3 secreted concentrations of G-CSF, FGF-basic and EGF similar to M^1 and M^2, EPO comparable with M^2, and a lower level of BDNF, but more than 25-fold higher level of IGF-1. As for VEGF, this growth factor, identified only in quarter of patients, was produced by both M^2 and M^3, but not M9-1 and was significantly higher in M^3- than in M^2 cultures.
Production of classical neurotrophic factors including CNTF, IGF, HGF, PDGF, NGF, BDNF, GDNF, and NT-3 by macrophages have been shown in numerous studies [3,7,13]. Evaluation of two of these factors (BDNF and IGF-1) in cultures of distinct macrophage subtypes in our study supported previous data and demonstrated comparable production of these
factors by inflammatory M9I and anti-inflammatory M^2. Moreover we have shown for the first time that in spite of a lower level of BDNF, M^3 were characterized with exclusively high secretion of IGF-1.
IGF-1 is a potent neurotrophic factor. Its pleiotropic effects range from classical trophic actions on neurons such as housekeeping or anti-apoptotic/pro-survival effects to modulation of brain-barrier permeability, neuronal excitability, or new neuron formation. IGF-1 is also known to significantly improve axon growth and remyelination [2,4]. The finding that IGF-1 is secreted abundantly by M^3 may point toward an important potential role for these macrophages in neuroprotection and regeneration.
In addition to neurotrophic factors, generated macrophages produced significant levels of VEGF. Detection of VEGF (in 7-day macrophage supernatants) only in part of the tested donors could be connected with an earlier peak of VEGF production. Nevertheless, in detectable cases VEGF was predominantly produced by both M^2 and M^3. VEGF has direct neuroprotective effects on motoneurons, induces neurogene-sis and angiogenesis and its reduced levels cause neurodegeneration in part by impairing neural tissue perfusion [31,38].
Other factors, such as EPO, G-CSF, FGF-p, and EGF, produced by M9-3 and M91/M92 subsets could also underlay the neuro-regenerative macrophage potential. Erythropoietin functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. This cytokine promotes both neuroprotection and neuroregeneration in various models of CNS injury and disease and is considered to be a promising candidate as neuroprotective agent [29,15]. G-CSF appears to have anti-apoptotic effect and stimulate differentiation of adult neural stem cells [26]. EGF is a motility factor for microglial cells and is shown to enhance the differentiation, maturation and survival of a variety of neurons in the central nervous system [36]. FGF-basic promotes the survival and neurite growth of brain neurons in vitro and in vivo, suggesting that it functions as a neurotrophic factor. In addition FGF acutely modulates synaptic transmission in the hippocampus, suggesting that it has a role similar to a neurotransmitter or neuromodulator [1].
Several groups have confirmed the therapeutic potential of activated microglia and monocyte derived macrophages in the injured spinal cord [3,23-25]. The success of these pre-clinical models prompted a Phase I clinical trial that was completed without any adverse effects. Implantation of macrophages preincubated with dermis was well tolerated. Of the eight patients with complete spinal cord injury, three recovered clinically significant neurological motor and sensory function [16].
Recent study of this group showed that augmenting the naive monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Notably, the enhancement of motor functions was associated with anti-inflammatory activity of infiltrating macrophages, mediated by inter-leukin 10 [28].
In this aspect, the M^3 subset described in our study is characterized by low pro-inflammatory/immunogenic properties and high regenerative potential and therefore may represent new candidates for cell therapy in CNS injuries.
Acknowledgements
The authors declare no competing interests. References
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© The Authors. This article is provided under the following license: Creative Commons Attribution 3.0 Unported, http://creativecom-mons.org/licenses/by/3.0/
Please cite this article as follows: Elena R. Chernykh, Ekaterina Ya. Shevela, Ludmila V. Sakhno, Marina A. Tikhonova, Yaroslav L. Petrovsky, Alexander A. Ostanin. The generation and properties of human M2-like macrophages: potential candidates for CNS repair? Cell Ther Transplant. 2010;2:e.000080.01. doi:10.3205/ctt-2010-en-000080.01
Ссылка: Клеточная терапия и трансплантация, 2010;2:e.000080.01. doi:10.3205/ctt-2010-en-000080.01
М2-подобные макрофаги у человека: потенциальные кандидаты для стимуляции репаративных процессов в ЦНС?
Елена Р. Черных, Екатерина Я. Шевела, Людмила В. Сахно, Марина А. Тихонова, Ярослав Л. Петровский, Александр А. Останин
Резюме
Регуляция иммунного ответа представляется перспективной стратегией в области восстановления повреждений центральной нервной системы (ЦНС). При этом важная роль в качестве кандидатов для клеточной терапии отводится макрофагам. Используя культуральные условия с низким содержанием сыворотки, мы разработали протокол генерации противовоспалительных, М2-подобных, макрофагов из моноцитов периферической крови и сравнили эти клетки (обозначенные как М3) со «стандартными» провоспалительными (Мф1) и противовоспалительными (Мф2) макрофагами, генерированными, соответственно, в присутствии GM-CSF и M-CSF.
Основное внимание было прежде всего сосредоточено на способности макрофагов стимулировать пролиферацию Т-клеток, а также продукцию макрофагами различных цитокинов, хемокинов и ростовых факторов. Дефицит сывороточных факторов не сказывался негативным образом на количестве генерированных макрофагов. Напротив, наибольший выход клеток наблюдался в культурах M3. По своим свойствам M3 макрофаги больше походили на Мф2, чем на Мф1. Так, в отличие от Мф1, макрофаги Мф2 и, особенно, M3 отличались относительно низким уровнем экспрессии CD86 и не стимулировали пролиферативный ответ Т-клеток. В противоположность провоспалительным Мф1 нестимулированные M3 продуцировали гораздо меньшие уровни провоспалительных (IL-ф, TNF-a, IL-6, IL-18, IL-12) и Th1/ Th2 цитокинов (IFN-y, IL-2, IL-4), вместе с тем - более высокий уровень IL-10. Более того, концентрации IL-ф и провоспалительных хемокинов IL-8 и MCP-1 в супернатантах M3 были снижены не только по сравнению с Мф1, но также и с Мф2 культурами. Подобно Мф1 и Мф2, М3 обладали способностью продуцировать нейротрофические (BDNF, IGF-1), ангиогенные (VEGF) и другие ростовые факторы с нейропротективной и регенераторной активностью (EPO, G-CSF, FGF-basic, EGF). При этом уровень продукции IGF-1 макрофагами 3-его типа превышал секрецию этого фактора Мф1 и Мф2 более чем в 25 раз.
Суммируя полученные данные, можно заключить, что генерируемые M3 клетки представляют M2-подобные макрофаги с высоким регенераторным потенциалом.
Ключевые слова: поляризация макрофагов, цитокины, хемокины, ростовые факторы, восстановление повреждений ЦНС
Ссылка: Клеточная терапия и трансплантация, 2010;2:e.000080.01. doi:10.3205/ctt-2010-en-000080.01
