Abstract
Background: Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Materials and Methods: Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Results: Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. Conclusion: The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs.
Macrophages are phagocytic cells that play an essential role in providing innate immunity by internalizing pathogens and releasing cytokines. Macrophages are currently of great interest because they possess the possibility of killing and eliminating cancer cells (1-3). Activation of macrophages is important for their function. In response to various signals, macrophages undergo various modifications in their signaling pathways and develop into various sub-types of activated forms (4, 5). However, the details of the activation mechanism are yet to be determined. Macrophages are usually classified into M1 and M2 macrophages (5); however, heterogeneity of the activated macrophages was reported in 2014 (6). The authors suggested that activated states of human macrophages can be classified into 7 types based on their activation signals.
Recently, we established a novel assay protocol for assessing the macrophage-activating factor (MAF) activity using the monocytic cell lines U937 and THP-1 (7, 8). Thus, this is the first step in standardization of the MAF assay protocol. Characterization of these macrophages, which are derived from monocytic cell lines, is valuable for understanding the function of MAFs. In the present study, we compared gene-expression profiles of these monocytic cell line-derived macrophages using gene expression microarray analysis, focusing on the activation mechanism, and examined their responses against several MAFs by in vitro experiments.
Materials and Methods
Cells and cell culture. The U937 and THP-1 cell lines were maintained in RPMI-1640 medium supplemented with 10% foetal bovine serum (Hyclone, Logan, UT, USA), 3% L-glutamine and 10% sodium hydrogen carbonate. Cells were cultured in a 5% CO2/95% air fully humidified atmosphere at 37°C. To induce differentiation into macrophage-like cells, U937 and THP-1 cells were seeded onto 35-mm culture dishes (5.0×105 cells/dish) and incubated for 72 h and 24 h, respectively, with 10 ng/ml 12-o-tetradecanoyl-13-acetate (TPA; Sigma-Aldrich, St. Louis, MO, USA). TPA-treated U937 cells were then pre-treated with serum-free RPMI-1640 medium for 2 h (sensitization) before the phagocytosis assay.
Gene expression profiles. The gene expression profiles of U937 cells, THP-1 cells, differentiated and sensitized U937-derived macrophages (sU937) and differentiated THP-1-derived macrophages (dTHP) were analysed by Hokkaido System Science (Sapporo, Japan) using the SurePrint G3 Human GE Microarray Kit 8x60k (Agilent, Santa Clara, CA, USA), with version 1 being used for U937 cells and version 2 for THP-1 cells. For inter-array comparison, the relative amount of gene expression was calculated as normalized intensity value. This method of normalization was the 75-percentile shift, which uses the 75th percentile signal value as a standard. However, quantitative comparison between different versions of microarrays is not valid.
Reagents. Human recombinant interferon (IFN)-γ, human recombinant interleukin (IL)-4 and human recombinant IL-10 were obtained from Wako (Osaka, Japan). LPS from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich (Saint Louis, MO, USA).
Phagocytosis assay. The media for sU937 macrophages and dTHP macrophages were replaced by fresh medium containing MAF and 45 μg magnetic beads (Dynabeads® Protein G; Invitrogen, Oslo, Norway). Macrophages were photographed under a bright field and phase contrast microscopes at 30, 60 and 240 min after MAF addition. All the internalized and non-internalized beads were counted in these photographs. Phagocytic activities of macrophages were evaluated as the internalized beads ratio (IBR). This index was calculated by the following formula:
Results
Sub-type assignment of differentiated macrophages. In 2014, Murray et al. (6) suggested the classification of activated states of human macrophages into 7 types: M(IL-4), M(IL-10), M(GC), M(GC+TGF-β), M(LPS), M(LPS+IFN-γ) and M(IFN-γ). They described several marker genes for each subtype. In the present study, expression of each Murray's subtype-marker gene in sU937 and dTHP was analysed by gene expression microarray analysis (Table I). Both macrophages expressed ALOX5AP, CCL4, CCL5, IL1B, IL4R, IRF5, MMP9, RGS1, STAT1, TGFB1, TGFBR2 and TNF. The marker genes of all sub-types were expressed, while some genes of all sub-types, such as IRF4, SOCS1, MMP12, SMAD2, IL6 and CCL18, were hardly expressed. Thus, these macrophages could not be assigned into a single sub-type based on the marker gene expression. Although sU937 and dTHP have similar expression profiles for these marker genes, CD163, CXCL10, MARCO and MMP1 were dominantly expressed in sU937, whereas CD40, KYNU, STAB1 and TGM2 were dominantly expressed in dTHP. This suggested that the two macrophage types had different phenotypes.
Activated states of macrophages predicted by the cytokine-signalling pathway. Signalling pathways of cytokines are well-characterized (4, 5). When a certain cytokine signal is activated, all the genes involved in that pathway, including the receptors, adapters and transcription factors, are expressed. The expression of these factors of each signalling pathway was examined by gene expression microarray analysis (Table II). The gene with normalized intensity value more than 1.5 was considered to be expressed. According to this, IL-4, IL-10 and LPS signaling pathways could be activated in both sU937 and dTHP. However, in the IFN-γ pathway, although IFNGR1, IFNGR2, STAT1 and STAT2 were expressed, the expression of the adaptor proteins JAK1 and JAK2 was very low. These results also suggested that the activated state of the differentiated macrophages could not be assigned to a specific sub-type.
The response of differentiated macrophages to the activation signals. The results of the response of the macrophages are summarized in Figure 1. When the phagocytic activity of sU937 was measured with and without IL-4, the IBR values were 22.4±5.1% and 20.1±6.6%, respectively. The probability value (p-value) of the t-test of this difference was 18.3 (n=3). Thus, sU937 was not activated by IL-4. However, the phagocytic activity of dTHP was elevated from 61.3±2.8% to 72.5±3.9% by addition of IL-4. The p-value of this difference was 0.009 (n=3). Therefore, it showed that IL-4 activated the dTHP.
The phagocytic activities of sU937 with and without IL-10 were 26.8±19.9% and 25.2±3.4%, respectively, and the p-value of this difference was 0.63 (n=3). When the dTHP was cultured with and without IL-10, the phagocytic activities were 51.5±5.9% and 62.0±6.3%, respectively, and the p-value of this difference was 0.04 (n=3). Thus, the sU937 were not activated, whereas dTHP were activated by IL-10 under 95% confidence limits.
Finally, we examined the phagocytic activities of macrophages in the presence of LPS, IFN-γ or both and in the absence of activating factors. The phagocytic activities of sU937 under these conditions were 22.4±5.1%, 18.4±18.7%, 20.8±28.0% and 20.1±15.7%, respectively. The p-values of these differences with and without activating factor(s) were 0.19, 0.72 and 0.40, respectively. On the other hand, the phagocytic activities of dTHP under these conditions were 60.7±1.0%, 68.3±8.5%, 65.8±8.5% and 68.9±5.3%, respectively. The p-values of the difference with and without activating factor(s) were 0.14, 0.27 and 0.03, respectively. Thus, only simultaneous addition of LPS and IFN-γ to the dTHP resulted in activation under 95% confidence limits.
Discussion
Both in vitro and in silico analyses showed that neither sU937 nor dTHP could be assigned to a single sub-type of Murray's classification criteria (6). At the same time, both analyses revealed differences between sU937 and dTHP. These results simply suggested that these differentiated macrophages were unknown sub-types and might not exist at all in vivo for the reason that these macrophages were differentiated from cancer cell lines (9, 10). However, these cells being human cells is an important fact as human cell line-derived macrophages could represent the molecular mechanism of human macrophages more precisely than mouse cells, which are used for many MAF assays (11), since human cells share the same gene set or genome set. U937- and THP-1 cell line-derived macrophages could offer a new stable assay system for MAFs because the conditions for macrophages can be reproduced. Moreover, as these monocytic cell lines can be fully differentiated into macrophages by the addition of TPA (12), preparation of these macrophages could be rapid and easy.
Expression profile of marker genes in differentiated macrophages*.
At the same time, the characters of sU937 and dTHP were unique. Gene expression microarray analyses showed that the gene expression profiles were different. For example, although MMP9 was greatly expressed in both macrophage lines, MMP1 was almost specific for sU937; especially in the in vitro experiments, responses against the activation signals were largely different. Although differentiated macrophages were predicted to be activated by most of the activation signals from the pathway analysis, dTHP fitted this prediction but sU937 did not, as it was not activated. As reported in our previous articles (7, 8), sU937 could be drastically activated by another activating factor, serum MAF (prepared by the β-galactosidase and neuraminidase treatment of human serum; 11). The dU937, which was not pre-treated with serum-free medium, actively phagocytized beads (7). These results suggested several interesting points regarding the activation mechanisms of macrophages. (i) Foetal bovine serum contains some factors that are necessary for macrophage activation. (ii) Without such factor(s), some inhibitory effects were exerted on the phagocytic activity of macrophages. (iii) Activation mechanism of serum GcMAF was different from other MAFs. (iv) The culture condition easily changed the activation states of macrophages. Therefore, comprehensive studies on sU937 and dTHP are needed to clarify the activation mechanisms of macrophages with the ultimate goal to interfere in the development of effective cancer immunotherapy.
Phagocytic activities of U937- and THP-1-derived macrophages treated with MAFs. sU937 (A, C, E) and dTHP (B, D, F) were cultured in the presence of MAFs. MAFs used in this experiment are as follows: 20 ng/ml IL-4 (A, B), 20 ng/ml IL-10 (C, D), 1 μg/ml LPS and/or 5 ng/ml IFN-γ (E, F). Phagocytic activity was evaluated as the internalized beads ratio (IBR). Error bars represent SD (n=3). *: Insignificant differences between with and without MAFs (A, C, E). **Significant differences between with and without MAFs (B, D). §Only the difference between with and without LPS+IFN-γ was significant (F).
Gene expression of molecules in signalling pathway*.
Acknowledgements
We are grateful to Dr. M. Mette for critical reading of this manuscript. This work was partly supported by The Naito Foundation Subsidy for Dispatch of Young Researchers Abroad and the grant form Hyogo Scientific and Technology Association.
Footnotes
This article is freely accessible online.
- Received April 3, 2015.
- Revision received May 8, 2015.
- Accepted May 11, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
