LPS and/or IFN-γ in vitro, stimulate cells of the murine RAW264.7 line along the differentiation pathway of secreting macrophages : induction of TNFα and NO production
We first made a detailed kinetics analysis of the response of RAW 264.4 cells to in vitro stimulation by LPS, or IFN, or both. We confirmed that these products stimulated the differentiation of the cells toward the phenotype of activated macrophages releasing NO and secreting TNFα. In our experiments, RAW 264.7 cells were exposed in vitro to either 50 units/ml of IFN-γ (IFN50), or 5 μg/ml LPS (LPS5), or a mixture of both stimulating products. Cell free culture supernatants were harvested at 0, 6, 24 and 48 H and assessed for the production of TNFα, nitrites/nitrates and citrullin. Titrating TNFα production by ELISA, the strongest stimulation was observed with either LPS or LPS + IFN, leading to abundant secretions of TNFα as early as 6 H after the beginning of the culture, with a subsequent plateau at 24 and 48 H. The quantities of TNFα released by 0.25 × 106 cells initially seeded were in the range of 1400–1700 pg/ml at 24 and 48 H. Stimulation by IFN alone was less efficient inducing roughly only half of the above TNFα production (table ). In the same supernatants, the production of NO was evaluated by quantifying citrulline and the total nitrite/nitrate with the Griess reagent. Both products were titrated in quantity (15–30 μM range and 30–70 μM range respectively) in 24 and 48 H culture supernatants of RAW 264.7 cells stimulated by either LPS or LPS+IFN. Smaller quantities were titrated in supernatants of cells stimulated by IFN alone (Table ). Parallel experiments were performed to monitor NOs2 (iNOs) mRNA induction and NOs2 (iNOs) protein synthesis in RAW 264.7 cells exposed in vitro to either 50 units/ml of IFN-γ (IFN50), or 5 μg/ml LPS (LPS5), or a mixture of both stimulating products. Cells were pelleted at 0, 6, 24 H and 48 H. NOs2 (iNOs) mRNA was evaluated by semi-quantitative RT-PCR (Figure ), and NOs2 (iNOs) protein intracellular level by immunoblot (Figure ). NOs2 (iNOs) was undetectable both at the mRNA and protein levels in unstimulated RAW 264.7 cells. It was strongly induced at times 6, 24 and 48 H with the three stimulation protocols (Figure &). Only slight variations were noted depending of the stimulations used; IFN-γ alone being as previously, the weakest inducing stimulus compared to LPS and LPS + IFN-γ.
Differentiation of RAW 264 7 cells toward secreting-activated macrophages following culture with LPS, IFN-γ or LPS+IFN-γ
Figure 1 A/ Growth rate and survival of RAW 264.7 cells cultured in medium, or medium supplemented with LPS or IFN-γ or LPS+IFN-γ. RAW 264.7 cells were cultured (4 × 106 cells in 20 ml) in medium (• œ) or medium supplemented (more ...)
Death induction of RAW 264.7 cells stimulated by IFN-γ and/or LPS in vitro, with selection of a fraction of surviving activated RAW 264.7
In vitro exposure of cells of the murine macrophage cell line RAW 264.7 to either 5 μg/ml LPS (LPS5), or 50 units/ml of IFN-γ (IFN50), or a mixture of both stimulating products, led to a substantial reduction of the growth rate and number of surviving cells after 24 H or 48 H in culture (Figure ). The strongest deleterious effects were observed culturing the cells with either LPS alone or both IFN and LPS, resulting in the recovery of only approximatively 20% viable cells as estimated by MTT assay (or trypan blue test) at 24 H, and 13% at 48 H. IFN alone induced less cytoxicity with 54% and 50% viable cells recovered at 24 H and 48 H respectively (Figure ). The surviving cells subsequently recovered an in vitro growth rate comparable to untreated parental RAW 264.7 cells in culture (not shown). Additional experiments indicated however that those surviving activated macrophages had acquired peculiar resistance capacity to exogenous oxydative and nitrosative stress. More experiments were performed to characterize this resistance phenotype and the differentiation induced cellular metabolic events responsible for this resistance.
Nitrosative stress resistance of RAW 264.7 macrophages surviving LPS, or IFN, or LPS+ IFN, stimulation
We next established that the selected population of activated macrophages surviving in vitro stimulation by either LPS, or IFN, or LPS+ IFN, had acquired a nitrosative stress resistance phenotype. RAW 264.7 cells, recovered from 24 H stimulated cultures with LPS, or IFN, or LPS+ IFN, or from unstimulated cultures (control parental cells), were washed and seeded in fresh culture medium in the presence of 1 mM of the NO releasing compound diethylenetriamine nitric oxide (DETA-NO). This concentration had been established previously to reproducibly induce the apoptotic death of about 50% unstimulated parental RAW 264.7 cells after 48 H in vitro (Figure ). The susceptibility of LPS, or IFN, or LPS+ IFN activated macrophages to the NO releasing compound DETA-NO, was evaluated in vitro by measuring the number of metabolically active cells (reducing Formazan = MTT assay) or trypan blue negative cells, recovered at the indicated time. We established previously that the MTT test provides a reproducible quantitative estimate of cell viability giving comparable results with the trypan blue dye exclusion method. As can be seen in Figure , unselected parental RAW 264.7 cells were susceptible to DETA-NO death induction in vitro, with only 55% cells surviving after 24 H, and 45% after 48 hours. By contrast, LPS, or IFN, or LPS+ IFN activated RAW 264.7 macrophages were less susceptible to DETA-NO death induction in vitro, with variations depending upon the stimulation protocol.(Fig ). Activated-macrophages recovered from cultures stimulated with LPS alone were almost completely resistant to the nitrosative stress. Activated-macrophages recovered from IFN, or IFN + LPS stimulated cultures, exhibited an intermediate NO resistance, with about 70% and 60% metabolically active cells, or trypan blue negative cells, recovered after 24 or 48 H in culture with DETA-NO.
Figure 2 A / Susceptibility of RAW 264.7 undifferentiated cells to various doses of the NO donor compound DETA-NO in culture. RAW 264.7 cells seeded at 5.104 per well in 96 wells plates were cultured in medium alone (●) or medium supplemented with 125 (more ...)
Upregulation of redox protection/detoxification systems in LPS-differentiated NO resistant RAW 264.7 cells
LPS (and/or IFN) induced NO resistance of differentiated RAW 264.7 macrophages could result from an up-regulation of their cellular redox protection/detoxification systems. We therefore evaluated the LPS (and/or IFN) induced modulation of a number of redox proteins, critical in the three major systems protecting cells against oxydative and nitrosative stress : the superoxide dismutase/catalase system (Cu/Zn SOD, Mn SOD and catalase); the glutathione system (glutathione (GSH), γ-glutamyl cystein synthase (γ-GCS), glutathione peroxidase (Gpx), gluthatione reductase (GR); and the thioredoxin system (thioredoxin (Trx), thioredoxin reductase (TR). The transcriptional modulation of several of these redox proteins was evaluated by semi-quantitative RT-PCR on 24 hours cell extracts, using the specific primers described in table . As shown in Figure , Cu/Zn SOD, Mn SOD, and catalase, were significantly upregulated (130 to 200 % increased) in NO resistant activated RAW 264.7 macrophages obtained following 24 hours stimulation with LPS (and/or IFN). By contrast, γ-glutamyl cystein synthase (γ-GCS) and glutathion peroxidase (Gpx) were only marginally increased with IFN, while glutathione reductase (GR), thioredoxin (Trx) and thioredoxin reductase (TR) mRNA were not significantly affected. This gene transcription study was further documented by measuring cellular concentrations of glutathione (GSH) in lysates of activated RAW 264.7 macrophages, recovered 6, 24 or 48 hours after LPS (and/or IFN) stimulations. As can be seen in Figure , glutathione level was approximatively 3.5 folds increased at 6 hours in LPS stimulated RAW 264.7 cells, compared to basal level in unstimulated control RAW 264.7 cells. This LPS induced high glutathione level was still detected at 24 and 48 hours (2.6 and 5.2 fold increased respectively). IFN50 and IFN50/LPSs stimulations also increased intra cellular glutathione levels in RAW 264.7 cells, with the same kinetics than LPS alone, although with variable efficiency (Figure ). Additional experiments were performed to quantify the cellular specific activity of several other redox protection proteins in lysates of RAW 264.7 macrophages, recovered 6, 24 or 48 hours after LPS (and/or IFN) stimulations. As can be seen in table , catalase activity was approximately two folds increased at 6 and 24 hours after LPS stimulation of RAW 264.7 cells, compared to basal level in unstimulated cells, (but not after IFN50 or LPS5/IFN50 treatments), returning to basal level at 48 H. A sharp seven fold increased SOD activity was observed at 6 H in RAW 264.7 cells stimulated with IFN50 and IFN50/LPS5, which was not observed at any other time point, nor with the other stimulation protocols. No significant changes in Gpx activity was observed in stimulated cells.
Pairs of synthetic primers used in RT-PCR to amplify the different molecular systems studied.
Figure 3 Transcriptional modulation of important redox proteins in RAW 264.7 either undifferentiated, or differentiated cells. RAW 264.7 cells were cultured (2.5 × 106 cells/well) in medium, or medium supplemented with either LPS (5 μg/ml – (more ...)
Figure 4 Glutathione concentrations in RAW 264.7 either undifferentiated, or differentiated cells. Cells were cultured for the indicated times in medium, or medium supplemented with 5 μg/ml LPS, 50 U/ml IFN-γ or a mixture of IFN-γ (50 U/ml) (more ...)
Activities of the redox enzymes catalase, Gpx and SOD, in differentiated NO resistant RAW 264 7 cells
LPS induced NO resistance of differentiated RAW 264.7 macrophages is abrogated by chemical inhibitors affecting the intra-cellular redox protection/detoxification systems
The causal relationship between the acquisition of NO resistance and the upregulation of a selective set of redox protection/detoxification systems in LPS differentiated RAW 264.7 macrophages was established using chemical inhibitors targeting those redox systems. The compounds used were relatively specific inhibitors affecting the SOD/catalase system and the glutathione system, the two redox protection/detoxification biochemical sets significantly up-regulated in LPS-differentiated macrophages. DETC is an inhibitor of both Mn SOD and Cu/Zn SOD, ATZ is a catalase inhibitor, and BSO reduces the intra-cellular level of glutathione by inhibiting the γ-glutamyl cysteine synthase. In our experiments, RAW 264.7 cells were first differentiated in vitro, as previously, by stimulation with an optimal dose of LPS (5 μg/ml) for 48 h. Selected-surviving-LPS-differentiated RAW 264.7 macrophages (and control unstimulated RAW 264.7 cells) were washed and incubated for an additional 2 hours period in the presence of the redox inhibitors (or not). Subsequently, nitrosative stress resistance of the treated cells was evaluated by culturing them in the presence of 1 mM DETA-NO and assessing cell viability after 24 hours (number of metabolically active cells reducing formazan = MTT test). The results are presented in Figure . LPS differentiated macrophages were 90–100% resistant to the toxic effects of NO released by 1 mM DETA-NO in vitro, while unstimulated cells were susceptible to NO, with 45% cells only surviving to the same in vitro treatment. Noteworthy, incubation of LPS differentiated cells in the presence of the redox inhibitors abrogated their NO resistance and restored their susceptibility to the toxic effects of DETA-NO. The SOD inhibitor DETC was the most effective compound. It completely reversed the NO resistance status of LPS-differentiated macrophages : only 10% of DETC-treated LPS-differentiated macrophages were recovered at 48 hours following exposure to DETA-NO; compared to 90% recovery of LPS-differentiated macrophages cultured without DETC (Figure ). The two others redox inhibitors BSO (γ-GCS inhibitor) and ATZ (catalase inhibitor), also very significantly abrogated NO resistance in LPS-differentiated macrophages (with only 35% and 30% viable cells recovery following DETA-NO exposure of LPS-differentiated macrophages treated with these two compounds, respectively) (Figure &). In addition we noted that the SOD inhibitor DETC substantially sensitized unstimulated RAW 264.7 cells to the toxic effects of DETA-NO : only 10% DETC treated unstimulated RAW 264.7 cells survived NO exposure, compared to 40% control unstimulated RAW 264.7 not incubated with DETC (Figure ).
Figure 5 LPS induced NO resistance in differentiated RAW 264.7 macrophages is abrogated by chemical inhibitors affecting the intra-cellular redox protection/detoxification systems. RAW 264.7 cells were first treated in vitro, as previously, culturing them in medium (more ...)