Effects of ferumoxides-protamine sulfate labeling on immunomodulatory characteristics of macrophage-like THP-1 cells

Abstract

Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFkappaB pathway activation, as shown by immunobloting; TNF-alpha secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide the model for studying the effects of in vivo clearance of iron particles via incorporation into the host's macrophages that may follow after in vivo application of any type of magnetically labeled mammalian cells. To better mimic the complex in vivo scenario, this model may be further exploited by introducing additional cellular and biological, immunologically relevant, components.

Figure 1. Prussian blue staining of FePro labeled THP-1 cells.

THP-1 cells were labeled overnight with ferumoxide-protamine sulfate complex and stained using Prussian blue method to visualize the intracellular iron incorporation (A). Non-stained THP-1 cells were used as a negative control (B).

Figure 2. Effect of FePro labeling on THP-1 cell differentiation.

THP-1 cells were labeled with FePro complexes and then differentiated in the presence of 100 nM of TPA for 24 h. Phase contrast photomicrograhs of non-labeled THP-1 cells before (A) and 24 h after incubation with 100 nM of TPA (B). FePro labeled THP-1 cell before (C) and 24 h after incubation with 100 nM of TPA (D).

Figure 3. Effect of FePro labeling on the THP-1 cells expression of differentiation induced cell surface markers.

The data depicts the levels of protein expression in non-labeled (A) and FePro labeled THP-1 cells (B) after 24 h incubation with 100 nM of TPA and following 4 h incubation with 100 ng/ml of LPS. After incubation, cells were stained with FITC conjugated anti-CD11b, PE conjugated anti-CD117 and PE-Cy5 conjugated anti-HLA DR and anti- CD86. Flow cytometric histograms of the non treated (control) cells (solid green lines), TPA treated (solid blue lines) and TPA and LPS treated (solid yellow lines) from one representative experiment are shown (n = 3). At least 10,000 live-gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype control shown as solid blue histograms.

Figure 4. Effect of FePro labeling on THP-1 cell ability to respond to LPS stimulation.

Effect of LPS on the levels of CD83 and CD54 cell surface expression (A), TNF-α production (B) and activation of NFκB signaling pathway (C) in non-labeled and Fe-labeled THP-1 cells. (A) After incubation with LPS, cells were stained with PE conjugated anti-CD54 and anti-CD83 antibodies. Flow cytometric histograms of the non treated (control) cells (solid green lines), TPA treated (solid blue lines) and TPA and LPS treated (solid yellow lines) from one representative experiment are shown (n = 3). At least 10,000 live-gated cells were analyzed for PE expression. Isotype control shown as solid blue histograms. (B) TNF-α protein levels in THP-1 cells supernatants after 4 h of stimulation with 100 ng/ml of LPS, determined by ELISA. Data expressed as means±SD. * p<0.05 compared to non-labeled, TPA differentiated and LPS stimulated control cells whose maximum response was set to be 100%. (C) Images of Western blots of nuclear protein fraction probed with anti- p65NFκB antibody (top panel) and cytoplasmic protein fraction probed with anti-IκBα antibody (lower panel). The data depicts the levels of protein in non-labeled and FePro labeled THP-1 cells after 24 h incubation with 100 nM of TPA and following 30 minutes with 100 ng/ml of LPS. Single representative experiment is shown (n = 3). The same patterns of relative NFκB and IκBα expression were observed in all three experiments.

Figure 5. Effect of FePro labeling on THP-1 cell chemotaxis in response to MCP-1 and Rantes.

THP-1 cells incubated in the presence of MCP-1 (A) and Rantes (B). Cell migration determined by fluorescence based assay, values expressed as units of fluorescence. Experiment included non-labeled and FePro labeled THP-1 cells incubated without the treatment (white bars), incubated in the presence of 10% FBS (grey bars) and 300 ng/ml of MCP-1 or 50 ng/ml of Rantes (black bars). Bars, means±SD. * p<0.05 compared to non-labeled control cells.

Figure 6. Effect of FePro labeling on THP-1 cell ability to modulate T cells.

The data depicts the levels of CD markers expression after 24 h of THP-1 cell-T cell co-culture. T cells were stained with FITC conjugated anti-CD3, PE/Cy5 conjugated CD4 and PE conjugated anti-CD8. Cells were gated based on the forward and side scatter characteristics (R1) and on the expression of CD3 marker (R2) (A). Flow cytometric histograms of T cells cultured without the presence of THP-1 cells (red line), T cells co-cultured with non-labeled THP-1 cells (blue line) and T cells co-cultured with FePro labeled THP-1 cells (green line) from representative experiment are shown. THP-1 cells induced modulation of T cell expression levels of CD4 and CD8 (B). Note the complete overlap of blue and green lines. At least 10,000 live-gated cells were analyzed for CD4 and CD8 expression.

Figure 7. Effect of FePro labeling on THP-1 ability to induce T cell proliferation.

Non-labeled and FePro labeled, gamma irradiated, THP-1 cells co-cultured with T cells for 7 days. MTT proliferation assay performed at day 3 (gray bars) and day 7 (black bars). Data are presented as a mean±SD of values of optical density measured at 570 nm wavelength. Statistical analysis was performed by comparing proliferation at day 7 with day 3 within the group (labeled or non-labeled THP-1 co-culture and labeled or non-labeled THP-1 cells only or T cells only) and by comparing proliferation at day 7 between the groups (labeled vs. non-labeled THP-1 co-culture). * = p<0.05 day 7 compared to day 3. No significant difference was observed between labeled and non-labeled conditions in co-culture and in THP-1 cells only samples.