Effect of Serum Deprivation Stress on Signal Induction Regulatory Protein-Alpha (SIRP-Alpha)-Mediated Erythrophagocytosis by Macrophages

Background

Macrophages are powerful phagocytic cells that are involved in inflammation, innate immune response, and repair of damaged tissues [1]. They also assist in erythrocyte synthesis and maturation [2]. Macrophages avoid phagocytosing self-cells through surface receptor interactions. One of these receptors is the signal induction regulatory protein alpha (SIRP-α) [3], which is expressed on the surface of macrophages and dendritic cells, and primarily acts to inhibit the phagocytosis of Cluster of Differentiation 47 (CD47)- expressing cells, such as erythrocytes [4]. CD47-SIRP-α interaction functions as a signal for self-cell recognition, hence foreign cells that lack CD47 are more readily phagocytosed than those expressing it. The expression of CD47 decreases in erythrocytes as they age, resulting in higher rates of phagocytosis [5]. In rare circumstances, abnormal macrophage behavior results in failure of self-cell recognition, leading to phagocytosis of erythrocytes, leukocytes, and platelets, a condition known as hemophagocytic lymphohistiocytosis (HLH) [6].

HLH can be primary (familial) when genetic mutations are involved, or can be secondary to infections, autoimmune conditions, or malignancies [7]. During inflammatory process, macrophages interact with different cells, including natural killer (NK) cells and cytotoxic T lymphocytes (CTL), which play an important role in regulating macrophage activity. Impaired NK and CTL functions during inflammation can cause loss of inhibition of macrophage activity and continued cytokine stimulation, which accounts for the pathophysiology of HLH [6,8].

Macrophage phagocytic function can also be influenced by other cytokines and cell signals. Xaus et al. found that lipopolysaccharides (LPS) can impede macrophage phagocytosis by inhibiting the major histocompatibility complex II (MHC II) expression [9]. Moreover, Bian et al. showed that LPS can increase phagocytosis towards self-cells by downregulation of SIRP-α [10]. On the other hand, Xaus et al. demonstrated that initial activation by interferon-gamma (INF-γ) rendered macrophages unresponsive to apoptotic signals by the induction of P21WAF1. Additionally, Martinet et al. showed that macrophage phagocytic activity against heat inactivated gram-positive and gram-negative bacteria was increased by nutrient deprivation, thus demonstrating the crucial role of macrophage environment in regulating phagocytic activity [11]. Our hypothesis is that erythrophagocytosis can occur as a result of failure of macrophage self-recognition of erythrocytes. In this study, we explored the possible correlation between SIRP-α expression and rate of phagocytosis.

Material and Methods

CELL CULTURE AND TREATMENTS:

RAW264.7 macrophage cells (ATCC, Manassas, VA) were seeded in 6-well plates in CM, composed of Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin (GE Healthcare Life Sciences, Logan, UT). For macrophages differentiation into M1, cells were incubated in a 5% CO₂ incubator at 37°C for 24 h, and 50 nM of PMA was then added. After 48 h, the medium was replaced with either CM+/− PMA or SFM+/− PMA, and cells were incubated at 37°C for an additional 24 h. IFN-γ (Sigma-Aldrich, St. Louis, MO) and LPS (Sigma-Aldrich, St. Louis, MO; 10 μM each) were added to both media and incubated for 24 h. The experiment was repeated 3 times for consistency.

CD47 STAINING:

RBCs with CD 47+/+ (tested for CD 47 positivity by fluorescence-activated cell sorter) were bought from Astarte Biologics (Bothell, WA). They were kept at 4°C for up to 1 month. For each experiment, 1 ml of RBCs from the main stock vial was centrifugated at 960 rpm for 8 min at a temperature of 4°C. Supernatant was aspirated and the cell pellet was gently re-suspended in 1 ml of phosphate-buffered solution (PBS) containing 5% of bovine serum albumin (BSA). We then added 100 ng/μl of the CD47 antibody (Ab; Clone miap301; BD Pharmingen, San Jose, CA). The cell suspension was gently mixed and incubated at 37°C in a 5% CO2 incubator for 1 h. Cells were then washed and re-suspended in 1 ml of PBS solution containing 5% of BSA. A 10-μl cell aliquot was placed onto the center of a glass slide, a cover-slip was placed over the top, and the CD47 staining was assessed using a Zeiss AxioVert 200M microscope (63X oil objective). Percentages of CD47-positive cells were calculated per field of view in more than 20 randomly selected fields (Figure 1A). The experiment was repeated 3 times.

RNA EXTRACTION AND QUANTITATIVE REVERSE TRANSCRIPTASE PCR (QRT-PCR):

Total RNAs were isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was then synthesized from 1 μg of RNA using the Verso cDNA kit (Thermo Scientific, Pittsburgh, PA). To measure SIRP-α cDNA, PCR was performed using the FastStart Universal SYBR Green Master (Roche, Mannheim, Germany) on a MyIQ real-time PCR system (BioRad, Hercules, CA) using SIRP-α-specific primers (Cat# PPM05351E-200, Qiagen, Valencia, CA), and following the defined program: 1× heating for 10 min at 95°C; 40× denaturing for 15 s at 95°C, followed by annealing/extension 1 minute at 60°C/60× extension for 20 s at 60°C. Transcript levels were normalized to the S15 housekeeping gene (Forward: 5′-CAACGGCAAGACCTTCAAC-3′/Reverse: 5′-GGCTTGTAGGTGATGG AGAAC-3′). The CT values were determined by automated threshold analysis with MyIQ version 1.0 software. SIRP-α fold change was then calculated using the ΔΔCt method. Each treatment sample was tested in triplicate. The size of the amplicon was then verified on a 2% agarose gel. The experiment was repeated 3 times for consistency.

:

The present protocol was optimized based on our previously published work [13]. Briefly, 2.2×105 RAW 246.7 cells were seeded in complete media onto 6-well plates and incubated at 37°C for 24 h, and 50 nM of PMA was then added. After 48 h, the medium was replaced with either CM+/− PMA or SFM+/− PMA. After 24 h, the media was replaced with the following treatments: 1. CM; 2. CM+PMA; 3. SFM+PMA; 4. CM+PMA+IFN-γ/LPS; 5. SFM+PMA+IFN-γ/LPS; 6. CM+PMA+IFN-γ/LPS+anti-SIRP-a Ab (5 μl of a 0.03 μg/5μl Ab solution); 7. SFM+PMA+IFN-γ/LPS+anti-SIRP-α Ab. The cells were incubated at 37°C for another 24 h. RBCs previously dyed (Cell Tracker Red CMTPX dye-Life Technologies) as recommended by the manufacturer (Figure 1B) were then added to the macrophages at a ratio 1: 10 (macrophages: RBCs) for 24 h. Confocal images were obtained using the Nikon T1-E microscope with A1 confocal and STORM super-resolution with a 60× objective (N.A. 1.4; oil; Z-stack). After imaging, Z-stacks were merged (Figure 2). Percent phagocytosis was determined by counting the number of macrophages containing red fluorescent over the total number of macrophages. Staining with Trypan blue (0.4% in PBS) was done to assess the viability of the RBCs before co-culturing with the macrophages, and 24 h after to verify that RBCs death was not due to the different treatments. The experiment was repeated 3 times for consistency.

STATISTICAL METHODS:

Data were analyzed using IBM SPSS Statistics for Windows version 23 (IBM Corp., Armonk, NY). Seven groups of different mixtures were identified and phagocytosis incidents were counted in each group. All data were summarized into frequencies and percentages. The chi-square test (χ²) was used to examine differences between groups of categorical data. All p-values were two-tailed. A p-value of <0.05 was considered significant. As for SIRP-α expression assay, the Mann-Whitney U test was employed.

Results

:

We measured SIRP-α mRNA expression under various conditions. SIRP-α expression was higher in SFM compared to CM conditions, and was also higher after adding IFN-γ/LPS (we used them because they are commonly produced in inflammation) to both CM and SFM (Figure 3, Table 1). The results of the phagocytosis assay showed that the rate of the phagocytosis was also higher in SFM conditions and IFN-γ/LPS combination conditions when compared to CM and conditions without IFN-γ/LPS, respectively (Tables 2, 3).

:

To determine the potential effect of SIRP-α in RBCs phagocytosis, we next used specific SIRP-α blocking antibodies and we compared its effect in stressed and unstressed conditions. The results showed that after adding anti-SIRP-α Ab to the SFM+PMA+Combo (representing stressed macrophages in serum free media), the phagocytosis rate significantly decreased by 28.4% (Table 2). However, adding those antibodies to the CM+PMA+Combo (representing stressed macrophages in complete media) did not affect the phagocytosis rate. A summary graph represents the relation between phagocytosis rate and SIRP-α mRNA expression in Figure 4.

Discussion

Conclusions

Self-recognition receptors like SIRP-α, when interacting with CD47, can normally inhibit phagocytosis during non-inflammatory conditions. However, in the presence of macrophage stressors (i.e., culturing in serum deprivation and induction by LPS/INF-gamma), SIRP-α expression increases along with phagocytosis rate. Moreover, blocking the SIRP-α receptor, under the effect of stressors (mainly serum deprivation), unexpectedly led to decreased erythrophagocytosis, which indicates that the SIRP-α receptor functions as pro-phagocytic mediator.