After 24 h, the media was replaced with the following treatments: 1. indicates that SIRP- receptor can have pro-phagocytic activity. assay The present protocol was optimized based on our previously NSC87877 published work . Briefly, 2.2105 RAW 246.7 cells were seeded in complete media onto 6-well plates and incubated at 37C for 24 h, and 50 nM of PMA was then added. After 48 h, the medium was replaced with either NSC87877 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-/LPS; 5. SFM+PMA+IFN-/LPS; 6. CM+PMA+IFN-/LPS+anti-SIRP-a Ab (5 l of a 0.03 g/5l Ab solution); 7. SFM+PMA+IFN-/LPS+anti-SIRP- Ab. The cells were incubated at 37C for another 24 h. RBCs previously dyed (Cell NSC87877 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 TFRC 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. Open in a separate window Figure 2 Role of SIRP- in the phagocytosis of RBCs by macrophages in vitroshowed that SIRP- expression on monocytes in autoimmune hemolytic anemia (AIHA) was higher than in a healthy population. When monocytes from AIHA were treated with dexamethasone, SIRP- expression was reduced without affecting erythrophagocytic activity. Although this study also indicates that erythrophagocytosis is independent on SIRP- expression , the that monocytes in their study were only treated with steroids and there were no stimulants or inducers used, in contrast to our study. Moreover, the possibility of pro-phagocytic activity of SIRP- was not ruled out by using anti-SIRP- antibodies. Takuro Kuriyama et al. hypothesized that HLH occurs as a result of SIRP-/CD 47 interaction, which is in accordance with our hypothesis. However, they found that hematopoietic stem cell (HSC) CD 47 NSC87877 expression was decreased in HLH in correlation with cytokine surge. They also concluded that SIRP- expression or mutation is irrelevant to HLH. It should be noted that their study focused mainly on granulocytes, megakaryocytes, and erythrocyte progenitors to prove their theory. However, the mature forms of these cells are also phagocytosed, which means that HLH pathogenesis can include other pathways. Furthermore, the SIRP- expression and mutations assay were analyzed using blood samples from 15 patients with HLH, but the authors did not mention whether those patients were receiving treatment, which makes a huge difference in data interpretation . In our study, we used mature erythrocytes from CD 47+/+ mice to exclude the possibility of phagocytosis being carried out due to immaturity. Also, as mentioned previously, the fact that SIRP- expression and the treatment anti-SIRP- antibody in non-stressed macrophages did not alter significantly the phagocytosis rate is compatible with these above-mentioned studies. Anti-SIRP- antibody was used as potential treatment against melanoma and renal cell carcinoma cells in a study done by Yanagita et al. . This study also proves that SIRP- has anti-phagocytic function, but since NSC87877 our results showed that inhibition of SIRP- in stressed macrophages led to decreased phagocytosis, this may indicate that SIRP- receptors go through morphological changes during phagocytosis and become pro-phagocytic. SIRP- phagocytic activity is supported by a study by Burger et al., which demonstrated that CD 47 receptors on aged RBCs go through conformational changes that eventually act as a target for phagocytosis by pulp macrophages in the spleen after binding to SIRP- . Another study, by Tada et al., revealed that apoptotic cells are engulfed by macrophages via CD 47/SIRP- interaction, which is mediated by phosphatidylserine . This study indicates.