Phagocytosis-Mediated Acute Hemolytic Events Induce Distinct Responses By Inflammatory Monocytes
Authors: Youssef Lyla A , Weisberg Stuart Phillip , Bandyopadhyay Sheila , Hod Eldad A. , Spitalnik Steven L 
Published: October 2019

Abstract
Introduction
Red blood cell (RBC) transfusions are a common therapy, with ~15 million RBC units administered annually in the United States. Studies in mice and dogs identified increased circulating levels of multiple inflammatory cytokines after transfusions of refrigerator storage-damaged RBCs. One chemokine identified was monocyte chemoattractant protein 1 (MCP-1; also known as CCL2). MCP-1 is the ligand for CCR2, which is expressed by several cell types including Ly6Chi (i.e., inflammatory) monocytes. One reserve site of Ly6Chi monocytes is the bone marrow, from which they emigrate into the circulation in a CCR2-dependent manner to traffic to sites of inflammation. Because the spleen is a primary site of RBC clearance, we aimed to identify splenic cells responsible for producing MCP-1 following transfusions of storage-damaged or antibody-coated RBCs.
Methods
Leukoreduced murine RBCs were prepared from wild-type (WT) C57BL/6 donors and refrigerator stored for 12-13 days. MCP-1 transgenic reporter mice were transfused with fresh, or storage-damaged ("old"), or fresh anti-RBC antibody-coated RBCs. Spleen, bone marrow, and blood were collected post-transfusion and cells were analyzed by multi-parameter flow cytometry. WT C57BL/6 mice were also transfused with fresh or old RBCs; splenocytes isolated post-transfusion were flow sorted followed by RNA isolation and RT-PCR.
Results
Splenic red pulp macrophages (VCAM1hi, F4/80hi, CD11blo) were primarily responsible for erythrophagocytosis after transfusions with old or antibody-coated RBCs. However, in each case, Ly6C hi, CD11b+ splenic inflammatory monocytes in MCP-1 reporter mice expressed MCP-1. MCP-1 expression by these cells was also confirmed in WT recipients by RT-PCR after flow sorting. Interestingly, only a small percentage of inflammatory monocytes ingested transfused RBCs. Furthermore, circulating inflammatory monocyte levels increased following transfusion of old or antibody-coated RBCs, accompanied by reduced levels in the bone marrow.
Conclusions
Although red pulp macrophages were the major cell type responsible for clearing transfused refrigerator-damaged or antibody-coated RBCs, a different splenic cell population (i.e., inflammatory monocytes) produced MCP-1. Thus, the splenic reserve of inflammatory monocytes produced an inflammatory response following phagocytosis-mediated acute hemolytic events. Increased numbers of circulating inflammatory monocytes and reduced numbers in the bone marrow suggest that these cells emigrated from the bone marrow, perhaps in response to MCP-1 signaling. Thus, one possible mechanism explaining our results is that erythrophagocytosis in the spleen induces an inflammatory response, triggering splenic inflammatory monocytes to synthesize MCP-1 and release it into the circulation; MCP-1 then binds CCR2 on bone marrow inflammatory monocytes, causing their egress into the circulation. The additional signals involved in these phenomena, along with their clinical relevance to transfusion medicine, require additional investigation.

Disclosures
No relevant conflicts of interest to declare.