Mechanistic insight in clearance of RBC by spleen macrophages

One of the most relevant aspects of RBC biology in the context of RBC transfusion, and one of the least understood, is the clearance of RBC in vivo. The mechanism of removal of RBC under homeostatic conditions is an issue of continuing debate. Moreover, various clinical conditions cause accelerated RBC clearance, which often leads to anemia. Examples of exaggerated breakdown of RBC are anemia of inflammation, in which random destruction of RBC by macrophages occurs, autoimmune haemolytic anemia and inborn RBC defects such as sickle cell disease. In these conditions RBC are frequently given as a cellular therapy to correct the anemia. However, there can be a high degree of removal of donor RBCs, which ranges from levels as high as 10 to 25% within 24 hours after transfusion, depending on the storage time. Furthermore, donor RBC removal can be influenced by the condition of the recipient, a phenomenon that is well recognised but ill understood.

In this context, our goal is to understand the rapid removal of donor RBC after transfusion. Although various improvements over time in the processing and storage of RBC have certainly had their positive effects on the recovery, safety, and efficacy of RBC as a transfusion product, there is clearly significant room for improvement. Most studies on the effects of RBC transfusions have been observational, and have not provided any mechanistic insight(s). For circulating RBC, the cell types primarily responsible for the uptake are the macrophages in the red pulp of the spleen and the Kupffer cells in the liver.

While there is a growing awareness that there is a great deal of heterogeneity among macrophages, the specialized properties of these particular macrophage subpopulations are only poorly understood. Fortunately, we have the opportunity to obtain fresh samples of healthy human spleens, derived from organ donors. This opened up the unique possibility to isolate these primary human red pulp macrophages and to study their interactions with RBC. By doing so, we have identified a new mechanism of recognition, based on the conformation dependent interaction of CD47 on the RBC and signal regulatory protein alpha (SIRPα) on red pulp macrophages of the spleen2. This illustrates that the availability of these spleen samples are instrumental in identifying pathways that can contribute to clearance of RBC. 

Monitoring clearance of RBC after transfusion

As indicated above, up to 25% of the donor RBC can be cleared within 24 hours after transfusion. This extensive breakdown of RBC is a burden for the patients that require transfusion. The patients that receive blood transfusions can be divided into different groups. First, there is a group of patients that receives red cell concentrates (RCCs) to correct a long lasting anemia, and is therefore repeatedly transfused. These patients build up iron deposits in their organs, mostly due to the primary cause of the anemia such as sickle cell disease or other types of inborn RBC defects, but also during myelodysplasia. This iron accumulation often leads to tissue damage and organ failure in the long run. Clearly, extensive breakdown of donor RBC comprises an additional burden on the iron metabolism of this group of patients.

Another group that receives RCCs is the patients that suffer from anemia of inflammation, for instance critically ill patients at the intensive care unit (ICU). Transfusion of RCCs in this patient group has recently received much attention, since it is believed that blood transfusion in critically ill patients can also be detrimental and may actually contribute to mortality and morbidity. Mechanisms underlying this detrimental effect are not fully understood, but rapid destruction of the transfused RBC both via hemolysis as well as phagocytosis of the donor RBC are generally believed to contribute to inflammation, especially in critically ill patients. Patients suffering from anemia of inflammation are a complex, yet very interesting, patient group in which to study RBC survival. The anemia seen in these patients is usually caused by both a defect in the production of red blood cells as well as a large increase in the breakdown of the circulating RBC. Macrophages in the liver and spleen of these patients are randomly phagocytosing and degrading RBC and other blood cells due to the systemic inflammation that these patients suffer from.

Clearly, it is to be expected that the clearance of donor RBC in this patient group will differ from the clearance seen in the first group, which do not actively degrade their circulating RBC. The last group of patients that receive red blood cell transfusions consists of patients who are actively bleeding, either due to surgery or to trauma. Again, the effects of RBC transfusion are extensively studied in this group of patients, but the rate of donor RBC clearance is not studied in detail. To be able to study donor RBC survival and phenoype in time, and to obtain clues on their removal, we have recently adopted a protocol to biotinylate donor RBC prior to transfusion. This procedure provides an easy and safe approach to “track” donor RBC after transfusion. We have recently used this approach succesfully in a model of autologous transfusion in healthy volunteers. In the future, we plan to perform similar studies in different cohorts of donor RBC recipients.

Immune adherence clearance (IAC), transfer of pathogens by red blood cells

Another line of research we are working on and which will be part of our research in the future is the interaction of RBC with pathogens and macrophages. RBC are equipped with the immune adherence receptor, complement receptor 1 (CR1), through which they can bind opsonized pathogens as well as immune complexes. This cargo can then be transferred to phagocytes, through shedding of part of the RBC membrane as microvesicle, and then subsequently degraded. In this manner RBC play a role in pathogen transport and as such contribute to host defense during infection, a process that has been termed immune adherence clearance. Although this model for immune adherence clearance is basically rather simple, we have found that during this process also direct interactions take place between the RBC and the phagocyte, such as monocytes and macrophages from the spleen, and these were until now completely unknown.

This firm adhesion between RBC that carry opsonized pathogens and phagocytes occurs prior to the transfer of the pathogen to the phagocyte. These interactions were found to be critical for efficient transfer. We were also able to demonstrate that for efficient transfer of the bacteria from the RBC monocytes/macrophages RBC a combination of adhesion molecules is required. The activation of RBC adhesion molecules by complement opsonized pathogen binding may have therapeutic potential for the treatment of infection. We are currently exploring the possibilities to treat bacteraemia, especially in patients suffering from sepsis, by removing RBC-pathogen complexes from the blood. 

Key publications

• A method for red blood cell biotinylation in a closed system. de Back DZ, Vlaar R, Beuger B, Daal B, Lagerberg J, Vlaar APJ, de Korte D, van Kraaij M, van Bruggen R. Transfusion. 2018 Apr;58(4):896-904.
• Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging. Klei TRL, de Back DZ, Asif PJ, Verkuijlen PJJH, Veldthuis M, Ligthart PC, Berghuis J, Clifford E, Beuger BM, van den Berg TK, van Zwieten R, El Nemer W, van Bruggen R. Blood Adv. 2018 Jan 3;2(1):14-24.
• Human and murine splenic neutrophils are potent phagocytes of IgG-opsonized red blood cells. Meinderts SM, Oldenborg PA, Beuger BM, Klei TRL, Johansson J, Kuijpers TW, Matozaki T, Huisman EJ, de Haas M, van den Berg TK, van Bruggen R. Blood Adv. 2017 May 26;1(14):875-886.
• Clearance of stored red blood cells is not increased compared with fresh red blood cells in a human endotoxemia model. Peters AL, Beuger B, Mock DM, Widness JA, de Korte D, Juffermans NP, Vlaar AP, van Bruggen R. Transfusion. 2016 Jun;56(6):1362-9.
• Loss of CD47 Makes Dendritic Cells See Red. van den Berg TK, van Bruggen R. Immunity. 2015 Oct 20;43(4):622-4.