Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis.

INTRODUCTION: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1ß in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. METHODS: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1ß. RESULTS: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1ß was measured in mice treated with rFc-µTP-L309C. CONCLUSION: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1ß production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Platelet vesicles are potent inflammatory mediators in red blood cell products and washing reduces the inflammatory phenotype.

BACKGROUND: Studies suggest that washing red cell concentrates (RCCs) to remove soluble mediators and/or inflammatory components, such as extracellular vesicles (EVs), may lead to better clinical outcomes. This study tested the hypothesis that non-red blood cell (RBC) generated vesicles in RCC are potent inflammatory mediators in vitro and washing RCCs can reduce these vesicles and subsequently decrease the inflammatory activity of RCCs. STUDY DESIGN AND METHODS: Sixteen RCCs were pooled and split into four groups based on pre-wash storage time (Day 2 or 14; n = 4/group). Each group was tested 24 hours and 7 days post-wash. Characteristics of RBCs and EVs, cytokines released by monocytes, and expression of human umbilical vein endothelial cells (HUVECs) adhesion molecules were assessed. RESULTS: All RCCs meet quality standards for hemolysis, hematocrit, and hemoglobin. Washing did not remove residual platelets from RCCs but led to a significant reduction in platelet-EV count regardless of the group. Supernatant of RCCs washed on Day 14 and stored for 24 hours had significantly lower concentrations of RBC-EVs and white blood cell EVs compared to unwashed controls. Supernatant of unwashed RCCs showed higher production of inflammatory cytokines/chemokines MCP-1, IL-8, and TNF-α, and heightened expression of HUVEC VCAM-1, which were significantly reduced by washing. Spiking washed RCC supernatants with platelet-EVs showed significant increase in IL-8, MCP-1, VCAM-1, and E-selection in groups washed on Day 14. CONCLUSIONS: Platelet-EVs in RCCs are associated with pro-inflammatory activity. As washing significantly reduced RCC immunomodulatory activity, implementation of this process may improve transfusion outcomes.

Red-blood-cell manufacturing methods and storage solutions differentially induce pulmonary cell activation.

BACKGROUND AND OBJECTIVES: Red-blood-cell (RBC) transfusion is associated with lung injury, which is further exacerbated by mechanical ventilation. Manufacturing methods of blood products differ globally and may play a role in the induction of pulmonary cell activation through alteration of the immunomodulatory property of the products. Here, the effect of different manufacturing methods on pulmonary cell activation was investigated in an in vitro model of mechanical ventilation. MATERIALS AND METHODS: Pulmonary type II cells were incubated with supernatant from fresh and old RBC products obtained via whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD) or whole blood-derived (WBD) methods. Lung cells were subjected to 25% stretch for 24 h. Controls were non-stretched or non-incubated cells. RESULTS: Fresh but not old AD products and WBF products induce lung cell production of pro-inflammatory cytokines and chemokines, which was not observed with WBD or RCF products. Effects were associated with an increased amount of platelet-derived vesicles and an increased thrombin-generating capacity. Mechanical stretching of lung cells induced more severe cell injury compared to un-stretched controls, including alterations in the cytoskeleton, which was further augmented by incubation with AD products. In all read-out parameters, RCF products seemed to induce less injury compared to the other products. CONCLUSIONS: Our findings show that manufacturing methods of RBC products impact pulmonary cell activation, which may be mediated by the generation of vesicles in the product. We suggest RBC manufacturing method may be an important factor in understanding the association between RBC transfusion and lung injury.

Screening of red blood cells for extracellular vesicle content as a product quality indicator.

BACKGROUND: The controversy around the quality and clinical impact of stored and differentially manufactured red cell concentrates (RCCs) from different donor groups is ongoing. Current studies are limited by the lack of quality measures suitable for routine screening of RCCs. As extracellular vesicles (EVs) are markers of cellular activation or degradation, this study investigated the utility of EV screening to characterize the effects of RBCs production methods and storage. STUDY DESIGN AND METHODS: RCCs were prepared by whole blood filtration or red blood cell (RBC) filtration methods, centrifuged to prepare a supernatant, and tested for EV content (dynamic light scattering or tunable resistive pulse-sensing techniques), hemolysis, ATP, and RBC deformability on Days 7, 21, and 42 of storage. To simulate nondestructive quality control (QC) testing, 1 RBC unit was tested in parallel with six 10-mL aliquots that were stored in small-volume containers. RESULTS: EV content showed a linear increase with storage time (p < 0.001) and correlated with supernatant hemoglobin and inversely with ATP or RBC deformability. The method of component manufacturing influenced the characteristics of the EVs during storage. A strong correlation between both EV testing methods' measure of total EV was observed. EV content in the six aliquots were consistent at each time point but statistically higher than in the original RCCs on and after 21 days of storage. CONCLUSIONS: EV content correlates with measures of hemolysis and other RBC quality indicators and could be implemented as a routine screening tool for nondestructive QC testing of RCCs.

Characteristics of Extracellular Vesicles in Red Blood Concentrates Change with Storage Time and Blood Manufacturing Method.

BACKGROUND: Extracellular vesicles (EVs) in blood products are potential effectors of inflammation and coagulation after transfusion. The aim of this study was to assess the impact of different blood manufacturing methods and duration of hypothermic storage on the EV subpopulations in relation to other in vitro quality parameters of red blood cell concentrate (RCC) products. METHODS: RCCs were produced using whole blood filtration (WBF) or red cell filtration (RCF) (n = 12/method), refrigerated for 43 days, and evaluated for EV size profile and concentration, red cell deformability, ATP and 2,3-DPG, hemolysis, and hematological indices. RESULTS: The total number of EVs increased significantly with storage in both methods, and WBF-RCCs contained the higher numbers of EVs compared to RCF-RCCs. The concentration of small EVs was greater in WBF-RCCs versus RCF-RCCs, with difference between the two methods observed on day 43 of storage (p = 0.001). Throughout storage, significant decreases were identified in ATP, 2,3-DPG, and EImax, while an increase in hemolysis was observed in both RCC products. CONCLUSION: The dynamic shift in the size and concentration of the EV subpopulations is dependent on the blood manufacturing method and length of storage. Better understanding of the potential clinical implications of these heterogeneous populations of EVs are needed.

Extracellular vesicle characteristics in stored red blood cell concentrates are influenced by the method of detection.

Extracellular vesicles (EVs), including microvesicles and exosomes, are small phospholipid vesicles (≤1µm in diameter) that are present in blood products, accumulate during storage, and have a potential transfusion-related immunomodulatory role. Knowledge of EVs in stored blood products is limited due to the challenges and difficulties in detecting these heterogeneous submicron-sized vesicles. The aim of this study was to assess the impact of different approaches to characterize EVs in stored RBC products. Quantification and size-profiling of EVs in leukoreduced red cell concentrates (RCCs) were examined on day 3, 7, 21, and 42 of storage using tunable resistive plus sensing (TRPS), flow cytometer (FC), and dynamic light scatting (DLS) methods. Using the TRPS method, the concentration of EVs<200nm significantly increased throughout storage (p<0.05). This change in exosome concentration was not detectable with FC or DLS due to limitations in their ability to resolve particles <200nm and/or accurately determine EV concentration. Both the TRPS and FC demonstrate that the concentration of EVs≥200nm significantly increases in RCCs by day 42/43 compared to EVs present on day 3 (p<0.001). As the DLS measures the average size of particles in suspension, only an increase in the zeta-average size was observed during storage. EV size and concentration in RBC products is significantly influenced by the length of storage. Overall, this study shows that combining technologies may be important to improve the characterization and study of EVs in stored RCCs.

Extracellular vesicles in transfusion-related immunomodulation and the role of blood component manufacturing.

There is an emerging interest in the risks posed by the ability of blood transfusion to modulate the immune system of recipients. Observational trials suggest that RBC transfusions may be associated with increased morbidity and mortality, however studies demonstrating the deleterious consequences of transfusion-related immunomodulation have had conflicting results. Efforts to understand the biological mechanisms responsible for TRIM are under way, and are focusing on the role that the extracellular vesicles (EVs) that accumulate in a red cell concentrate (RCC) during storage may play. EVs are heterogeneous submicron-sized vesicles that vary in size, composition and surface biomarkers. The biophysical and biochemical parameters of EVs reflect their mechanism of formation and cell sources. RCCs have been shown to contain a mixed population of EVs and not all EVs in RCC are solely from the constituent RBCs. The concentration of the different EVs (the RBC EVs and the non-RBC EVs), their composition, as well as their effects on the quality of the blood product vary depending on the manufacturing methods used to produce the RCC units. This article will review current evidence of the role of extracellular vesicles in transfusion-related immunomodulation and will discuss the impact that different methods used to collect, manufacture and store blood have on the composition and characteristics of EVs in RCCs.

(NZW × BXSB) F1 male mice: An unusual, severe and fatal mouse model of lupus erythematosus.

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune/inflammatory disease. The heterogeneity and complexity of clinical presentation has made it challenging to study or treat this syndrome. The (NZW×BXSB) F1 lupus-prone male mouse model of this disease is potentially useful to study mechanism and treatment modalities, but there is a lack of information about this model's characterization and disease progression. Therefore, the aim was to examine this lupus model's physical/clinical disease presentation and its immunological status. Materials and methods: Clinical and physical status were observed in 8- and 16-week-old male and female (± 1 week) (NZW/LacJ x BXSB/MpJ) F1 mice (n = 8 per group). Young males (8 ± 1 week) without disease and female (16 ± 1 week) mice served as controls. Physical changes, quantitative values of autoantibodies, and blood cell parameters were determined. Necropsy and post-mortem histopathology were also performed. Results: With aging (≥ 12 weeks), significant increases in severe abdominal distension/swelling, inability to walk, paleness of paws and significant weight increase were observed compared to controls (p < 0.05). The necropsy examination showed abdominal distension associated with serous effusion and histological examination identified severe edema and multi-organ abnormalities (spleen, lymph nodes, and kidney). Significant increases in anti-double-stranded DNA antibody (anti-dsDNA) was seen in old/sick compared to female (p = 0.0002) or young male (p = 0.0036) mice. Old mice developed immune thrombocytopenia compared to female (p = 0.0056) and young male (p = 0.0007) mice. Anti-platelet was detectable in old, sick mice. The mortality rate increased with aging; more than 35% of male mice died during this study between the ages of 13-18 weeks. Conclusion: We found that the (NZW/LacJ x BXSB/MpJ) F1 male mice spontaneously exhibit, over varying lengths of time, extremely severe and fatal clinical disease symptoms. This model may be too severe to be helpful in investigating SLE and testing potential treatment modalities.

Blood manufacturing methods affect red blood cell product characteristics and immunomodulatory activity.

Transfusion of red cell concentrates (RCCs) is associated with increased risk of adverse outcomes that may be affected by different blood manufacturing methods and the presence of extracellular vesicles (EVs). We investigated the effect of different manufacturing methods on hemolysis, residual cells, cell-derived EVs, and immunomodulatory effects on monocyte activity. Thirty-two RCC units produced using whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD), and whole blood-derived (WBD) methods were examined (n = 8 per method). Residual platelet and white blood cells (WBCs) and the concentration, cell of origin, and characterization of EVs in RCC supernatants were assessed in fresh and stored supernatants. Immunomodulatory activity of RCC supernatants was assessed by quantifying monocyte cytokine production capacity in an in vitro transfusion model. RCF units yielded the lowest number of platelet and WBC-derived EVs, whereas the highest number of platelet EVs was in AD (day 5) and in WBD (day 42). The number of small EVs (<200 nm) was greater than large EVs (≥200 nm) in all tested supernatants, and the highest level of small EVs were in AD units. Immunomodulatory activity was mixed, with evidence of both inflammatory and immunosuppressive effects. Monocytes produced more inflammatory interleukin-8 after exposure to fresh WBF or expired WBD supernatants. Exposure to supernatants from AD and WBD RCC suppressed monocyte lipopolysaccharide-induced cytokine production. Manufacturing methods significantly affect RCC unit EV characteristics and are associated with an immunomodulatory effect of RCC supernatants, which may affect the quality and safety of RCCs.

Impact of technical and assay variation on reporting of hemolysis in stored red blood cell products.

BACKGROUND: Hemolysis of RBCs is an important measure of product quality and is influenced by donor factors, blood component manufacturing and storage. Percent hemolysis is determined using hematocrit (Hct), supernatant Hb (SHb) and total Hb (THb), each of which can be measured using a variety of methods. METHODS: Sixteen members of an international collaborative were surveyed to understand equipment and procedural variation in hemolysis testing. In a laboratory-based evaluation, we examined how hemolysis was impacted by: measurement of Hct, SHb, THb and number and force of centrifugations for SHb preparation. The number and size of extracellular vesicles (EVs) was also examined. RESULTS: There was no consensus in equipment or procedures used by international laboratories to measure hemolysis. The centrifugation force used to prepare samples influenced SHb concentration when a single or double (p=0.0001) centrifugation step was used. The number and force of centrifugation related directly to the ability to remove EVs and EV-bound Hb from samples. Hemolysis varied significantly from 0.16% to 0.32% (mean of 0.22%) depending on the combination of methods or centrifugation conditions used to test expired samples. CONCLUSION: Method and preparative procedures have a critical impact on measurement of hemolysis in RCC.