In Contrast to Platelet Microparticles, Red Cell Microparticles Are Anti-Inflammatory, Judging By Effect on CD11b Expression on Neutrophils
Authors: McCauley Robert Forestal , Jy Wenche , Horstman Lawrence L. , Yaniz Miriam , Gonzalez Martha Q. , Ahn Henry Y 
Published: November 2019

BACKGROUND: It has been shown that platelet microparticles (PMP) exert pro-inflammatory activity. It has also been suggested that red cell MP (RMP) are similarly pro-inflammatory, but the evidence is less clear. This issue has clinical significance for several reasons. For example, there is evidence that both PMP and RMP have hemostatic activity [Horstman et al, Semin Thromb Hemost, 2019] and that infusion of RMP is a viable strategy for control of excessive bleeding [Jy et al,Thromb Haemost, 2013]. For these reasons we undertook assay of inflammatory potential of PMP and RMP by the method of CD11b expression on neutrophils, using N-Formylmethionyl-leucyl-phenylalanine (fMLP) as a positive control.

METHODS: RMP were isolated from leukoreduced, type O+ packed RBCs obtained from three different donors at storage time points of 3 and 6 weeks (RMPStor). In addition, RMP were prepared by high-pressure extrusion from type O+ packed RBCs stored for less than one week (RMPxt). Platelets were isolated from healthy volunteers and sonicated to produce PMP. MP concentrations were determined using flow cytometry. RMP were identified by PE-labeled anti-glycophorin and PMP were identified by FITC-labeled anti-CD41. Each MP species was added to separate 500 µL aliquots of fresh whole blood collected from healthy volunteers for equal final concentrations of 20,000/µL, 40,000/µL, 80,000/µL, and 200,000/µL. To one of each pair, fMLP was added for a final concentration of 1 µM. Fresh, whole blood treated with sterile saline was used as a negative control. A positive control was obtained by adding fMLP to whole blood without MP treatment. The blood samples were then incubated for one hour under gentle shaking. Two-color flow cytometry with PE-Cy5-anti-CD11b and FITC-anti-CD45 MAbs were employed to measure the ratio of CD11b to CD45 expression on neutrophils (identified through FS, SS, and CD45+ FL gating). The differences in CD11b expression were compared using the paired t test. The mean values presented below were determined using the results from at least 5 replicate experiments in which the two groups were directly assayed against one another using neutrophils from a single donor, resulting in slightly different mean values for each group depending on the comparison being analyzed.

RESULTS: For all RMP species, there was no significant difference between concentration and their effect on neutrophil CD11b expression. In contrast, the concentration of PMP positively correlated with an increase in CD11b expression (R2=0.78). At a MP concentration of 200,000/µL, there was no statistical difference when comparing neutrophil CD11b expression in the negative control vs. incubation with RMPxt (27.5 vs. 27, p=0.7), or the negative control vs. incubation with RMPStor Wk 6 (29.0 vs. 28.4, p=0.6). However, neutrophil CD11b expression was lower when incubated with RMPStor Wk 3 compared to the negative control (25.1 vs. 28.8, p&lt;0.03). In comparison, incubation with PMP increased CD11b expression to nearly 200% of the negative control (44.2 vs. 22.1, p&lt;0.0001). All phenotypes of RMP inhibited fMLP-induced CD11b expression. Compared to the positive control, RMPxt decreased CD11b expression by 12% (73.4 vs 64.6, p=0.0003), RMPStor Wk 3 decreased CD11b expression by 19% (77.5 vs. 62.8, p&lt;0.0001), and RMPStor Wk 6 decreased CD11b expression by 12% (77.3 vs. 68.4, p=0.0002). Conversely, PMP potentiated fMLP-induced CD11b expression by 20% when compared to the positive control (90.7 vs. 72.7, p&lt;0.007).

CONCLUSION: In sum, PMP exhibited robust pro-inflammatory activity, but RMP did not. On the contrary, RMP exhibit modest but significant anti-inflammatory activity in this assay method. It remains necessary to elucidate the mechanism and to assess if these findings apply in vivo.

Disclosures

Jy: RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding. Horstman:RxMP Therapeutics: Patents & Royalties: US patents No. 8,150,632 and 9,155,764. Ahn:RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding.