In this study, we showed that aged PRBC-derived plasma was more potent than fresh PRBC-derived plasma at inducing superoxide production in neutrophils. Antibody array analysis showed that this oxidative burst was accompanied by increased protein tyrosine phosphorylation. Some of the key proteins phosphorylated were members of the NF-kB family (p105, IKK and p50). We showed upregulation of non-muscle myosin IIA, MYH9, in plasma-treated neutrophils. MYH9 upregulation was reversed by NADPH oxidase inhibitor, DPI. We also showed that aged plasma promoted neutrophil migration and induced morphological changes and phagocytosis. These processes were reversed by pretreating neutrophils with blebbistatin, a myosin type II inhibitor.
TRALI is initiated by an immune priming step, followed by exposure to specific biological response modifiers via transfusion, causing an interaction between neutrophils, platelets and lung endothelium.(4)
Early research indicated the presence of a priming agent present in PRBCs, WB and platelet concentrates (PCs), which activated this enzyme system.(25)
This priming activity was observed in aged blood, but not in fresh blood. Further analysis showed the presence of lysophosphatidylcholines as well as a lipid priming activity in WB, PRBCs and PCs.(2)
Since fresh frozen plasma did not contain this lipid priming activity, such bioactive lipids which appeared after a few days of storage, were thought to be generated via cellular breakdown.(2)
Our results agreed with these studies and showed higher levels of superoxide production in neutrophils incubated with aged PRBC-derived plasma when compared with fresh PRBC-derived plasma. Since phagocyte activation is characterized by oxidative burst (increased production of superoxide anions), these data suggested that aged plasma would be more efficient than fresh plasma at inducing phagocytosis. We used a yeast phagocytosis assay and showed higher levels of phagocytic activity in neutrophils exposed to aged plasma when compared to fresh plasma. Interestingly, Lyn, a member of the Src family tyrosine kinases, was previously shown to play an important role in phagocytosis by alveolar macrophages via regulation of respiratory burst.(26)
It will be interesting to more fully characterize the molecular mechanisms underlying increased phagocytosis in neutrophils treated with aged PRBC-derived plasma.
Migration of cells is a complex process, which results in the generation of motor forces through cytoskeleton reorganization. We previously showed that TNF induced migration of T cells by regulation of the myosin-actin cytoskeletonsome.(22)
There are conflicting reports on the effect of transfusion on cell migration and chemotaxis. Supernatant from standard RBC preparations was recently shown to prime polymorphonuclear cells (PMNs) and induce chemotaxis.(27)
These effects were directly correlated with storage time. Other reports indicated that supernatants from stored RBCs could inhibit fMLP-induced neutrophil chemotaxis as well as induce neutrophil migration.(28)
RBC transfusions were also recently shown to inhibit neutrophil chemotaxis.(29)
Our data showed that aged PRBC-derived plasma was more efficient than fresh PRBC-derived plasma at inducing protein phosphorylation in neutrophils. Phosphorylation and activation of mitogen-activated protein kinase, p38, was previously shown to be required for priming of respiratory burst in neutrophils.(30)
Early studies also showed enhanced respiratory burst in neutrophils stimulated with granulocyte-colony stimulating factor (G-CSF), which resulted in activation of transcription factor p80c-rel.(31)
In this study, we showed that among the proteins that were phosphorylated, were members of the NF-kB family (p105, p50 and IKK). Since NF-kB activation has been long suspected to play a role in cell migration, presumably by transcriptional regulation of genes involved in migration,(32)
our data suggest a regulatory role for NF-kB in plasma-induced neutrophil migration.
Non-muscle myosin II has previously been implicated in the migration of tumor cells and leukocytes(18,33)
and has been shown to participate in the redistribution of adhesion receptors in the immunological synapse.(34,35)
Myosin IIA-deficient T-cells exhibit decreased interstitial migration.(36)
Furthermore, the H chain of myosin II is known to play a role in regulation of pseudopod formation and chemotaxis in Dictyostelium discoideum.(37)
Interestingly, MYH9, an isoform of the non-muscle type II myosin H chain, has been shown to be important for spontaneous (matrix-induced) as well as ligand-induced migration of leukocytes. In this study, we showed upregulation of MYH9 expression in neutrophils incubated with aged PRBC-derived plasma. These data are consistent with the increased migration observed in these cells. We used blebbistatin, a specific myosin type II inhibitor, to look at the role of MYH9 in neutrophil migration. Blebbistatin has previously been shown to inhibit cell migration and cell morphological changes during cytokinesis.(16,38)
We showed a significant abrogation of plasma-induced cell migration and morphological changes in neutrophils which were pretreated with blebbistatin.
Taken together, our data showed that neutrophils treated with PRBC-derived plasma expressed significant amounts of MYH9 protein, while resting neutrophils did not. It will be interesting to investigate the kinetics of MYH9 upregulation and cell migration in response to PRBC-derived plasma. A number of studies have investigated possible mediators of neutrophil activation and transfusion-related immunomodulation. H2O2 was shown to amplify the innate immune response in certain inflammatory disorders, by stimulating NOX2-mediated superoxide production in neutrophils via Ca(2+)/c-Abl signaling pathway.(39)
Based on our results, we suggest that NLR-42D PRBCs induced superoxide production in neutrophils, leading to tyrosine phosphorylation and activation of transcription factors such as NF-kB, which may regulate MYH9 transcription. MYH9 and ICAM-1 were previously suggested to play a role in T cell migration.(18)
Our study, showing inhibition of neutrophil migration in response to blebbistatin, a myosin type II inhibitor is further validation that MYH9 plays a role in neutrophil migration. Furthermore, inhibition of MYH9 expression in response to DPI, an NADPH inhibitor, suggested that superoxide production plays a role in NLR-42D PRBC-induced MYH9 expression. The use of a no-plasma control group as well as the LR-42D negative control group in every experiment served to ensure that the difference between the fresh and aged plasma observed in our study were indeed authentic and not due to artifactual priming.
One limitation of our present study was that we did not use pooled donated blood samples. Individual differences may therefore play a role in the interpretation of the results. We would like to validate our results using larger sample sizes and pooled samples. We also did not explore if exposure of neutrophils to activators of oxidative burst could upregulate MYH9 expression. In our future studies, we would like to perform a finer dissection of the molecular mechanisms dictating the pathogenesis of transfusion-driven acute lung injury in vitro and in vivo.
To the best of our knowledge, we are the first to demonstrate a functional role of MYH9 in the directional migration of immune cells. Our findings indicate that transfusion with aged PBRC-derived plasmas resulted in a number of biologic effects such as (i) increased protein tyrosine phosphorylation, capable of triggering different signal transduction pathways in neutrophils, (ii) increased MYH9 expression in neutrophils, and (iii) modulation of phagocytosis, cell migration and morphological changes in neutrophils. Plasma-induced changes in migration and morphology were likely mediated via MYH9 and were abrogated by pretreatment of neutrophils with blebbistatin, a specific inhibitor of type II myosin. Our data provide an insight into the cellular and molecular mechanisms of transfusion-related injury and suggest that blebbistatin is a potential therapeutic agent for blood transfusion related disorders.