Augmented erythrocyte band-3 phosphorylation in septic mice

Abstract

Infection-induced RBC dysfunction has been shown to play a role in the modulation of host response to injury and infection. The underlying biochemical mechanisms are not known. This study investigated alterations in RBC band-3 phosphorylation status and its relationship to anion exchange activity in vitro as well as under in vivo septic conditions induced by cecal ligation and puncture (CLP) in mice. Pervanadate treatment in vitro increased band-3 tyrosine phosphorylation that was accompanied by decreased RBC deformability and anion exchange activity. Following sepsis, band-3 tyrosine phosphorylation in whole RBC ghosts as well as in cytoskeleton-bound or soluble RBC protein fractions were elevated as compared to controls. Although anion exchange activity was similar in RBCs from septic and control animals, band-3 interaction with eosin-5-maleimide (EMA), which binds to band-3 lysine moieties, was increased in cells from septic animals as compared to controls, indicating that sepsis altered band 3 organization within the RBC membrane. Since glucose-6-phosphate dehydrogenase is a major antioxidant enzyme in RBC, in order to assess the potential role of oxidative stress in band-3 tyrosine phosphorylation, sepsis-induced RBC responses were also compared between WT and (G6PD) mutant animals (20% of normal G6PD activity). Band-3 membrane content and EMA staining were elevated in G6PD mutant mice compared to WT under control non-septic conditions. Following sepsis, G6PD mutant animals showed lessened responses in band-3 tyrosine phosphorylation and EMA staining compared to WT. RBC anion exchange activity was similar between mutant and WT animals under all tested conditions. In summary, these studies indicate that sepsis results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization without grossly affecting RBC anion exchange activity. The observations also suggest that factors other than oxidative stress are responsible for the sepsis-induced increase in RBC band-3 tyrosine phosphorylation.

Figure 1

Relationship of band-3 tyrosine phosphorylation, RBC deformability, and sulfate uptake. RBCs were incubated in the absence or presence of pervanadate (0-2.0 mM) and tested for protein tyrosine phosphorylation, cell deformability and sulfate uptake as described in the materials and methods section. Part A: Protein extracts from RBC ghosts were subjected to SDS polyacrylamide electrophoresis and probed with anti phospho-tyrosine antibody (Panel A upper row) or band-3 content using an anti rat band-3 antibody (Panel A lower row). Part B: RBC elongation in response to increasing shear stress in the presence of pervanadate (2.0 mM). Part C: Time dependence of sulfate uptake and the effect of DIDS (10 μM). Part D: The effect of pervanadate (2.0 mM) on RBC sulfate uptake. Mean ± SEM, from 3-6 independent assays, *significant difference compared to vehicle.

Figure 2

Sepsis increases band-3 tyrosine phosphorylation in RBCs. RBC ghosts obtained from control and septic mice were sub-fractionated and then subjected to SDS PAGE and analyzed for band-3 content using antibodies against band-3 (A) or tyrosine-phosphorylated proteins (B). The intensity of tyrosine phosphorylated band 3 was normalized to total band-3 content obtained in the same lane for each fraction (PYB3/B3). Mean ± SEM from 6-8 animals in each treatment group. * Significant difference compared to control. Part C depicts a typical finding in ghosts (lane 1), unbound (lane 2) and cytoskeleton-bound (lane 3) fractions from control and septic animals as indicated.

Figure 3

Anion transport in RBCs from septic mice. Sulfate uptake in RBCs from control and septic mice was determined as described in the materials and methods section. Time course of sulfate uptake in RBCs (Part A) and the effect of prevailing concentration of DIDS (part B) in RBCs from control and septic mice are shown. Extrapolation of the DIDS inhibitory curve indicates an intercept with the X-axis approximately at 2.5 uM in both control and septic animals (dotted line). Mean ± SEM from 3 animals in each group.

Fig 4

Comparison of band-3 tyrosine phosphorylation between WT and G6PD mutant animals. In separate sets of experiments band-3 tyrosine phosphorylation status was compared between WT and G6PD deficient animals. RBC ghost and cell fractions were analyzed as described for Fig 2. Part A shows results after probing with band-3 antibody; part B indicates tyrosine-phosphorylated band-3. Mean ± SEM from 6 animals in each group. Significant difference, *compared to WT control; ^#compared to WT septic.

Fig 5

Band-3 EMA reactivity in RBCs. Whole RBC suspensions were prepared from control and septic; WT and G6PD deficient animals and incubated in the presence of EMA followed by flow cytometry analysis as described in the materials and methods section. Part A, summary of results from five animals in each group, mean ± SEM. Part B shows a representative finding. * Significant difference compared to WT control.