Aaron M. Cheng

Stored Packed Red Blood Cell Transfusion Up-regulates Inflammatory Gene Expression in Circulating Leukocytes

Summary Background Data:The transfusion of more than 6 units of packed red blood cells (PRBCs) within the first 12 hours of injury is the strongest independent predictor of multiple organ failure (MOF). This suggests that stored blood contains bioactive factors that may modify the immunoinflammatory response. Methods:To simulate postinjury major transfusions ex vivo, we obtained whole blood from 4 healthy adults and divided it into four 7-mL groups (I–IV). Group I was not diluted. Group II had 7 mL of 0.9% sterile saline (SS) added. Group III received 3.5 mL each of leuko-reduced stored PRBC and SS (simulating a major transfusion). Group IV received 3.5 mL each of SS and a hemoglobin-based oxygen carrier (PolyHeme) to evaluate the effects of hemoglobin alone. The hemoglobin content in groups III and IV was measured to be equal. Total leukocyte RNA was purified, and its gene array profiles were obtained. Results:Of the 56,475 oligonucleotide probe sets interrogated, 415 were statistically different (P < 0.001). Fourteen of the 415 probe sets were inflammatory-related. The PRBC group had a significantly different expression profile compared with the others and included up-regulation of the interleukin-8, toll-like receptor 4, cryropyrin, prostaglandin-endoperoxide synthase-2, and heparinase genes. Conclusions:PRBCs activate inflammatory genes in circulating leukocytes, which may be central to the pathogenesis of the adverse inflammatory responses that lead to postinjury MOF.

Bioactivity of postshock mesenteric lymph depends on the depth and duration of hemorrhagic shock

ABSTRACT Mesenteric hypoperfusion due to circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Postshock mesenteric lymph (PSML) has been shown to contain proinflammatory mediators elaborated from the ischemic gut. We hypothesize that the relative bioactivity of PSML depends on the depth and duration of circulatory shock. To first determine the timing of PSML bioactivity, we subjected rats to hemorrhagic shock (30 mm Hg × 45 min) and then resuscitation with 50 vol% of shed blood and normal saline (4× shed blood) over 2 h. Mesenteric lymph was collected hourly up to 6 h after shock. Superoxide release was measured from human neutrophils (polymorphonuclear neutrophils [PMNs]) incubated with lymph fractions collected from each of the hourly time points. Rats were then subjected to four different shock variations: (1) 30 mm Hg × 45 min, (2) 30 mm Hg × 15 min, (3) 45 mm Hg × 45 min, and (4) 45 mm Hg × 15 min, and were resuscitated. PSML flow depends on depth of shock, but not duration of shock or resuscitation volume. Maximal PSML bioactivity, as measured by PMN priming for the respiratory burst, occurred during the third postshock hour, which correlated with peak lymph flow rate. PSML bioactivity was greatest with 30 mm Hg × 45 min, followed by 30 mm Hg × 15 min, 45 mm Hg × 45 min, and 45 mm Hg × 15 min. Hemorrhagic shock provokes the release of bioactive agents in PSML that is dependent on both depth and duration of shock.

Exportin 1 inhibition attenuates nuclear factor-κB-dependent gene expression

Activation of nuclear factor (NF)-&kgr;B is mediated by signal-induced phosphorylation of I&kgr;B&agr;, subsequent I&kgr;B&agr; degradation, and then translocation of unbound NF-&kgr;B to the nucleus. Termination of gene expression occurs when I&kgr;B&agr; binds NF-&kgr;B subunits (Rel A) in the nucleus. Leptomycin B specifically inhibits export of I&kgr;B&agr; and the inactive I&kgr;B&agr;/Rel A complex via the nuclear export protein exportin 1. We hypothesized that inhibition of I&kgr;B&agr; nuclear export would increase nuclear I&kgr;B&agr; and attenuate NF-&kgr;B inflammatory gene expression in pulmonary microvascular endothelial cells. We found that inhibition of exportin 1 causes nuclear accumulation of both endogenous NF-&kgr;B (Rel A) and I&kgr;B&agr;. IL-1&bgr; causes nuclear accumulation of NF-&kgr;B (Rel A) but does not increase nuclear I&kgr;B&agr;. Inhibition of exportin 1 before IL-1&bgr; prevented an increase in the nuclear ratio of NF-&kgr;B (Rel A) to I&kgr;B&agr; and decreases NF-&kgr;B DNA binding. Furthermore, inhibition of exportin 1 attenuates IL-1&bgr;-induced phosphorylation of I&kgr;B&agr; without affecting I&kgr;B kinase phosphorylation. Lastly, inhibition of exportin 1 attenuates monocyte chemoattractant protein, IL-8, and intercellular adhesion molecule expression in response to IL-1&bgr; stimulation. We suggest that the decrease in cell activation due to exportin 1 inhibition is a result of termination of NF-&kgr;B DNA binding due to increased concentration of I&kgr;B&agr; in the nucleus.

Transfusion-induced leukocyte IL-8 gene expression is avoided by the use of human polymerized hemoglobin.

BACKGROUNDnRed blood cell (pRBC) transfusion is an independent risk factor for multiple organ failure (MOF); a maladaptive immuno-inflammatory response is implicated. Interleukin-8 (IL-8) is one putative mediator of this response. We previously observed that injured patients resuscitated with pRBCs have increased plasma IL-8 compared with those given human polymerized hemoglobin (PolyHb). To further elucidate the mechanisms responsible for this difference in IL-8, we devised an ex-vivo transfusion model. We hypothesize that pRBC transfusion induces increased IL-8 gene expression that is avoided by the use of PolyHb.nnnMETHODSnHuman volunteer blood was incubated alone (RB) or with a major transfusion (50% exchange) of either post-storage leukoreduced O-pRBCs (RB + pRBC) or PolyHb (RB + PolyHb) for 30 minutes at 37 degrees C. Total leukocyte (TL) or polymorphonuclear leukocyte (PMN) total RNA was isolated and IL-8 mRNA quantified. Results are reported as amol IL-8 mRNA/microg total RNA +/- SEM. Stats: ANOVA with Bonferroni/Dunn post hoc analysis.nnnRESULTSnSimulated transfusion of pRBCs increased TL IL-8 mRNA (RB=0.28 +/- 0.10 amol/microg total RNA, RB + pRBC=2.24 +/- 0.25 amol/microg total RNA, p <0.01), whereas PolyHb did not (B + PolyHb=0.82 +/- 0.30 amol/microg total RNA). PolyHb IL-8 mRNA was less than pRBC transfused (p <0.01). In PMNs, simulated transfusion of pRBCs increase IL-8 mRNA (RB=3.17 +/- 1.05 amol/microg total RNA, RB + pRBC=7.60 +/- 1.79 amol/microg total RNA, p <0.01), whereas PolyHb did not (RB + PolyHb=4.53 +/- 1.64 amol/microg total RNA).nnnCONCLUSIONSnStored pRBCs induces increased TL and PMN IL-8 gene expression, whereas human polymerized hemoglobin, in lieu or pRBCs, avoids this increase. These experimental results corroborate our previous clinical studies and further encourage the study of PolyHb as a resuscitation strategy to decrease postinjury MOF.