Lysophosphatidylcholines (lyso-PCs), generated during blood storage, are etiologic in a two-insult, sepsis-based model of transfusion-related acute lung injury (TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do not activate neutrophils (PMNs). We hypothesized that priming of PMNs alters their reactivity such that a second priming agent causes PMN activation and endothelial cell damage. PMNs were primed or not with LPS and then treated with lyso-PCs, and oxidase activation and elastase release were measured. For coculture experiments, activation of human pulmonary microvascular endothelial cells (HMVECs) was assessed by ICAM-1 expression and chemokine release. HMVECs were stimulated or not with LPS, PMNs were added, cells were incubated with lyso-PCs, and the number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 and release of ENA-78, GRO alpha, and IL-8. PMN-mediated HMVEC damage was dependent on LPS activation of HMVECs, chemokine release, PMN adhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates the microbicidal arsenal, and PMN-mediated HMVEC damage was the result of two insults: HMVEC activation and PMN oxidase assembly.

A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca(2)(+)-dependent signaling. The lyso-PC mix (0.45-14.5 micro M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca(2)(+). Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca(2)(+) functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca(2)(+) chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca(2)(+)-dependent manner that involves the activation of a pertussis toxin-sensitive G-protein.

Secretory phospholipase A(2) (sPLA(2)) produces lipids that stimulate polymorphonuclear neutrophils (PMNs). With the discovery of sPLA(2) receptors (sPLA(2)-R), we hypothesize that sPLA(2) stimulates PMNs through a receptor. Scatchard analysis was used to determine the presence of a sPLA(2) ligand. Lysates were probed with an antibody to the M-type sPLA(2)-R, and the immunoreactivity was localized. PMNs were treated with active and inactive (+EGTA) sPLA(2) (1-100 units of enzyme activity/ml, types IA, IB, and IIA), and elastase release and PMN adhesion were measured. PMNs incubated with inactive, FITC-linked sPLA(2)-IB, but not sPLA(2)-IA, demonstrated the presence of a sPLA(2)-R with saturation at 2.77 fM and a K(d) of 167 pM. sPLA(2)-R immunoreactivity was present at 185 kDa and localized to the membrane. Inactive sPLA(2)-IB activated p38 MAPK, and p38 MAPK inhibition attenuated elastase release. Active sPLA(2)-IA caused elastase release, but inactive type IA did not. sPLA(2)-IB stimulated elastase release independent of activity; inactive sPLA(2)-IIA partially stimulated PMNs. sPLA(2)-IB and sPLA(2)-IIA caused PMN adhesion. We conclude that PMNs contain a membrane M-type sPLA(2)-R that activates p38 MAPK.

Transfusion of autologous blood is associated with fewer complications, although all untoward events of transfusion may not be negated with this strategy. We report a case of acute pulmonary insufficiency and hypotension following transfusion of autologous packed red blood cells (PRBCs) in a patient, who was undergoing major surgery. Anti-HLA class-I and class-II and anti-granulocyte antibodies were measured in the unit and in the recipient. Neutrophil (PMN)-priming activity was measured as the augmentation of the formyl-Met-Leu-Phe-activated respiratory burst. No immunoglobulins were identified; however, significant lipid-priming activity was present in the implicated, autologous PRBC unit that primed PMNs from both healthy people and the recipient. In addition, lipids, identical to those that accumulate during PRBC storage, caused significant hypotension when infused into rats at similar concentrations found in stored PRBCs. We conclude that the observed transfusion-related acute lung injury reaction with significant hypotension may be the result of two independent events: the first is related to inherent host factors, in this case major surgery, and the second is the infusion of lipids that accumulate during the routine storage of PRBCs.

Background Although transfusion is a life-saving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells induce pro-inflammatory activation of human pulmonary microvascular endothelium (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Methods Ten units of red blood cells (RBCs) were collected and 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). Another 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10–40%]FINAL of the supernatants day (D)1-D42 of storage, lipid extracts and purified lipids. Endothelial surface expression of ICAM-1, IL-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. Results The supernatants [10–40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20–40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused co-precipitation of β-arrestin-1 with BLT2, PKCβI, and PKCδand served as the first event in a two-event rodent model of ALI. In conclusion, lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

Silliman CC. Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils. Am J Physiol Cell Physiol 297: C886 -C897, 2009. First published March 18, 2009 doi:10.1152/ajpcell.00416.2008.-Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMNmediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to Ͼ98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET ϩ )] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAFmediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.

Interstitial deletions in the terminal region of chromosome 6 are rare. We describe three new cases with subtle interstitial deletions in the q24-q26 region of the long arm of chromosome 6. The karyotypes were analyzed at a 550 band level. Patient1 is a 9-month-old boy with an interstitial deletion, del(6)(q24.2q25.1), developmental delay, low birth weight, hypotonia, heart murmur, respiratory distress, craniofacial and genital anomalies. This is the first report of a case with deletion del(6)(q24.2q25.1). Patient 2 is a 17-year-old young man with an interstitial deletion del(6)(q25.1q25.3), developmental delay, short stature, mental retardation, autism, head, face, chest, hand and feet anomalies and a history of seizures. For the first time autism was described as a manifestation in 6q deletions. Patient 3 is baby boy with a de novo interstitial deletion, del(6)(q25.1q26), anomalies of the brain, genital organs, limbs and feet. This is the first report of a case with deletion, del(6)(q25.1q26). In all three patients, fluorescence in situ hybridization (FISH) using chromosome 6 painting probe ruled out an insertion. The ESR (6q25.1) and TBP (6q27) probes were used to confirm the breakpoints. Since TBP signal is present in all cases, it confirmed an interstitial deletion proximal to this probe. Patient 1 has a deletion of the ESR locus; Patient 2 and 3 have signals for the ESR locus on both chromosomes 6. Therefore the deletion in Patients 2 and 3 are between ESR and TBP loci distal to that of Patient 1. FISH validated the deletion breakpoints assessed by conventional cytogenetics.

A. Tumor necrosis factor-␣ causes release of cytosolic interleukin-18 from human neutrophils.