Jon A. Buras

Gastrointestinal Ischemia-Reperfusion Injury Is Lectin Complement Pathway Dependent without Involving C1q

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

Role for the Alternative Complement Pathway in Ischemia/Reperfusion Injury

The terminal complement components play an important role in mediating tissue injury after ischemia and reperfusion (I/R) injury in rats and mice. However, the specific complement pathways involved in I/R injury are unknown. The role of the alternative pathway in I/R injury may be particularly important, as it amplifies complement activation and deposition. In this study, the role of the alternative pathway in I/R injury was evaluated using factor D-deficient (-/-) and heterozygote (+/-) mice. Gastrointestinal ischemia (GI) was induced by clamping the mesenteric artery for 20 minutes and then reperfused for 3 hours. Sham-operated control mice (+/- versus -/-) had similar baseline intestinal lactate dehydrogenase activity (P = ns). Intestinal lactate dehydrogenase activity was greater in -/- mice compared to +/- mice after GI/R (P = 0.02) thus demonstrating protection in the -/- mice. Intestinal myeloperoxidase activity in +/- mice was significantly greater than -/- mice after GI/R (P < 0.001). Pulmonary myeloperoxidase activity after GI/R was significantly higher in +/- than -/- mice (P = 0.03). Addition of human factor D to -/- animals restored GI/R injury and was prevented by a functionally inhibitory antibody against human factor D. These data suggest that the alternative complement pathway plays an important role in local and remote tissue injury after GI/R. Inhibition of factor D may represent an effective therapeutic approach for GI/R injury.

A2B Adenosine Receptor Blockade Enhances Macrophage-Mediated Bacterial Phagocytosis and Improves Polymicrobial Sepsis Survival in Mice

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

Endothelial nuclear factor-κB translocation and vascular cell adhesion molecule-1 induction by complement inhibition with anti-human C5 therapy or cGMP analogues

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism.

Objective:This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)–induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. Design:Prospective, experimental study. Setting:University experimental research laboratory. Subjects:C57BL/6 and C57BL/6 IL-10−/− mice. Interventions:Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b+ peritoneal macrophages. Measurements and Main Results:Survival was monitored for 100 hrs after CLP ± HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. Conclusion:HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10−/− mice were not protected by HBO2 treatment.

A Keratin Peptide Inhibits Mannose-Binding Lectin

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-d-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (KD) of 5 × 10−5 mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10–50 μg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.

CpG Oligodeoxynucleotide Protection in Polymicrobial Sepsis Is Dependent on Interleukin-17

CpG oligodeoxynucleotides (ODNs) may prevent mortality from infection. We have identified a therapeutic benefit in treating sepsis with phosphorothioate ODN sequences containing the CpG motif. Sepsis was induced in rats by cecal ligation and puncture (CLP), and treatment with CpG ODNs reduced sepsis mortality from 80% to 15% during a 108-h period. Protection from mortality was dose dependent. Bacterial load in peritoneal fluid was reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Lung injury, as determined by total myeloperoxidase activity, was also reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Indirect evidence suggests that CpG-induced expression of interleukin (IL)-23 as levels of p40--but not p35--were significantly increased in both plasma and peritoneal lavage fluid in CpG ODN-treated versus non-CpG ODN-treated rats 24 h after CLP. Anti-IL-17 antibody inhibited the CpG-mediated prevention of mortality. These data suggest that IL-17 may mediate CpG-inducible host defenses during intraabdominal sepsis.

Anoxia and reoxygenation of human endothelial cells decrease ceramide glucosyltransferase expression and activates caspases

Endothelial oxidative stress induces cellular activation and sometimes death. Endothelial death can occur via necrosis or apoptosis. Understanding the mechanisms involved in cellular activation and death may lead to therapeutics designed to increase death or preserve cellular function. In the present study, brief periods of anoxia (3 h) followed by varying lengths of reoxygenation (0–5 h) lead to a time‐dependent increase in human umbilical vein endothelial cell (HUVEC) caspase activity. Furthermore, ROCK‐1 cleavage, which is dependent on caspase‐3 activity, was also increased in cells undergoing oxidative stress compared with normoxic cells. Microarray data demonstrated that glucosylceramide synthase (GCS; glucosylceramide transferase), but not acid sphingomyelinase, was modulated by anoxia and reoxygenation. We confirmed that GCS mRNA and protein expression were significantly decreased in a time‐dependent fashion following oxidative stress by real‐time polymerase chain reaction and Western blot, respectively. Treatment of normoxic cells with the GCS‐specific inhibitor, d,l‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), increased caspase activity to the same degree as cells undergoing oxidative stress. Fumonisin B1, the N‐acyl‐sphinganine dehydrogenase (e.g., ceramide synthase) inhibitor significantly attenuated caspase activity in HUVECs undergoing oxidative stress. These data suggest that alterations in GCS expression following brief periods of oxidative stress in human endothelial cells lead to increased caspase activity.

A Practical Approach to Animal Models of Sepsis

The underlying mechanisms active during the pathogenesis of sepsis are not clearly defined; however, several animal models of sepsis have been used in an attempt to understand these complex cellular and molecular interactions that result in disease. There are three general categories of sepsis models, the host barrier disruption model, the chemical shock model, and the exogenous infection model. Host barrier disruption sepsis models initiate infection with the release of endogenous bacteria into normally sterile compartments. The chemical shock model employs intravenous or intraperitoneal administration of a toll-like receptor (TLR) agent, such as lipopolysaccharide (LPS or endotoxin) or zymosan, to initiate a state of proinflammatory cytokine-induced shock. The exogenous infection model of sepsis utilizes administration of an exogenous viable pathogen, typically bacteria, directly (by an intravenous or intraperitoneal route) into the host. Each of these models has particular strengths and weaknesses with respect to their ability to mimic the clinical progression of sepsis in human patients.