No abstract

Complement and FcγR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcγRIIB, increased levels of activating FcγRIII, and highly induced FcγR-mediated TNF-α and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory FcγR system in vitro, and analysis of the various C5aR-, FcγR-, and Gi-deficient mice verifies the importance of Gαi2-associated C5aR and the FcγRIII-FcγRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcγRIII-positive cells into C5aR- and FcγRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcγRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcγRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Gαi2-dependent signal for modulating the two opposing FcγR, FcγRIIB and FcγRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Complement activation plays a key role in mediating apoptosis, inflammation, and transplant rejection. In this study, the role of the complement 5a receptor (C5aR) was examined in human renal allografts and in an allogenic mouse model of renal transplant rejection. In human kidney transplants with acute rejection, C5aR expression was increased in renal tissue and in cells infiltrating the tubulointerstitium. Similar findings were observed in mice. When recipient mice were treated once daily with a C5aR antagonist before transplantation, long-term renal allograft survival was markedly improved compared with vehicle-treatment (75 versus 0%), and apoptosis was reduced. Furthermore, treatment with a C5aR antagonist significantly attenuated monocyte/macrophage infiltration, perhaps a result of reduced levels of monocyte chemoattractant protein 1 and the intercellular adhesion molecule 1. In vitro, C5aR antagonism inhibited intercellular adhesion molecule 1 upregulation in primary mouse aortic endothelial cells and reduced adhesion of peripheral blood mononuclear cells. Furthermore, C5aR blockade markedly reduced alloreactive T cell priming. These results demonstrate that C5aR plays an important role in mediating acute kidney allograft rejection, suggesting that pharmaceutical targeting of C5aR may have potential in transplantation medicine.

A synthetic 7-mer, HHHRHSF, was recently identified by screening a phage display library for binding to the Tie-2 receptor. A polyethylene-oxide clustered version of this peptide, termed vasculotide (VT), was reported to activate Tie-2 and promote angiogenesis in a mouse model of diabetic ulcer. We hypothesized that VT administration would defend endothelial barrier function against sepsis-associated mediators of permeability, prevent lung vascular leakage arising in endotoxemia, and improve mortality in endotoxemic mice. In confluent human microvascular endothelial cells, VT prevented endotoxin-induced (lipopolysaccharides, LPS O111:B4) gap formation, loss of monolayer resistance, and translocation of labeled albumin. In 8-wk-old male C57Bl6/J mice given a ∼70% lethal dose of endotoxin (15 mg/kg ip), VT prevented lung vascular leakage and reversed the attenuation of lung vascular endothelial cadherin induced by endotoxemia. These protective effects of VT were associated with activation of Tie-2 and its downstream mediator, Akt. Echocardiographic studies showed only a nonsignificant trend toward improved myocardial performance associated with VT. Finally, we evaluated survival in this mouse model. Pretreatment with VT improved survival by 41.4% (n = 15/group, P = 0.02) and post-LPS administration of VT improved survival by 33.3% (n = 15/group, P = 0.051). VT-mediated protection from LPS lethality was lost in Tie-2 heterozygous mice, in agreement with VT's proposed receptor specificity. We conclude that this synthetic Tie-2 agonist, completely unrelated to endogenous Tie-2 ligands, is sufficient to activate the receptor and its downstream pathways in vivo and that the Tie-2 receptor may be an important target for therapeutic evaluation in conditions of pathological vascular leakage.

IntroductionAngiopoietin-1 (Angpt1), the natural agonist ligand for the endothelial Tie2 receptor, is a non-redundant endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leukocyte-endothelium interactions. Here we evaluate the efficacy of a novel polyethylene glycol (PEG)-clustered Tie2 agonist peptide, vasculotide (VT), to protect against vascular leakage and mortality in a murine model of polymicrobial abdominal sepsis.MethodsPolymicrobial abdominal sepsis in C57BL6 mice was induced by cecal-ligation-and-puncture (CLP). Mice were treated with different dosages of VT or equal volume of phosphate-buffered saline (PBS). Sham-operated animals served as time-matched controls.ResultsSystemic administration of VT induced long-lasting Tie2 activation in vivo. VT protected against sepsis-induced endothelial barrier dysfunction, as evidenced by attenuation of vascular leakage and leukocyte transmigration into the peritoneal cavity. Histological analysis revealed that VT treatment ameliorated leukocyte infiltration in kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression. VT-driven effects were associated with significantly improved organ function and reduced circulating cytokine levels. The endothelial-specific action of VT was supported by additional in vitro studies showing no effect of VT on either cytokine release from isolated peritoneal macrophages, or migratory capacity of isolated neutrophils. Finally, administration of VT pre-CLP (hazard ratio 0.39 [95% confidence interval 0.19-0.81] P < 0.001) and post-CLP reduced mortality in septic mice (HR 0.22 [95% CI 0.06-0.83] P < 0.05).ConclusionsWe provide proof of principle in support of the efficacious use of PEGylated VT, a drug-like Tie2 receptor agonist, to counteract microvascular endothelial barrier dysfunction and reduce mortality in a clinically relevant murine sepsis model. Further studies are needed to pave the road for clinical application of this therapeutic concept.

IgG Fc receptors (FcγRs, especially FcγRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcγRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcγRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcγRs, and costimulation of ICs with rhC5a results in strong enhancement of FcγRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcγRIII and suppression of inhibitory FcγRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcγRII/III receptor pair to connect complement and FcγR effector pathways in immune inflammation

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.