Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation

MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBL-associated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling. © 2014 Elsevier Ltd. All rights reserved

C1 esterase inhibitor reduces lower extremity ischemia/reperfusion injury and associated lung damage

BACKGROUND:Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS:Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle. CONCLUSIONS:C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application

Plasma levels of cytokines, chemokines and growth factors (in pg/ml) at baseline and after 24 h of reperfusion.

<p>EPO indicates Erythropoietin; CXCL1, Chemokine (C-X-C motif) Ligand 1; IFN-gamma, Interferon-gamma; IL, Interleukin; MCP-1, Monocyte chemotactic protein-1; MIP, Macrophage inflammatory protein; RANTES, Regulated and normal T cell expressed and secreted; TNF-α, Tumor necrosis factor-α; VEGF, Vascular endothelial growth factor; M-CSF, Macrophage colony-stimulating factor. Values are mean ± SD. P<0.05*; P<0.01** by ANOVA with Dunnett's post test vs. NaCl. Multiplex analysis of the shown markers was performed using a standard rat 24-plex panel from Bio-Rad. ^†IL-1β, IL-2, IL-6, IL12p70, IL-13, G-CSF, GM-CSF were below detection level and are not listed.</p

Deposition of C3b/c and factor B in muscle and lung tissue.

<p>(A, D, G and J) Quantification data of C3b/c and factor B deposition in muscle and lung tissue, respectively. (B and C) Representative immunofluorescence images of C3b/c deposition in muscle tissue. (<b>E</b> and <b>F</b>) Representative immunofluorescence images of C3b/c deposition in lung tissue. Counterstaining with DAPI (blue channel, only shown for muscle tissue), C3b/c visible in the red channel (CY3). (<b>H</b> and <b>I</b>) Representative immunofluorescence images of factor B deposition in muscle tissue. (<b>K</b> and <b>L</b>) Representative immunofluorescence images of factor B deposition in lung tissue. Counterstaining with DAPI (blue channel), factor B visible in the green channel (Alexa 488). One-way ANOVA followed by Dunnett's post hoc test for significance vs. NaCl controls was used. Error bars indicate mean ± SD. *P<0.05; **P<0.01; ***P<0.001.</p

Frequency of apoptotic cells in muscle and lung tissue.

<p>(A and H) Quantitative analysis of TUNEL staining in muscle and lung tissue, respectively. (<b>B–G</b>) and (<b>I–N</b>) Representative immunofluorescence images of TUNEL staining of reperfused muscle and lung, respectively. TUNEL-positive cells are shown in red (<b>B, D, F, I, K, M</b>), corresponding DAPI staining of all nuclei in blue (<b>C, E, G, J, L, N</b>). One-way ANOVA followed by Dunnett's post hoc test for significance vs. NaCl controls was used. Error bars indicate mean ± SD. *P<0.05; **P<0.01; ***P<0.001.</p

Deposition of C1q, MBL and C4b/c in muscle tissue.

<p>(A, G and J) Quantification data of C1q, MBL and C4b/c deposition in muscle tissue. (B–F) Representative immunofluorescence images of C1q deposition depending on treatment. (H and I) Representative immunofluorescence images of MBL deposition in muscle tissue. Counterstaining with DAPI (blue channel), C1q or MBL visible in the red channel (CY3). (K and L) Representative immunofluorescence images of C4b/c in muscle tissue, C4b/c visible in the green channel (Alexa 488). One-way ANOVA followed by Dunnett's post hoc test for significance vs. NaCl controls was used. Error bars indicate mean ± SD. *P<0.05; **P<0.01; ***P<0.001.</p

Effect of C1 INH on edema formation, muscle viability and histological assessment of muscle damage.

<p>(A) Analysis of edema in the gastrocnemic muscle of both the contralateral- and reperfused legs. NaCl treated rats were compared with C1 INH, APT070 as well as vehicle treated and normal rats. C1 INH treatment reduced muscle wet weight/dry ratio for C1 INH as compared to NaCl controls. (B) Representative images of edema formation after 24 h reperfusion for treatment with NaCl (left) and C1 INH (right). (C) Edema formation in the lung. C1 INH treatment led to a reduced lung wet/dry weight ratio as compared to NaCl controls. (D) Viability of the gastrocnemic muscle as assessed by MTT. C1 INH treatment improved muscle viability as compared with NaCl controls. (<b>E</b>–<b>H</b>) Hematoxylin/eosin stained histological samples of reperfused gastrocnemic muscle. Representative images are shown for NaCl (<b>E</b>) and vehicle (<b>F</b>) controls as well as C1 INH (<b>G</b>) and APT070 (<b>H</b>) treatment. One-way ANOVA followed by Dunnett's post hoc test for significance vs. NaCl controls was used. Error bars indicate mean ± SD. *P≤0.05; **P<0.01; ***P<0.001.</p