Serum MASP-1 in complex with MBL activates endothelial cells.

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca2+ signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL-MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response

MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms

The migration of PMNs activated by the rMASP-1-treated HUVEC supernatant.

<p>HUVEC cells were treated with 2 µM rMASP-1, or left untreated for 30 minutes. Then, the medium was changed to rMASP-1 free HBSS for 2.5 hours and then MASP-SN and UNT-SN were collected. Residual rMASP-1 concentration of MASP-SN was checked by LPAPR-AMC fluorescent substrate assay, where the Control column shows the rMASP-1 concentration of the rMASP-1 treated HUVEC supernatant before changed to HBSS (<b>A</b>). PMNs were isolated from venous blood collected from 2 healthy volunteers. 10?5 cells were seeded in 3-µm pore size transwell inserts and placed for an hour into the wells containing the MASP-SN, UNT-SN or HBSS with/without 2 ng/ml IL-8. The percentage of transmigrated/total cells was calculated as the mean (+/−SEM) of 3 different chemotaxis assays (<b>B</b>). The significance of the differences among rMASP-1 and other treatments is shown. *: p<0.05, ns: non-significant.</p

Screening for cytokine production induced by rMASP-1, and the dose dependence of IL-6, IL-8, and MCP-1.

<p>The cells were treated with 2 µM rMASP-1 for 6 hours (mRNA level) or 24 hours (protein level). Seven cytokines were assessed by qPCR and xMAP technology. (0: no effect, +: significant induction, (+): tendency for induction, not confirmed by ELISA.) (<b>A</b>) The cells were treated with 0, 0.25, 0.5, 1, 2 µM rMASP-1 for 24 hours, and then, the supernatants were collected. IL-6 (<b>B</b>), IL-8 (<b>C</b>), and MCP-1 (<b>D</b>) production were determined by sandwich ELISA kits according to the manufacturers' protocol. The values were calculated as the mean (+/−SEM) of three independent experiments.</p

rMASP-1 treatment of HUVEC activates CREB and JNK signaling pathways.

<p>The cells were treated or not treated with 2 µM rMASP-1 for 25 minutes and then, fixed with ice cold methanol-acetone (1∶1), labeled overnight at 4°C with 1∶200 diluted rabbit anti-human Phosopho-CREB antibody, and stained with goat anti-rabbit (1∶500) Alexa 568 (left column) and Hoechst nuclear staining (right column). The figure depicts one out of 5 similar, independent experiments. The bars represent 50 µm (<b>A</b>). The images were obtained by Olympus IX-81 inverted fluorescence microscope equipped with 40× UPLF objective (NA = 0,75), and an Olympus DP70 digital camera. The mean intensity of red fluorescence in the nuclear and perinuclear region was evaluated using the AnalySIS software. The mean (+/−SEM) differences between the indicated regions of 5 experiments are shown (<b>B</b>). Cells were seeded on 6-well plates and treated for 25 minutes. Cells were then lysed and Western-blots were performed. The membranes were probed for CREB and phospho-CREB (<b>C</b>) or JNK and phospho-JNK (<b>D</b>). Two representative phospho-CREB and phospho-JNK images are shown (linear intensity adjusted). The graphs were calculated from unadjusted values of phospho- and total protein ratios from 3 independent experiments. The significance of the differences among rMASP-1 and the other treatments is shown. *: p<0.05, **: p<0.01, ***: p<0.001, ns: non-significant.</p

The kinetics of IL-6 and IL-8 production induced by rMASP-1.

<p>HUVECs were treated with 2 µM rMASP-1, or with 10 ng/mL TNFα (only at protein level) for different periods. The samples were analyzed for IL-6 (<b>A, B, C</b>) and IL-8 (<b>D, E, F</b>) by qPCR (<b>A, D</b>) and ELISA (<b>B, C, E, F</b>). qPCR results were plotted as percentage of non-treated control. The results of ELISA were calculated from the standard curve, and plotted as concentration values (<b>B, E</b>). To compare the effects of rMASP-1 and of TNFalpha, we calculated the magnitude of the increase of the values (treated/non-treated) at each time-point (<b>C, F</b>). All data are presented as the mean (+/−SEM) of three independent experiments.</p

The signaling pathways of IL-6 and IL-8 production induced by rMASP-1.

<p>The cells were pre-incubated with signaling pathway inhibitors for 30 minutes and then treated with 2 µM rMASP-1 for 3 (<b>A, B</b>) or 24 (<b>C, D</b>) hours. Supernatants were collected and analyzed with commercial IL-6 (<b>A, C</b>) and IL-8 (<b>B, D</b>) ELISA kits. C1-Inhibitor (C1Inh) was applied differently; it was premixed with rMASP-1 for 30 minutes and then, the mixture was added to the cells. Each panel was calculated as the mean (+/−SEM) of 3 independent experiments using HUVECs from different donors. The significance of the differences among rMASP-1 and other treatments is shown. *: p<0.05, **: p<0.01.</p