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 Ca2+-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.


Introduction
The immune system responds to various pathogens through different sets of immune mechanisms. The predominant, and most effective initial mechanisms for eliminating bacterial or fungal infection are the complement system and neutrophil granulocytes.
The complement system is part of the innate immune system. It can recognize, identify, and eliminate invading pathogens and altered host cells. The complement system can be activated through three different routes: the classical, the lectin, and the alternative pathways. The pattern-recognition molecules of the lectin pathway -mannan-binding lectin (MBL), collectin 11 (CL-K1), and the three ficolins (H-, L-, and M-ficolin) -circulate in the blood in complexes with MBL-associated serine proteases (MASP-1, 2 and 3), and mannose-binding lectin associated proteins (MAp19 and MAp44). When MBL, CL-K1, or ficolins recognize a microorganism, the activation of MASP-1, as a promiscuous protease [1], leads to several distinct outcomes: 1) activation of the complement system via cleavage of MASP-2 [2,3], 2) cleavage of kininogen and the release of bradykinin [4], 3) cleavage of fibrinogen and factor XIII (transglutaminase) [5], 4) activation of endothelial cells via protease activated receptor 4 (PAR-4, a member of GPCR family) signaling, as we have previously described [6]. Briefly, we showed that rMASP-1 can cleave PAR-1, 2, and 4 with different efficacy, and PAR-4 activation leads to Ca 2+ -signaling, the nuclear translocation of NFkappaB, and the phosphorylation of p38-MAPK. The cleavage of endothelial PAR-1 and PAR-4 by thrombin causes changes in endothelial cell morphology, as well as in the release of vasoactive substances and cytokines [7]. In general, cytokine-generation during the inflammatory response requires the involvement of the p38-MAPK, JNK, NFkappaB or cAMP responding elementbinding protein (CREB) signaling pathways [8][9][10][11][12].
The endothelium can generate anti-inflammatory cytokines such as IL-1ra (receptor antagonist), as well as pro-inflammatory cytokines (e.g. IL-1alpha, IL-6, IL-8, MCP-1 and TNFalpha) in response to various stimuli [8,[10][11][12][13]. TNFalpha, IL-1alpha, and IL-6 are the most important mediators of the acute phase response, and of fever. Furthermore, TNFalpha can regulate the levels of the proteins required for antigen presentation. IL-1alpha is a regulator of Th1/Th2 balance, whereas IL-6 is a potent survival factor of plasma cells, and participates in IgA class switching. IL-1ra is a natural inhibitor of the pro-inflammatory IL-1beta cytokine [11]. IL-8 and MCP-1 (monocyte chemoattractant protein) are chemokines, which control the migration of selected leukocyte subsets into inflamed tissues. IL-8 and MCP-1 are chemoattractants for neutrophil granulocytes and monocytes, respectively [14]. IL-6, IL-8 and MCP-1 are secreted with a low constitutive rate by endothelial cells; however, they can also be stored in different granular structures (Weibel-Palade bodies and type-2 chemokine-containing organelles) [15]. Upon pro-inflammatory stimuli, both rapid degranulation, and de novo protein synthesis may result in the elevated secretion of these cytokines. Although IL-6, IL-8, and MCP-1 are regulated similarly in most cases, there are also dissimilarities in their secretion. One major difference is the lacking P-CREB binding site in the promoter region of MCP-1, but notwithstanding, the expression of IL-6 and IL-8 can be driven by CREB [9,[16][17][18]. Differential chemokine production, together with the adhesion molecule pattern, can be the most important regulators of leukocyte trafficking driven by the endothelium. The effector function of neutrophil granulocytes (polymorphonuclear cells, PMN) is a multi-step process. Chemotaxis precedes transmigration through endothelial cell junctions, the production of reactive oxygen species, and microbial killing.
In this study, we described that a unique cytokine profile, produced by rMASP-1-stimulated HUVECs, is able to induce chemotaxis of neutrophil granulocytes, as a novel link between the complement system and the endothelial cell-mediated regulation of the neutrophil response.

Preparation and culture of human umbilical vein endothelial cells (HUVECs)
Cells were harvested from fresh umbilical cords obtained during normal delivery of healthy neonates (according to Helsinki Protocol, Semmelweis University Institutional Review Board specifically approved this study, (permission number: TU-KEB64/2008), and all participants provided their written informed consent to participate in this study), by collagenase digestion as described by Oroszlan et al [20]. HUVECs were kept in gelatin-precoated flasks in AIM-V medium (Invitrogen) completed with 1% FCS, 2 ng/ml human recombinant epidermal growth factor (R&D Systems), 250 pg/ml human recombinant bendothelial cell growth factor (BioSource/Invitrogen), and 7.5 U/ ml heparin, hereinafter referred to as Comp-AIM-V. Each experiment was performed on at least three independent primary HUVEC cultures from different individuals.

Measurement of CREB phosphorylation by immunofluorescence microscopy
HUVECs were treated with/without 2 mM of rMASP-1, in 100 ml Comp-AIM-V for 25 minutes, fixed and stained with rabbit-anti-human phospho-CREB (P-CREB, 1:200, Cell Signaling Technology Inc.) antibody followed by Alexa568 conjugated goat-anti-rabbit IgG (1:500) and Hoechst 33342 (1:50000, Molecular Probes/Invitrogen) as described previously [8]. All analyses were performed using the original, unmodified images; for visualization, the pictures were modified according to a standardized procedure, using Adobe Photoshop CS, without gamma-correction.

Analysis of CREB and JNK phosphorylation by Western blotting
Confluent cell cultures in 25 cm 2 flasks were treated with rMASP-1, thrombin, TNFalpha or IL-1beta for 25 minutes. After washing with ice-cold PBS, cells were lysed in buffer containing 30 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 20 mM NaF, 1 mM PMSF, 1 mM Na 3 VO 4 and 2% protease inhibitor cocktail (BD Bioscience). For the P-CREB analysis, the buffer described above was supplemented with 1% Triton-X100. Samples were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes, and probed as we described previously [8]. For CREB and phospho-CREB staining, we used the same antibodies as for microscopy.

mRNA analysis
Variously treated HUVECs were lysed and stored in TRIH reagent. Total RNA purification, reverse transcription and LightCyclerH analysis were performed as previously described by Megyeri et al [6]. The primers (Table 1) were designed from NCBI database, and produced by Bio Basic Canada Inc. GAPDH and bactin gene-specific primers were synthesized according to the published cDNA sequences. The purity and the size of the PCR products were checked by melting curve analysis and agarose gelelectrophoresis.

Measurement of cytokine production by sandwich ELISA
Confluent layers (10 5 cell/well) of HUVECs were cultured in 96-well plates for 24 hours in 100 ml Comp-AIM-V medium in the presence of rMASP-1 or other endothelial cell activating factors. IL-1alpha, IL-1ra, IL-6, IL-8, MCP-1, and TNFalpha were measured by sandwich ELISA kits according to the manufacturer's protocol (R&D Systems). We also analyzed the production of IL-6 and IL-8 at 1, 3, 6, 10, and 24 hours.

Analysis of IL-6, IL-8 production in presence of pathway inhibitors
Confluent layers of HUVECs were pre-incubated for 30 minutes with the following pathway inhibitors: SP600125, 25 mM -JNK; SB203580, 2 mM -p38-MAPK; Wortmannin, 100 nM -PI3-K; Bay-11 7082, 5 mM -NFkappaB and U0126, 1 mM -MEK1/2. The effective, non-toxic dose of different pathway inhibitors was determined in our preliminary experiments, and was similar to the literature data. For rMASP-1 treatment, we used the purified C1-Inhibitor -an endogenous, irreversible inhibitor of MASP-1 -as negative control. rMASP-1 was pre-incubated with C1-Inhibitor for 30 minutes before treatment. The IL-6 and IL-8 content of the supernatant were determined at 3 and 24 hours as described beforehand.

Measurement of rMASP-1 concentration and rMASP-1/ C1-Inhibitor disintegration
We measured the activity of rMASP-1 using LPAPR-AMC fluorescent substrate as previously described [6]. Concentration of rMASP-1 was calculated from the slopes of the kinetic curves. The detection limit of rMASP-1 was 0.5 nM.

Neutrophil preparation
Human neutrophils were isolated from venous blood of healthy volunteers by Ficoll gradient centrifugation, followed by hypotonic lysis of RBCs [22]. Cells were resuspended in Ca 2+ /Mg 2+ -free HBSS supplemented with 20 mM HEPES, pH 7.4, and used immediately. All experiments on human neutrophil granulocyte samples were approved by the institutional review board of Semmelweis University. All participants provided their written informed consent to participate in this study.

Preparation of HUVEC supernatants for experiments with neutrophil granulocytes
To avoid any possible direct effects of rMASP-1 on neutrophil granulocytes, HUVECs were treated or not with 2 mM rMASP-1 for 30 minutes then the medium was replaced by rMASP-1 free HBSS containing 0.5 mM CaCl 2 and 1 mM MgCl 2 for additional 2,5 hours. Then the HBSS supernatants (MASP-SN -rMASP-1 treated HUVEC supernatant, or UNT-SN -untreated HUVEC supernatant) were collected and stored frozen (280uC) until use.

Superoxide production
To measure reactive oxygen species production, a lucigeninbased chemiluminescence method was used [23]. Briefly, the cells were activated with MASP-SN or UNT-SN for 3 hours. In other experiments, we used HUVEC supernatants as preconditioning stimuli for 20 minutes and then, the response to 100 mM phorbol 12-myristate 13-acetate (PMA) or 1 mM N-formyl-methionylleucyl-phenylalanine (fMLP) was recorded.

Statistical analysis
Data were compared using Student's t-test or one-way ANOVA with Tukey's post-test (GraphPad Prism 5.01 software, GraphPad, http://www.graphpad.com/). A p-value less than 0.05 was considered significant. Data are presented as means 6 SEM unless otherwise stated.

Activation of CREB and JNK by rMASP-1
Previously, we reported that rMASP-1 could activate p38-MAPK, NFkappaB, and Ca-mobilization in HUVECs. We also demonstrated that the cleavage of PARs might be the initiator of rMASP-1 signaling [6]. Since PARs also utilize -through Gprotein coupled signaling -other pathways to induce proinflammatory cytokines, first we examined CREB and JNK activation by rMASP-1.
We found that rMASP-1 treatment could activate CREB phosphorylation in HUVECs, compared with untreated cells (Figure 1/A,B). We confirmed our results by Western Blot analysis. The effect of rMASP-1 on CREB phosphorylation was comparable with that of thrombin and of IL-1beta, used as positive controls. The effect of rMASP-1 could be completely inhibited by co-incubation with the C1-Inhibitor, which by itself did not induce CREB phosphorylation (Figure 1/C). rMASP-1 was also able to induce JNK phosphorylation, and the C1-Inhibitor abolished this effect too. (Figure 1/D).

Screening of cytokine production
Endothelial cells can produce several pro-, and anti-inflammatory cytokines. Therefore, we treated HUVECs with rMASP-1 for 6 hours and then, determined specific cytokine mRNA levels by qPCR. The level of IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha mRNA was increased, whereas the amount of IL-1alpha mRNA did not change (Figure 2/A). In addition, we treated HUVECs with rMASP-1 for 24 hours, and measured the cytokine profile with xMAP technology. IL-6 and IL-8 production increased significantly. MCP-1 level was moderately, but not significantly higher than in untreated controls, whereas the production of IL-1alpha, TNFalpha, IL-1ra did not change (Figure 2/A).
rMASP-1 dose dependence on IL-6 and IL-8 production rMASP-1 treatment induced IL-6, and IL-8 production in a dose-dependent manner. In concentrations as low as 250 nM, rMASP-1 caused a significant increase in the levels of both cytokines (one-way ANOVA). Although endothelial cells secrete large amounts of MCP-1, rMASP-1 did not increase the production of the latter any further (Figure 2/B-D).

Comparison of rMASP-1 with other endothelial cell activators
Several internal and external factors can trigger cytokine production in endothelial cells. A precise comparison of rMASP-1 with these factors (including fine dose-and time-dependence) was beyond the scope of our experiments. Nevertheless, we performed a simple analysis, where the effects of a single, predetermined, optimized dose of histamine (50 mM), bradykinin (20 mM), thrombin (300 nM) [6], TNFalpha (10 ng/mL), IL-1beta (1 ng/mL), and LPS (100 ng/mL) [8] were compared with those Kinetics of IL-6 and IL-8 production IL-6 and IL-8 synthesis and secretion are regulated at multiple levels in endothelial cells. To determine whether the transcription or the exocytosis of pre-formed granules is the main contributor to their secretion, we performed kinetic measurements at mRNA and protein levels. HUVECs were treated with 2 mM rMASP-1, and samples for mRNA extraction were harvested at 1, 2, 6, and 10 hours. The mRNA level of IL-6 increased rapidly and peaked between 1 and 2 hours (Figure 4/A). The increase of the mRNA level of IL-8 was slower (the peak was around 2 hours), but greater (Figure 4/D). Then, HUVECs were treated with 2 mM rMASP-1, and supernatants for ELISA were collected at 1, 3, 6, 10, and 24 hours. TNFalpha was used as a positive control. Untreated HUVECs produced both IL-6 and IL-8 at a basal level. rMASP-1 and TNFalpha induced IL-6 with very similar kinetics (Figure 4/ B,C). The kinetics of IL-8 production was, however, different in response to rMASP-1 and to TNFalpha. While rMASP-1 promptly induced IL-8 secretion (relative production compared to untreated cells peaked between 1 and 3 hours), the maximum effect of TNFalpha was at 6 hours ( Figure 4/E,F).
Unraveling the signaling pathways that contribute to IL-6 and IL-8 production induced by rMASP-1 Cytokine production can be triggered through various signaling pathways, most of which can be activated also by rMASP-1. Thus, we used several commercially available signaling pathway inhibitors to identify the most important pathways required for IL-6 and IL-8 production. HUVECs had been pre-incubated with the inhibitors for 30 minutes and then, treated with 2 mM rMASP-1 for 3 or 24 hours. The expression of both IL-6 and IL-8 could be inhibited by NFkappaB, p38-MAPK, or JNK at 3 hours of rMASP-1 activation ( Figure 5/A,B), whereas only the p38-MAPK inhibitor was able to block the production of both cytokines significantly, at 24 hours ( Figure 5/C,D). ERK 1/2 and PI3kinase inhibitors had no blocking effect at any time. PI3-kinase inhibitor, however, increased the IL-6 production induced by rMASP-1 at 3 hours. Furthermore, C1-Inhibitor blocked the expression of both cytokines when it was pre-incubated with rMASP-1 for 30 minutes before adding to the cells. Interestingly, while the blocking effect of the C1-Inhibitor on IL-6 persisted for 24 hours, it had no effect on IL-8 production when applied for 24 hours. Theoretically, this could happen if the rMASP-1/C1-Inhibitor complex disintegrates within a few hours (unlike C1-Inhibitor complexed with C1r/s, which is very stable), and rMASP-1 regains its activity. To test this hypothesis, we explored the disintegration of the rMASP-1/C1-Inhibitor complex. rMASP-1 was incubated with or without equimolar amounts of C1-Inhibitor for 24 hours, and small aliquots were tested with a fluorometric substrate-cleavage assay at different times. We found that rMASP-1 enzymatic activity decreased only slightly during the 24-hour incubation period (initial reaction rates of 20 nM

Activation of neutrophil granulocytes by the supernatant of rMASP-1 treated HUVECs
Since the advent of high-sensitivity of ELISA assays, the biological significance of cytokine production can be overestimat-  ed by the statistical methods. Thus, we tested whether the shortterm activation of HUVECs with rMASP-1 was able to induce cytokines in an amount sufficient to stimulate neutrophil granulocytes (PMN). To assess the quick response of PMNs, we monitored superoxide (O 2 2 ) production, and performed chemotactic measurements. To avoid the direct effect of rMASP-1 on PMNs, media with or without rMASP-1 were replaced after 30 minutes and further incubated in fresh, rMASP-1 free HBSS for additional 2,5 hours before collection (MASP-SN or UNT-SN, respectively), as it was described in Materials and Methods, and we verified that the MASP-SN was rMASP-1 free (Figure 6/A). First, we assessed superoxide production over 20 minutes by lucigeninbased chemiluminescence. We found that both supernatants induced extremely weak O 2 2 production, and MASP-SN did not differ from UNT-SN (1.35% vs. 1.55%, p = 0.31, where the maximal superoxide production, generated by 100 mM PMA, was regarded as 100%). PMNs were also preconditioned with MASP-SN or UNT-SN for 20 minutes and then, stimulated with PMA or fMLP. MASP-SN priming had no effect on the O 2 2 generation of PMNs compared to UNT-SN using either stimulator (15.52% vs. 15.96% for fMLP, p = 0.68 and 70.42% vs. 71.13% for PMA, p = 0.76).
Next, we performed a chemotactic assay, where PMNs were pipetted into transwell inserts and placed into 24-well plates loaded with HBSS (with Ca 2+ /Mg 2+ , as a negative control), MASP-SN, UNT-SN, or IL-8 (2 ng/mL, as a positive control). Compared with HBSS, even the untreated HUVEC supernatant induced chemotaxis, and this was further increased by MASP-SN. The chemotactic effect of MASP-SN was similar to that of 2 ng/mL IL-8 ( Figure 6/B).

Discussion
We found that rMASP-1 treatment activated JNK and CREB phosphorylation. The most important pro-inflammatory cytokines including IL-1beta and TNFalpha can simultaneously activate different MAPKs and NFkappaB. MASP-1 and these three pro-inflammatory activators appear to use similar pathways; however, in case of MASP-1, the MAPK pathways may be more effective than NFkappaB signaling. CREB, another important transcription factor of pro-inflammatory cytokine production [9,18,25], can be phosphorylated and activated via two main routes: by cAMP activated protein kinase A, and by p38-MAPK [9,26]. Since we have found previously that p38-MAPK pathway is readily activated by rMASP-1, the phosphorylation of CREB is not unexpected [6]. Thrombin, a serine protease similar to MASP-1 [27], also induces CREB phosphorylation, which supports that PAR signaling can lead to the activation of the CREB transcription factor [28].
The balance between the pro-and anti-inflammatory phenotypes is partially dependent on the cytokine pattern produced by endothelial cells. The pro-inflammatory cytokines IL-1alpha, IL-6, IL-8, MCP-1 and TNFalpha are representative of the endothelial phenotypic changes occurring during inflammation [29]. To counterbalance the effect of pro-inflammatory cytokines, the endothelium can produce anti-inflammatory cytokines, e.g. IL-1receptor antagonist, which can downregulate the inflammatory processes [30]. First, we screened the cytokine pattern of endothelial cells in response to rMASP-1 by bead-array and qPCR, to determine the most important targets for future studies. rMASP-1-triggered endothelial cells produced IL-6 and IL-8 at mRNA and protein levels, which switched the endothelial cells to a pro-inflammatory phenotype.
We described that the rMASP-1-induced production of the IL-6 and IL-8 cytokines was dose dependent. Interestingly, although we showed that rMASP-1 is able to increase the expression of IL-1ra, MCP-1 and TNFalpha at mRNA level, we could not detect any dose-dependent production of these chemokines at protein level. Increased mRNA levels without protein secretion predicts that MASP-1 may act in synergy with pro-inflammatory factors to produce IL-1ra, MCP-1 and TNFalpha. MASP-1 is one of the most abundant proteases in the plasma; its mean systemic concentration is 11 mg/ml [31] (143 nM). We found that the measurable effect of rMASP-1 on endothelial cytokine production was as low as 250 nM. However, at the site of complement activation and microvessel injury, local MASP-1 concentration may greatly exceed 143 nM. In these in vivo physiological/ pathophysiological conditions, therefore, MASP-1 concentrations may be similar to those tested in our in vitro experiments.
One-by-one comparisons of the production of cytokines may be misleading and useless, because the in vivo concentrations of endothelial cell activators are different. An alternative approach is to evaluate complex cytokine patterns [8], focusing on the relative proportions of the production of individual cytokines. The three most-cited factors acting as inflammatory signals and triggering cytokine production by endothelial cells are LPS, TNFalpha, and IL-1beta [29]. Compared with these, rMASP-1 has a lesser overall effect on cytokine production, and no effect whatsoever on MCP-1 production, whereas it induces significant IL-6 and IL-8 secretion. In contrast, LPS, TNFalpha, and IL-1beta stimulate IL-6, IL-8, and MCP-1 production, although to different extents [8]. Histamine, bradykinin, and thrombin are also well-described endothelial activators with pro-inflammatory effects, and additional actions that are not related to the inflammatory process [32]. Histamine and thrombin induced weak IL-6 and IL-8 expression, and left MCP-1 expression unchanged (data not shown). Thus, as regards the cytokine pattern, rMASP-1 is more similar to these two factors than to LPS, IL-1beta, and TNFalpha, or bradykinin.
Monocyte/granulocyte recruitment occurs at the site of inflammation within minutes, and the prompt release of relevant mediators from the endothelium underlies this rapid mechanism [33]. To control the migration of different cell subsets, the endothelial cells store granules of different cytokine composition [34]. MCP-1 and IL-8, the two well-characterized chemoattractants for monocytes and neutrophil granulocytes, are stored separately, and excreted in different quantities and with dissimilar velocities [34,35]. The rapid, thrombin-induced secretion of IL-8 takes place without de novo protein synthesis, while the proinflammatory, endothelial activators such as TNFalpha and IL-1beta cause consistent and prolonged IL-8 production [15,33,35]. IL-6 may co-localize with IL-8, but their separate storage has also been described [15]. We found that rMASP-1 and TNFalpha induced similar IL-6 excretion. However, rMASP-1 evoked much faster release of IL-8 than TNFalpha, and this implicates an instant chemotactic role for MASP-1-induced endothelial cells in the early immune response.
To better understand the secretion of cytokines, we tried to identify the signaling pathways involved in MASP-1-induced IL-6 and IL-8 production. The p38-MAPK inhibitor could abolish IL-6 and IL-8 secretion at both 3 and 24 hours, whereas the NFkappaB and JNK inhibitors were effective only at 3 hours. The ineffectiveness of JNK and NFkappaB inhibitors at 24 hours could be explained by the degradation of pathway inhibitors. However, Cherla et al. showed that these inhibitors were able to block LPSinduced IL-8 production completely at 24 hours [36]. It implicates that IL-6/IL-8-containing granules may translocate to Weibel-Palade bodies (WPBs) and subsequently undergo exocytosis in response to rMASP-1, as it has been described in case of IL-1beta [37], and this translocation as well as the process of exocytosis may be triggered via several pathways [38]. The p38-MAPK pathway, by contrast, appears to have a major role in the de novo synthesis of IL-6 and IL-8, which become the predominant cytokines after 24 hours. These cytokines carry several transcription factorbinding sites at their promoter region, of which NFkappaB, AP-1 and CREB they share in common. The MCP-1 promoter region also has binding sites for NFkappaB and AP-1, but not for CREB [16]. Since rMASP-1 did not induce MCP-1 expression, the difference in the presence of CREB binding sites between MCP-1 and IL-6/IL-8 promoters may emphasize the importance of CREB activation in rMASP-1 induced IL-6 and IL-8 production. PI3-kinase has only a weak effect on rMASP-1 induced IL-6 secretion at 3 hours. It is not surprising that this effect was an increased cytokine production instead of inhibition because Kim et al. described that PI3-kinase can suppress the NFkappaB pathway in HUVECs [39] and we showed that NFkappaB plays a role in the early IL-6 production by rMASP-1. Unexpectedly, although the C1-Inhibitor partially blocked IL-6 production at 24 hours, it had no effect on IL-8 secretion at this time. Since we showed that the rMASP-1/C1-Inhibitor complex did not disintegrate within 24 hours, an alternative mechanism should be presumed. As the C1-Inhibitor complex -but not the active C1-Inhibitor -binds to low-density lipoprotein receptor-related proteins (LRPs) [40] expressed on endothelial cells [41], it appears plausible that the rMASP-1/C1-Inhibitor complex may induce IL-8 production via LRPs after 24 hours of activation.
The differential expression of IL-6/IL-8 and of MCP-1 prompted us to assume that MASP-1-induced endothelial cells preferentially influence neutrophil functions. Neutrophil granulocytes are the first line of cellular defense against bacterial and fungal pathogens. Their activation begins within the blood vessels, whereby chemotaxis is induced by several pro-inflammatory chemoattractants, including IL-8, fMLP, and C5a [42]. This rapid process is followed by transmigration, further activation then phagocytosis and ROS generation. The rMASP-1-induced HUVEC supernatant did not trigger or prime O 2 2 production, whereas it had a significant effect on the rapid chemotaxis of neutrophils. Moreover, this effect was similar to that of 2 ng/mL IL-8, which is nearly the same dose measured in the rMASP-1treated HUVEC supernatant.
In our study, we assessed the effects of rMASP-1 on HUVECs as the most widely used endothelial cell model system. However, Figure 6. The migration of PMNs activated by the rMASP-1-treated HUVEC supernatant. HUVEC cells were treated with 2 mM 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 (A). PMNs were isolated from venous blood collected from 2 healthy volunteers. 10ˆ5 cells were seeded in 3-mm 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 (+/2SEM) of 3 different chemotaxis assays (B). The significance of the differences among rMASP-1 and other treatments is shown. *: p,0.05, ns: non-significant. doi:10.1371/journal.pone.0087104.g006 further experiments are required to compare MASP-1 responsiveness of endothelial cells with different origin.
MASP-1, a crucial element of the complement lectin pathway [2], is immediately activated upon microbial infection, enabling the downstream mediators of the complement system to opsonize and/or kill microbes. Our findings implicate that rMASP-1 can promptly attract neutrophil granulocytes by activating endothelial cells. This action may link the complement lectin pathway to targeted neutrophil activation, as two highly effective mechanisms of innate antimicrobial immunity.