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Originally published online as doi:10.1189/jlb.1105683 on April 14, 2006

Published online before print April 14, 2006
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(Journal of Leukocyte Biology. 2006;80:107-116.)
© 2006 by Society for Leukocyte Biology

C1q and MBL, components of the innate immune system, influence monocyte cytokine expression

Deborah A. Fraser*, Suzanne S. Bohlson*, Nijole Jasinskiene*, Nenoo Rawal{dagger}, Gail Palmarini*, Sol Ruiz*,1, Rosemary Rochford{ddagger} and Andrea J. Tenner*,§,2

* Departments of Molecular Biology and Biochemistry and
§ Pathology, Center for Immunology, University of California, Irvine;
{dagger} Department of Biochemistry, University of Texas Health Center at Tyler; and
{ddagger} Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse

2 Correspondence: Departments of Molecular Biology and Biochemistry and Pathology, Center for Immunology, 3205 McGaugh Hall, University of California, Irvine, CA 92697. E-mail: ATenner{at}uci.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has recently been recognized that the innate immune response, the powerful first response to infection, has significant influence in determining the nature of the subsequent adaptive immune response. C1q, mannose-binding lectin (MBL), and other members of the defense collagen family of proteins are pattern recognition molecules, able to enhance the phagocytosis of pathogens, cellular debris, and apoptotic cells in vitro and in vivo. Humans deficient in C1q inevitably develop a lupus-like autoimmune disorder, and studies in C1q knockout mice demonstrate a deficiency in the clearance of apoptotic cells with a propensity for autoimmune responses. The data presented here show that under conditions in which phagocytosis is enhanced, C1q and MBL modulate cytokine production at the mRNA and protein levels. Specifically, these recognition molecules of the innate immune system contribute signals to human peripheral blood mononuclear cells, leading to the suppression of lipopolysaccharide-induced proinflammatory cytokines, interleukin (IL)-1{alpha} and IL-1ß, and an increase in the secretion of cytokines IL-10, IL-1 receptor antagonist, monocyte chemoattractant protein-1, and IL-6. These data support the hypothesis that defense collagen-mediated suppression of a proinflammatory response may be an important step in the avoidance of autoimmunity during the clearance of apoptotic cells.

Key Words: human • macrophages • complement


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The innate immune system provides a powerful first line of host defense against invading pathogens with molecules of the innate immune system directly involved in the recognition and elimination of pathogens. It is clear, however, that the innate immune system is not only responsible for the induction of acute responses necessary for elimination of pathogens but is also able to activate appropriate adaptive immune responses via ligand interaction and secretion of cytokines and chemokines [1 ].

C1q and mannose-binding lectin (MBL), as well as surfactant protein A (SPA), SPD, and ficolin, are members of a unique family of proteins, known as the defense collagens. This family of macromolecules is characterized by a conserved, collagen-like region of repeating Gly-X-Y triplets contiguous with a noncollagen-like sequence [2 ]. In general, the globular carboxyl terminus recognizes specific pathogen-associated molecular patterns such as mannose-containing carbohydrates on pathogen surfaces (recognized by MBL), triggering an appropriate, immediate, protective response. For example, C1q, MBL, and ficolin function to eliminate invading microorganisms by activating the classical pathway (C1q) or the lectin pathway (MBL and ficolin) [3 ] of the complement system by transmitting a signal from the recognition domains of the globular heads to their collagen-like domains, which autoactivates their associated serine proteases (C1r2s2 or mannan-binding lectin-associated serine proteases [4 ]). It has also been shown that members of the defense collagen family, C1q, MBL, SPA, SPD, and ficolin, can enhance or initiate the phagocytosis of suboptimally opsonized targets [5 6 7 ] (and unpublished data). All defense collagens that have been tested (C1q, SPA, MBL) have shown a qualitatively and quantitatively similar enhancement of monocyte phagocytosis of targets that are suboptimally opsonized with immunoglobulin G (IgG) or complement receptor type 1 ligands, C4b and C3b [5 , 8 , 9 ], and the six amino acid sequence required for this functional stimulation has been identified within the collagen-like domain [10 ]. This rapid enhancement of phagocytic activity is triggered when the defense collagen is bound to the particle to be ingested [11 ] or presented to the cell in a multivalent manner as when immobilized on a surface [5 ]. This may be a critical mechanism in host defense, particularly at early stages of infection/disease when little or no adaptive response is yet present [2 , 8 , 12 ].

Recent evidence suggests an additional role for these pattern recognition molecules in the recognition and removal of apoptotic cells [13 14 15 16 ]. C1q and MBL have been shown to bind directly to apoptotic cell surfaces and apoptotic cell blebs via their globular heads [17 , 18 ]. Interaction of the collagen-like tails with the phagocyte surface triggers apoptotic cell ingestion via macropinocytosis [15 ]. Indeed, the importance of the role of defense collagens in clearance of apoptotic cells is highlighted by studies in vivo of mice deficient in C1q, SPD, and MBL [16 , 19 , 20 ], in which mice exhibit impaired clearance of apoptotic cells. Deficiency of C1q is also a risk factor for the development of autoimmunity in humans and mice [21 22 23 24 ]. These data are consistent with the hypothesis that a deficiency in rapid clearance of apoptotic cells, which can result in extracellular disintegration of the cell and release of intracellular components, may contribute to "breaking tolerance" by facilitating an immune response to intracellular constituents (i.e., promoting autoimmunity) [25 ].

It is now evident that cytokines and phagocytic antigen-presenting cells play a critical role in directing the type and extent of an immune response to perceived "danger" [26 , 27 ]. Mice deficient in defense collagens SPA or SPD exhibited enhanced inflammatory responses in the lung to a variety of stimuli [28 29 30 ], which could be a result of uncleared pathogens or the absence of additional signals presented to the cells by the defense collagen. Indeed, overexpression of SPD leads to decreased inflammation (reviewed in ref. [31 ]). In addition, a mounting body of evidence suggests that apoptotic cells are able to actively suppress an inflammatory response. That is, apoptotic cells inhibit the production of inflammatory mediators and promote secretion of anti-inflammatory and immunoregulatory cytokines such as interleukin (IL)-10 by monocytes, macrophages, and dendritic cells (DC) [32 33 34 ]. Although the role of apoptotic cell opsonins, such as the defense collagens, in facilitating a rapid phagocytic response has been investigated by a number of groups, the influence of defense collagens on cytokine production by phagocytic cells has only been explored recently [35 , 36 ]. The studies reported here were undertaken to begin to dissect the role of defense collagens in modulating the cytokine expression by phagocyte cells, specifically examining the effect of C1q and MBL on monocyte cytokine production under conditions in which phagocytosis is enhanced.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and reagents
HL-1 medium (serum-free) was purchased from Cambrex Laboratories (Walkersville, MD), fetal calf serum from Hyclone (Logan, UT), and L-glutamine from Mediatech Cellgro (Herndon, VA). The human serum albumin (HSA) used for the elutriation buffer was obtained from FFF Enterprises as prepared for the American Red Cross by Baxter Healthcare Corp. (Westlake Village, CA). Lipopolysaccharide (LPS; from Escherichia coli 0127:B8) was obtained from Sigma Chemical Co. (St. Louis, MO; Cat. #L-3129), and ultra-pure LPS (from E. coli O111:B4) was obtained from List Biological Laboratories (Campbell, CA). {alpha}32P Deoxyuridine triphosphate was obtained from Amersham Biosciences (Piscataway, NJ). All other reagents used, except where noted otherwise, were obtained in the highest quality available from Sigma Chemical Co. Pyrogen-free water (MilliQ-Plus) was used for all laboratory buffers and reagent preparation.

Protein isolation
C1q was isolated from plasma-derived human serum by the method of Tenner et al. [37 ] and modified as described [38 ] or purchased from Advanced Research Technologies (now Complement Technology, Inc., Tyler, TX). The preparations used were fully active, as determined by hemolytic titration, homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and free of endotoxin to below 1 pg/mL in the C1q concentrations used in these studies, as assayed by the limulus amebocyte lysate assay (BioWhitaker, Walkersville, MD). Protein concentration was determined using an extinction coefficient (E1%) at 280 nm 6.82 for C1q [39 ]. MBL was purified from human plasma by the method of Tan et al. [40 ] and modified as follows: MBL was purified further by ion-exchange chromatography using fast protein liquid chromatography. The purity of MBL was determined by SDS-PAGE, which showed a single 32-kDa band in the presence of reducing agents. Protein concentration of MBL was determined using E1% at 280 nm 7.2 [41 ]. All proteins were stored at –70°C.

Monocytes
Human peripheral blood monocytes were isolated by counterflow elutriation using a modification of the technique of Lionetti et al. [42 ] as described [43 ]. All blood samples were collected in accordance with the guidelines and approval of the University of California Irvine (UCI) Institutional Review Board. Blood units were collected into citrate acid dextrose adenine (CPDA1) at the UCI General Clinical Research Center. Greater than 90% of the cells in each preparation were monocytes, according to size analysis on a Coulter Channelyzer. In some experiments, the elutriated cell preparations were incubated with antibodies to CD93 (IgM R3, as described [44 ], or from Chemicon, Temecula, CA) and antibodies from eBioscience (San Diego, CA)—CD14 (61D3), CD56 (MEM-188), and CD3 (Okt3)—and analyzed using the FACSCalibur (Becton Dickinson, San Jose, CA) and the CellQuest program. These cells (82–94%) were monocytes as measured by positive staining for CD93 [45 ] (previously shown to correspond to monocytes [46 ]), and <1% of these cells were CD3-positive or CD56-positive, and thus, the elutriated cell preparations are considered monocyte populations. Monocytes were cultured in serum-free HL-1 medium supplemented with 1% L-alanyl L-glutamine (Gibco-BRL, Grand Island, NY) for cytokine expression assays or resuspended immediately in phagocytosis buffer (RPMI 1640, 25 mM Hepes, 5 mM MgCl2). LabTek chambers (Nunc, Rochester, NY) were coated with C1q, MBL, or other control or test proteins (8 µg/mL) in coating buffer (0.1 M carbonate, pH 9.6) and incubated at 37°C for 2 h. After washing chambers twice with phosphate-buffered saline (PBS), monocytes (2 ml 106/ml per one-well chamber slide for cytokine expression assays and 250 ul 2.5x105/ml for phagocytosis assays) were added to chambers, centrifuged at 70 g for 3 min, and cultured for various periods of time at 37°C in 5% CO2 air. Where indicated, LPS (10–30 ng/ml) was added directly to the monocytes. In some experiments, intracellular cytokine staining was carried out on monocytes after 18 h of incubation followed by the addition of Monensin, a protein transport inhibitor (eBioscience) for 2 h. Cells were stained using phycoerythrin (PE)-conjugated anti human IL-1ß and appropriate isotype control, according to the manufacturer’s instructions (eBioscience), and analyzed by flow cytometry. These cells were also stained for the monocyte marker CD14.

Phagocytosis assay
Phagocytosis assays were performed essentially as described previously [47 ]. Sheep erythrocytes, opsonized suboptimally with IgG (EA), were used as targets and prepared as described previously [48 ]. After adherence of monocytes for the times indicated (5–60 min), 107 targets in 100 µl were added to each well and after centrifuging at 70 g for 3 min, incubated for an additional 30 min at 37°C 5% CO2. Uningested targets were lysed, and cells were fixed in 1% glutaraldehyde in PBS. Cells were visualized using a modified Giemsa stain (Sigma Chemical Co.), and at least 200 cells/well were counted. Percent phagocytosis is the number of cells ingesting at least one target/total number of cells scored x100. Phagocytic index is the number of ingested targets per 100 cells counted.

RNase protection assay (RPA)
Total RNA was extracted from monocytes using the TRIzolTM reagent (Gibco-BRL) following the manufacturer’s direction. Any nonadherent monocytes were recovered by centrifugation of cell supernatants and extracted as well. The RPA was performed following the manufacturer’s protocol (BD Biosciences PharMingen, San Diego, CA). Briefly, a human cytokine template set, hck2, containing IL-12p40, IL-10, IL-1{alpha}, IL-1ß, IL-1 receptor antagonist (IL-1Ra), IL-6, and L32, or a custom probe set, which also included tumor necrosis factor {alpha} (TNF-{alpha}), was used in an in vitro transcription reaction to synthesize an {alpha}32P-labeled anti-sense RNA probe set. Labeled antisense RNA was hybridized overnight with the total RNA, and unprotected (single-stranded) RNA was then digested by addition of RNase T1. Protected fragments were analyzed by electrophoresis in 5% acrylamide/8 M urea gels. Dried gels were placed in a Molecular Dynamics storage phosphor screen (Molecular Dynamics, Sunnyvale, CA) and were visualized using a Molecular Dynamics phosphorimager. Band intensities were measured by ImageQuaNT software for signal quantitation.

Luminex multiplex cytokine assay
The levels of IL-1{alpha}, IL-1ß, IL-1Ra, IL-6, IL-10, TNF-{alpha}, IL-12p70, and monoycte chemoattractant protein-1 (MCP-1) were measured in cell culture supernatants in a multiplex cytokine assay, which was carried out using a human cytokine LINCOplex kit (Linco, St. Charles, MO), according to the manufacturer’s protocol. Briefly, cytokine capture antibody-coupled latex microbeads were provided with distinct ratios of two fluorophores for each cytokine to be tested. Assay supernatants were centrifuged to remove cellular debris and incubated with the anticytokine bead sets in triplicate, followed by incubation with a detection antibody coupled to PE.

The microbeads and reporter molecule were read on a Luminex100 v.1.7 (Luminex, Austin, TX). Determination of cytokine concentrations from the mean fluorescence values obtained was calculated from standard curves of each cytokine tested using Miraibio Master Plex QT software (Miraibio, Alameda, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
C1q and MBL suppress LPS-mediated up-regulation of specific proinflammatory cytokine mRNA
Defense collagen-triggered enhancement of phagocytosis has been well-described (reviewed in ref. [2 ]) and is demonstrated with a photomicrograph of a representative experiment shown in Figure 1A . To clarify the time required to induce this enhanced uptake, human monocytes were adhered to C1q or control protein HSA for 5, 15, 30, or 60 min at 37°C prior to addition of erythrocytes suboptimally opsonized with IgG. After an additional 30-min incubation with the targets, phagocytosis was scored as described in Materials and Methods. The results demonstrate that C1q enhances the ability of phagocytic cells to ingest suboptimally opsonized targets after as little as 5 min of exposure to multimeric C1q (Fig. 1B) . To assay longer-term effects of these innate recognition molecules, the effect of C1q and MBL on mRNA expression of selected cytokines was examined in monocytes under similar conditions.


Figure 1
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  		Figure 1. C1q and MBL rapidly enhance phagocytosis of suboptimally opsonized targets compared with HSA control. (A) Photomicrographs of a typical experiment in which human monocytes were adhered to C1q, MBL, or HSA for 30 min prior to addition of suboptimally opsonized early antigen (EA)-IgG targets. After an additional 30 min, cells were fixed, stained, and photographed. (B) Quantified data from an assay, in which monocytes were adhered to C1q (dotted line) or HSA (solid line) for the time indicated, followed by addition of EA-IgG targets for 30 min. Values are the mean of duplicate wells in which at least 200 cells were scored. Error bars are SD. (A and B) Data are from individual experiments, representative of many ([5 , 8 , 10 , 49 50 51 ] and data not shown).

 
RNA was extracted from monocytes incubated for 2 h on HSA-, C1q-, or MBL-coated wells and for up to 18 h, and cytokine mRNA expression was measured by RPA as described in Materials and Methods. A phosphorimage scan of a representative experiment is shown in Figure 2A , and analysis of cytokine band intensity was normalized to the L32 ribosomal mRNA shown in Figure 2B . mRNA from cells cultured in C1q-coated wells showed a small but reproducible increase in the basal levels of IL-1{alpha}, IL-1ß, IL-6, IL-1Ra (Fig. 2A and 2B) , and TNF-{alpha} (data not shown) at 2 h but returned to the background level (HSA control equivalent) by 18 h. RPA analysis showed that exposure of monocytes to LPS resulted in an induction of IL-1{alpha}, IL-1ß, IL-6, and IL-1Ra mRNA, detectable at 2 h, and greatly increased at 6 (data not shown) and 18 h (Fig. 2A and 2B) . However, in cells adhered to C1q, the stimulatory effect of LPS on IL-1{alpha}, IL-1ß, and TNF-{alpha} mRNA levels was inhibited at 18 h (Fig. 2A and 2B , and data not shown). Similar results were seen with multiple donors (three for 2 h and six for 18 h). When the fold reduction of LPS-induced cytokine mRNA was calculated for cells adhered to C1q compared with control cells within each experiment, significant reductions in mRNA levels for IL-1{alpha}, IL-1ß, and TNF-{alpha} were evident (Fig. 2C) . Cells incubated on MBL-coated slides showed a similar, significant decrease in IL-1{alpha} and IL-1ß mRNA (TNF-{alpha} was not assayed). Decreases in IL-1Ra and IL-6 mRNA levels were also seen for monocytes exposed to LPS and incubated on C1q- and MBL-coated wells for 18 h, but these decreases were not statistically significant (Fig. 2C) . Although IL-12p40 mRNA was seen to be up-regulated in cells that were stimulated with C1q, mRNA for IL-12p35, required for formation of intact IL-12p70, was not detected in any samples tested (data not shown).


Figure 2
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  		Figure 2. C1q and MBL down-modulate LPS-induced proinflammatory cytokine mRNA synthesis by human monocytes cultured in serum-free (HL-1) media. Human monocytes were added to Labtek chambers coated with 8 µg/mL C1q, MBL, or HSA and stimulated with LPS. Cytokine mRNA expression was examined using an RPA. (A) Representative RPA gel phosphorimage from monocytes from an individual donor adhered to HSA (H) or C1q (Q) after 2 and 18 h of incubation with 0, 10, or 30 ng/ml LPS. (B) Quantitation of band intensity from the individual experiment in A of C1q (dotted line)- or HSA (solid line)-treated cells normalized to L32. (C) The average fold difference in levels of normalized cytokine mRNA in multiple experiments from cells incubated with C1q or MBL and activated with 30 ng/mL LPS compared with the LP5-treated HSA control within individual experiments (n=6 for C1q, n=3 for MBL) ± SD. *, P < 0.05; **, P < 0.005, ANOVA.

 
The suppressive effects of C1q on these proinflammatory cytokine mRNA levels were observed using three different C1q preparations, suggesting that the inhibition was caused directly by C1q and not contaminating molecules. Furthermore, the pepsin-resistant, collagen-like fragment of C1q, which is known to bind to cells and induce enhanced phagocytosis [8 , 52 ], inhibited cytokine synthesis similar to the intact C1q molecule (data not shown). Experiments presented here were carried out with LPS from Sigma Chemical Co.; however, similar results were seen when repeated with ultra-pure LPS from List Biological Laboratories (data not shown). Cells incubated under these different conditions had distinct morphology (Fig. 3 ), appearing more rounded and less adherent on C1q than those plated on HSA, but the cells were viable, as assessed by trypan blue exclusion and expressed comparable levels of the housekeeping L32 mRNA (Fig. 2A and data not shown).


Figure 3
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  		Figure 3. Morphology of monocytes varies according to the culture conditions. Human monocytes isolated by elutriation were cultured in serum-free, defined media (HL-1) in control wells (A, C) or C1q-coated wells (B, D) in the absence (A, B) or presence (C, D) of 100 ng/ml LPS for 18 h. Original magnification, 100x.

 
C1q and MBL enhance mRNA levels of anti-inflammatory cytokine IL-10
Given multiple reports of IL-10 anti-inflammatory action, we assessed the effect of C1q and MBL on IL-10 mRNA levels, which were never detected in unstimulated monocytes. Although expression varied among donors, IL-10 mRNA was detected in cells adhered to C1q-coated surfaces. Addition of 10–30 ng/ml LPS resulted in detectable expression of IL-10 mRNA (at 18 h) in three individual human donors, and C1q further enhanced the IL-10 mRNA (at least twofold) compared with the HSA-coated control wells. A phosphorimage scan of one experiment in which IL-10 is detected is shown in Figure 4A . Data from three such experiments were used to calculate fold enhancement shown in Figure 4B . Similar to C1q, monocytes cultured on MBL-coated wells also show significantly enhanced production of IL-10 mRNA when compared with HSA-coated control wells (Fig. 4B , n=3).


Figure 4
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  		Figure 4. C1q and MBL enhance anti-inflammatory IL-10 mRNA production in human monocytes. Human monocytes were added to chambers coated with C1q, MBL, or HSA (8 µg/ml) and cultured for 18 h in the presence of LPS. Cytokine mRNA expression was examined using an RPA as described in Materials and Methods. (A) Phosphorimage from an individual experiment representative of three. (B) The average fold enhancement in levels of IL-10 mRNA (normalized to L32 mRNA) in cells activated with 30 ng/mL LPS and interacting with C1q or MBL compared with the LPS-activated HSA control within individual experiments (n=3) ± SD. *, P < 0.05, ANOVA.

 
C1q and MBL suppress LPS-induced levels of proinflammatory cytokines IL-1{alpha} and IL-1ß
To investigate if the effects of C1q and MBL on cytokine mRNA production correlated with cytokine protein expression, multiplex Luminex analyses of cytokine levels in monocyte culture supernatants were carried out, as described in Materials and Methods. Cytokine levels were low or undetectable in supernatants from monocytes cultured in serum-free media and adhered to C1q, MBL, or HSA in the absence of LPS for 2–18 h for all cytokines tested. However, detectable levels of proinflammatory cytokines IL-1{alpha} and IL-1ß were observed after 6 h stimulation with 30 ng/mL LPS, and levels of IL-1{alpha} and IL-1ß were increased greatly by 18 h. As anticipated from the levels of cytokine mRNA after 18 h, the amounts of IL-1{alpha} and IL-1ß were reduced greatly in the media of LPS-treated cells exposed to C1q or to MBL compared with control LPS-treated cells (Fig. 5A , n=5 for C1q and n=3 for MBL). When the fold reduction in cytokine concentration was calculated for cells adhered to C1q compared with control cells within each experiment, monocytes cultured with C1q or MBL showed highly significant (P<0.005) reductions in protein levels from the control (2.5- to threefold) for IL-1{alpha} and IL-1ß (Fig. 5B) . Intracellular IL-1ß staining followed by fluorescein-activated cell sorter analysis demonstrated that the cellular source of this cytokine was the monocyte population. That is, 53–75% of the cells in the culture stained positive for IL-1ß (n=3, data not shown), and those cells showed forward- and side-scatter consistent with the CD14-positive monocyte population, providing evidence that it is the monocyte population that is responsible for producing the IL-1ß.


Figure 5
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  		Figure 5. C1q and MBL down-modulate secretion of LPS-induced proinflammatory cytokines from human monocytes cultured in serum-free (HL-1) medium. Monocytes were added to one-well chamber slides coated with HSA (solid bars), C1q (open bars), or MBL (hatched bars; 8 µg/ml) and cultured for 6 or 18 h in the presence or absence of 30 ng/ml LPS. Cytokine protein expression was quantified using a multiplex cytokine assay. (A) Mean ± SD of cytokine concentrations in supernatant for 6 h (n=3) and 18 h (n=5 or 3 for C1q and MBL, respectively). (B) The average fold difference of cytokine concentration from cells incubated 18 h with 30 ng/ml LPS, adherent to C1q or MBL relative to HSA plus LPS (n=5 for C1q, n=3 for MBL) ± SD. *, P < 0.05, ANOVA; **, P < 0.005, ANOVA.

 
C1q and MBL enhance LPS-induced levels of secreted cytokines IL-10, MCP-1, IL-1Ra, and IL-6 but not TNF-{alpha} or IL-12p70
Multiplex cytokine analysis of supernatants from LPS-activated monocytes cultured in serum-free media showed detectable levels of IL-10, IL-1Ra, MCP-1, IL-6, and TNF-{alpha} for all donors tested after 6 h (n=3) and to a greater extent, 18 h (n=5) of incubation (Fig. 6A ). Consistent with the mRNA data, protein levels of IL-10 were increased eight- to 50-fold in cells, which were incubated on C1q- or MBL-coated wells compared with HSA-coated control wells after 18 h in the presence of LPS (Fig. 6B) . It is interesting that levels of MCP-1 showed a similar C1q-dependent increase, 25-fold, relative to HSA in LPS-treated cultures.


Figure 6
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  		Figure 6. C1q and MBL enhance IL-10, MCP-1, IL-1Ra, and IL-6 production in human monocyte. Monocytes were added to chambers coated with HSA (solid bars), C1q (open bars), or MBL (hatched bars; 8 µg/ml) and cultured in the presence of 30 ng/ml LPS. Cytokine levels in the supernatant were examined using a multiplex cytokine assay. (A) Average cytokine concentration in the supernatants collected at 6 h (n=3) or 18 h (n=5 and 3 for C1q and MBL, respectively) ± SD. (B) The average fold difference in LPS-stimulated cytokine levels from cells incubated for 18 h with C1q or MBL relative to HSA (n=5 for C1q, n=3 for MBL) ± SD. *, P < 0.05, ANOVA; **, P < 0.005, ANOVA.

 
In contrast to the lack of significant regulation of IL-1Ra and IL-6 mRNA by C1q and MBL, protein levels of IL-1Ra and IL-6 were increased in supernatants from defense collagen-treated cells. Although the actual amounts varied between donors, particularly for IL-1Ra, C1q treatment resulted in apparent increases for IL-1Ra (twofold) and increases of IL-6 (fourfold) in terms of fold enhancement within individual experiments compared with supernatants from cells cultured in HSA-coated control wells (Fig. 6B) . MBL-stimulated monocytes also produced the enhanced expression of IL-10, MCP-1, IL-1Ra, and IL-6 compared with control cells, similar to that seen with cells cultured on C1q-coated wells (Fig. 6B) .

Soluble TNF-{alpha} levels, triggered by LPS activation of monocytes for 6 and 18 h, were variably altered by interaction with C1q (1.3- to fourfold over LPS alone) or MBL (0.2- to 2.9-fold LPS alone; Fig. 6B ). Levels of IL-12p70 were at or below the detection sensitivity of the assay for all samples tested (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The basic understanding of the immune system has undergone a substantial paradigm shift in the past decade, as an awareness of the power and influence of the innate immune system has emerged. It is now being recognized that the nature of the first response to invasion (i.e., the innate immune response) has significant influence in determining the nature of the subsequent adaptive immune response. Soluble and membrane-bound pattern recognition molecules of the innate immune system assess the level of danger of a particular intrusion or injury and initiate a program of protection for the host [53 ]. Phagocytic cells are mediators of some of these changes, as when activated by pathogens, monocytes/macrophages/DC often initiate the synthesis of proinflammatory cytokines [54 ]. The cytokine environment then influences the subsequent specific immune responses. Conversely, soluble/humoral and cellular components of the innate system clear damaged cells or tissue debris and enlist systems of tissue repair. Thus, in the absence of perceived danger, the innate system appears to be able to discriminate removal of dying cells and avoid the induction of an adaptive immune response, which would result in autoimmunity and/or further tissue damage [32 ]. In this study, we demonstrate that C1q and MBL, recognition molecules of the complement system that also facilitate rapid ingestion of suboptimally opsonized particles, modulate the profile of cytokines released as a result of their interaction with phagocytic cells.

C1q and MBL modulated cytokine mRNA and protein levels in response to a microbial signaling molecule LPS, including diminution of proinflammatory IL-1{alpha} and IL-1ß (Fig. 2A and 2B , Fig. 5 ) and elevating levels of anti-inflammatory IL-10 (Fig. 4 and 6) . IL-1{alpha} and IL-1ß are responsible for many of the symptoms associated with inflammation, including fever, pain, erythema, and swelling (for review, see ref. [55 ]). Consistent with a program modulating proinflammatory events, protein levels of the IL-1Ra, which is able to abrogate the effects of IL-1{alpha} and IL-1ß in vivo by competing for receptor binding, were also raised an average of three- to fourfold by interaction of the defense collagens with LPS-activated monocytes.

Sustained LPS activation of monocytes in vitro can lead to the production of anti-inflammatory IL-10 with maximal levels at 24–48 h [56 ]. Here, stimulation with C1q or MBL further enhanced IL-10 mRNA by an average of 1.8 (MBL)- to twofold (C1q) (Fig. 4B) . However, the effect of C1q and MBL on IL-10 production by LPS-activated monocytes was more striking at the protein level (Fig. 6A) , with average enhancements of 22- and 25-fold for C1q and MBL, respectively (Fig. 6B) , relative to LPS stimulation alone, reflecting more than one level of regulation of cytokine production. IL-10 has been shown to influence proinflammatory cytokine production [56 ], but it is unlikely that the enhanced production of IL-10 in the C1q- and MBL-treated cultures is mediating the decrease in LPS-induced mRNA and protein levels of IL-1{alpha} and IL-1ß as a result of the temporal appearance of IL-10 protein (i.e., later than the depressed IL-1 levels) in these cultures. IL-10 exerts anti-inflammatory functions by inhibition of various interferon-{gamma}-stimulated monocyte functions associated with induction of an inflammatory response such as major histocompatibility complex type II expression [56 ], H2O2 production [57 ], and nitric oxide synthesis [58 , 59 ] and also up-regulates phagocytosis [60 ]. Studies with knockout mice support the critical role of IL-10 in limiting the inflammatory response in vivo [61 , 62 ].

Levels of MCP-1 are also elevated in cultures in which monocytes are exposed to C1q or MBL (Fig. 6B) , even in the absence of LPS activation (although to a lesser degree). MCP-1 is a potent chemokine, which attracts monocytes and T cells into the site of infection/immune activation but does not itself cause inflammatory activation of cells [63 ] in the absence of secondary signals. MCP-1 has also been shown to enhance phagocytosis under certain conditions [64 ] and therefore, may be recruiting additional phagocytic cells to facilitate clearance, as well as cells to survey the injury site for evidence of pathogenic/danger signals.

In contrast to IL-1, IL-6, although somewhat reduced at the mRNA level by C1q and MBL, is elevated in the cell media of LPS-activated monocytes adhered to C1q or MBL, with an observed threefold average enhancement. This discrepancy between IL-6 mRNA level and protein levels in the media again indicates alternative regulation, perhaps via post-translational events, or enhanced stability of the protein over time compared with that of the mRNA. IL-6 is classically labeled a proinflammatory cytokine and skews the immune response to a T helper cell type 2 (Th2) versus Th1 response [65 ]. However, it was also reported recently that IL-6 was crucial in tolerizing autoreactive B cells [66 ]. Furthermore, enhanced levels of IL-6 have also been implicated in promoting the differentiation of monocytes to macrophages rather than DC [67 ], which could dampen the presentation of autoantigens to autoreactive T cells.

MBL and SPA have been shown to influence cytokine production in a number of situations [36 , 68 69 70 71 72 ]. Others have reported seemingly conflicting results of the effects of C1q on cytokine and chemokine production by in vitro-derived DC [73 74 75 ]. Gardai, working with Voelker, Henson, and others [36 ], reported that C1q added to macrophages had limited to no effects on cytokine production or signaling pathways in contrast to SPA. However, the C1q in these assays was monomeric rather than immobilized or particle-bound and thus, would not be expected to influence these activities (whereas SPA is prone to aggregation with itself or with phospholipids). Roos and colleagues [75 ] reported a C1q-mediated, approximately twofold up-regulation of phagocytosis of apoptotic Jurkat cells in DC-SIGN +/CD14– human monocyte-derived DC and an increase of IL-6 and IL-10 from immature DC over basal levels, but the additional, critical comparison of the production of these cytokines relative to levels that trigger inflammatory sequelae was not addressed.

The data presented here support the hypothesis that C1q and MBL (and SPA) are influencing gene expression of phagocytic monocytes, including a down-regulation of expression of certain inflammatory genes. This down-modulation may provide a crucial barrier to immune responses to self-antigens during uptake of apoptotic cells, as apoptotic cells bind C1q and MBL [15 ]. This profile of cytokines induced by C1q and MBL interaction with LPS-activated monocytes provides a cytokine milieu in which proinflammatory mediators are inhibited (IL-1{alpha} and IL-1ß), and anti-inflammatory mediators IL-10 and IL-1Ra are increased, contributing to the nonimmune-activating environment and/or promotion of resolution of the inflammatory response. This adds to the previously observed suppression of anti-inflammatory response from phagocytes after interaction with apoptotic cells [33 , 34 , 76 77 78 ] and the suppression of IL-12 biosynthesis and secretion of high levels of IL-10 by macrophages activated in the presence of immune complexes [79 , 80 ]. These studies provide new information about the ability of the innate immune system to direct appropriate responses for immunity or resolution of inflammation or injury.

Phagocytic cells use their multiple initial ligand receptor interactions to assess the level and type of danger of an intrusion or injury and thereby, initiate a specific program of events, which clears the initial foreign intruder and/or damaged cellular material and results in signals to other components of the immune system to induce an appropriate, protective response and/or to limit tissue damage and generation of immune responses to self-antigens. The end result depends not only on the milieu of signaling molecules present at a given site but also on the differentiation state of the phagocytic cells at the site of injury and the presence of relevant membrane receptors sensing the various stimuli. Such a mechanism allows for the optimal diversity and fine-tuning of the appropriate response—triggering a Th1 or Th2 adaptive immune response or facilitating repair and/or resolution of inflammation. Indeed, observations by Lacy-Hulbert and colleagues [81 ] have begun to delineate the qualitatively different responses, which result when apoptotic cells are phagocytosed by myeloid cells in the presence of Toll-like receptor ligands. With detailed knowledge about the processes and molecular interactions governing them, it should be possible to target these biosensors, including soluble tags and the cell surface molecules/complexes that are pattern recognition molecules or their receptors, to prevent or modulate autoimmunity as well as to direct more effective, protective responses, including more efficient, appropriate, and protective vaccine strategies.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant AI 41090 (A. J. T.) and HL073804 (N. R.). Support for obtaining human blood products used in this study was provided in part by Public Health Service Research Grant M01 RR00827 from the National Center for Research Resources. The authors are grateful to Ozkan Yazan and Mallary Greenlee for excellent technical assistance and Dr. Ed Nelson for helpful advice and comments. The authors thank the staff of the UCI General Clinical Research Center for obtaining human blood for monocyte purification.


   FOOTNOTES
 
1 Current address: Division of Biologicals and Biotechnology, Spanish Medicines Agency, 28220 Majadahonda, Madrid, Spain. Back

Received November 23, 2005; revised February 26, 2006; accepted March 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Activation of Complement Component C5: COMPARISON OF C5 CONVERTASES OF THE LECTIN PATHWAY AND THE CLASSICAL PATHWAY OF COMPLEMENT
J. Biol. Chem., March 21, 2008; 283(12): 7853 - 7863.
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T. Carter, M. Sumiya, K. Reilly, R. Ahmed, P. Sobieszczuk, J. A. Summerfield, and R. A. Lawrence
Mannose-Binding Lectin A-Deficient Mice Have Abrogated Antigen-Specific IgM Responses and Increased Susceptibility to a Nematode Infection
J. Immunol., April 15, 2007; 178(8): 5116 - 5123.
[Abstract] [Full Text] [PDF]

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D. A. Fraser, M. Arora, S. S. Bohlson, E. Lozano, and A. J. Tenner
Generation of Inhibitory NF{kappa}B Complexes and Phosphorylated cAMP Response Element-binding Protein Correlates with the Anti-inflammatory Activity of Complement Protein C1q in Human Monocytes
J. Biol. Chem.,