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PageArticles 1 of 46 in PresS. Am J Physiol Lung Cell Mol Physiol (October 27, 2006). doi:10.1152/ajplung.00127.2006 Complement Levels and Activity in the Normal and LPS-Injured Lung Molly S. Bolger1, DeAndre S. Ross1, Haixiang Jiang2, Michael M. Frank2, Andrew J. Ghio3, David A. Schwartz4, Jo Rae Wright1 1 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 2 Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 3 Human Studies Division, Environmental Protection Agency, Chapel Hill, North Carolina 4 NIEHS and NTP, Durham, North Carolina Copyright © 2006 by the American Physiological Society. Page 2 of 46 2 Abstract Complement, a complex protein system, plays an essential role in host defense through bacterial lysis, stimulation of phagocytosis, recruitment of immune cells to infected tissue, and promotion of the inflammatory response. Although complement is most well characterized in serum, complement activity is also present in the lung. Here we further characterize the complement system in the normal and inflamed lung. By Western blot, C5, C6 and Factor I were detected in bronchoalveolar lavage (BAL) at lower levels than in serum, whereas C2 was detected at similar levels in BAL and serum. C4 binding protein (C4BP) was not detectable in BAL. Exposure to Lipopolysaccharide (LPS) elevated levels of C1q, Factor B, C2, C4, C5, C6 and C3 in human BAL, and C3, C5, and Factor B in mouse and rat BAL. Message for C1q-B, C1r, C1s, C2, C4, C3, C5, C6, Factor B, and Factor H, but not C9 or C4BP, was readily detectable by RT-PCR in normal mouse lung. Exposure to LPS enhanced Factor B expression, decreased C5 expression, and did not affect C1q-B expression in mouse and rat lung. BAL from rats exposed to LPS had a greater ability to deposit C3b onto bacteria through complement activation than did BAL from control rats. In summary, these data demonstrate that complement levels, expression and function are altered in acute lung injury and suggest that complement within the lung is regulated to promote opsonization of pathogens and limit potentially harmful inflammation. Keywords Inflammation, Bronchoalveolar Lavage, Lipopolysaccharide, C3b Deposition, Complement expression Page 3 of 46 3 Introduction In spite of constant exposure to inhaled pathogens, allergens and noxious gases, the lung is normally free of infection and inflammation primarily due to a variety of defense systems that function locally to promote pathogen clearance. These defense systems include mucus, the mucociliary system, phagocytes, and opsonic proteins including surfactant proteins and complement (Wright 2005). The complement system is a family of proteins, most well characterized in serum, that contributes to host defense via a variety of mechanisms. For example, complement proteins enhance bacterial clearance by opsonizing bacteria and enhancing ingestion by phagocytes. Bacteria may also be directly lysed by complement proteins. In addition complement cleavage products act as chemoattractants to recruit immune cells to the site of infection where they can further contribute to pathogen clearance. It has also been demonstrated that complement activation products can contribute directly to vascular leakage, allowing serum proteins to enter the infected tissue from the bloodstream (Bossi, et al. 2004). The complement system is activated in response to invading pathogens through three different pathways – classical, alternative and lectin. Complement protein C1q binds to antibody on a pathogen surface to activate the classical pathway; cleaved C3 (C3b) binds directly to bacterial surfaces to activate the alternative pathway; and mannose binding lectin and ficolin bind directly to pathogen surfaces to activate the lectin pathway. The three pathways converge at C3, where the important opsonin, C3b, is formed and the pathway continues to C5, where the important chemoattractant and polymorphonuclear neutrophil (PMN) activator, C5a is formed. The terminal Page 4 of 46 4 complement proteins C6, C7, C8, and C9 form the membrane attack complex that can directly lyse bacteria. The major site of complement synthesis is the liver, which secretes complement proteins into the circulation (Alper, et al. 1980), although other tissues have been shown to produce complement proteins (reviewed in (Colten and Garnier 1998)), and tissue specific complement synthesis may be important for supporting an immediate, local immune response. For example, human and rat lung epithelial cells and alveolar macrophages have been shown to synthesize complement proteins in vitro (Ackerman, et al. 1978;Cole, et al. 1983;Strunk, et al. 1988). In addition, complement proteins have been detected in human lung washings (bronchoalveolar lavage, BAL), and the relative BAL levels of the different proteins in the normal lung appear to differ from levels in human serum (Reynolds and Newball 1974;Robertson, et al. 1976;Watford, et al. 2000). Complement activity has also been observed in the lung. Classical pathway activity was measured in human BAL using standard in vitro activation assays (Watford, et al. 2000). The importance of complement in the lung in vivo is supported by the fact that recurrent respiratory infections occur in many patients with deficiencies in complement proteins or complement receptors (Figueroa and Densen 1991). Additionally, mice infected intranasally with S. pneumonia are more likely to die if they do not express C1q (Brown, et al. 2002), and mice treated with cobra venom factor to deplete their complement system are less able to clear S. pneumonia or P. aeruginosa from their lungs (Gross, et al. 1978). Furthermore, in adult respiratory distress syndrome (ARDS), complement activation products measured in serum can be predictive of disease outcome (Langlois and Gawryl 1988;Langlois and Gawryl 1988). Although complement Page 5 of 46 5 has a role in lung host defense, its activity in the normal lung is reduced compared to serum even at same total protein concentrations (17-40% of serum activity) (Watford, et al. 2000), which may be due in part to complement inhibition by Surfactant Protein A (SP-A), a lung specific immune molecule (Watford, et al. 2001). Lung specific regulation of complement synthesis and activation may be important in maximizing the host response to inhaled pathogens while minimizing complement functions that could damage the delicate lung epithelium through promoting an inflammatory response. In particular, C5a has been shown to promote inflammation by causing a massive influx of neutrophils and protein into the alveolar space (reviewed in (Guo and Ward 2005)). Complement mediated tissue damage is normally modulated by a group of at least 11 regulatory proteins that control complement activation by inhibiting formation of active complement complexes or by cleaving specific active complement protein fragments (Liszewski and Atkinson 1998). C1inhibitor, which inhibits classical pathway activation through interaction with the C1 complex, has been detected in human BAL and is expressed in mouse lung (Vinci, et al. 2002;Watford, et al. 2000). However, little else is know about which complement regulatory proteins are present in the lung and how they may function to protect this delicate tissue. Complement is also regulated by changes in gene expression in response to infection or injury. The expression of several complement components increases during the acute phase, a period of systemic inflammation marked by increases in liver synthesis of certain proteins (Schutte, et al. 1975). In vitro, cytokines, hormones, and lipopolysaccharide (LPS) are able to modulate complement expression (Falus, et al. 1987;Falus, et al. Page 6 of 46 6 1990;Lake, et al. 1994;Lappin and Whaley 1991;Rothman, et al. 1989;Strunk, et al. 1985;Zach, et al. 1992) (and reviewed in (Volanakis 1995)). The goals of this study were to characterize further the complement proteins that are present in the normal lung and in a LPS model of acute lung inflammation and to determine if complement proteins are synthesized in the lung. Additionally, the hypothesis was tested that complement is regulated in the lung to enhance phagocytosis and to limit potentially harmful inflammation. Evidence is presented that levels of complement proteins in the lung differ from levels in the serum and that the lung responds to LPS-injury with changes in expression of specific complement components and with an enhanced ability to opsonize bacteria. Page 7 of 46 7 Materials and Methods Buffers Buffers were: isotontic VBS with metals and gelatin (GVBS++), prepared as previously described (Wagner, et al. 2001;Watford, et al. 2002); EDTA-GVBS- -, made without MgCl2 or CaCl2 and containing 0.01M EDTA; EGTA- GVBS++, containing 5 mM MgCl2 and 8 mM EGTA; Dulbecco’s Phosphate Buffered Saline (DPBS, Sigma- Aldrich, St. Lois, MO); and Tris Buffered Saline (TBS; 20 mM Tris-base, 137 mM NaCl, 3.8 mM HCl, pH 7.6). Human LPS exposure and bronchoalveolar lavage (BAL) Normal human bronchoalveolar lavage (BAL) was obtained from healthy human volunteers as previously described (Watford, et al. 2000). BAL was obtained from healthy human volunteers before and at various time points after exposure to aerosolized corn dust extract (delivering a dose of 0.4 µg endotoxin/kg body mass) as previously described (Deetz, et al. 1997). All BAL samples were aliquoted and stored at -80°C before analysis by Western blots. Serum collection Blood was collected via cardiac puncture (mouse and rat) or phlebotomy (human). Blood was allowed to sit at room temperature for 45 minutes and then on ice for 30 minutes. Clotted blood and cells were removed from serum by centrifugation at 2095 g for 10 minutes at 4°C. Serum was stored at -20ºC. Page 8 of 46 8 Animal models Pathogen free, male, C57/BL6 mice (Jackson Labs, Bar Harbor, Maine) were anesthetized by injection of ketamine and xylazine and a brief inhalation of isofluorane. An incision was made to expose the trachea and 50 µl of lipopolysaccharide (LPS from Escherichia coli 026:B6, Sigma-Aldrich) dissolved in sterile saline or only saline (as a vehicle control) was injected into the trachea using a 1/2 cc insulin syringe (28G1/2, Becton Dickinson, Franklin Lakes, NJ). A dose of 100 µg LPS/kg mouse body mass was used. After injection the incision was sutured and the mice were allowed to recover. Next, after various lengths of time post-LPS exposure, mice were euthanized by intraperitoneal injection of pentobarbital and exsanguination. Pathogen free, Sprague-Dawley, male rats (Taconic Farms, Hudson, NY) were anesthetized by inhalation of halothane. The trachea was visualized using a laryngoscope, a cannula was inserted, and 200 µl of sterile saline with or without LPS was instilled through the trachea at a dose of 100 µg LPS/kg rat body mass. Rats were allowed to recover, and then after various lengths of time, euthanized by intraperitoneal injection of pentobarbital and transection of the renal artery. Western Blots Human, mouse or rat BAL, normal human, mouse or rat serum, purified human complement proteins (C1q, C2, C4, C5, C6, Factor B, C4 Binding Protein (C4BP) and Factor I from Advanced Research Technology (now Complement Technology, Inc., Tyler, TX)), and normal human, mouse or rat IgG (Sigma-Aldrich) were separated by electrophoresis on a 15% SDS PAGE gel (7.5% for C4BP). For gels with normal human Page 9 of 46 9 BAL, equal total protein was added in lanes containing normal human serum or BAL. For human mouse or rat BAL from LPS-treated individuals, equal volumes of BAL fluid were loaded onto the gel for each time point. Samples were transferred overnight from the gel to nitrocellulose membrane (Protran®, Schleicher and Schuell BioScience, Keene, NH). Blots were blocked with TBS-Tween (0.1% Tween -20 (Sigma-Aldrich a) in TBS) containing 5% dry milk for 1 hour at room temperature. Blots were washed 3 times with TBS-Tween and then incubated with primary antibody (at concentrations listed in Table 1) in TBS-Tween containing 1% dry milk for either 2 hours at room temperature (C4, C5 for human samples, Factor I and SP-A), 2 hours at 4°C (C1q, C6 and Factor B), or 4°C overnight (C2, C3, C5 for mouse and rat samples, and C4BP). Anti- human C3, C5, and Factor B antibodies were found to cross-react with mouse and rat samples; treatment of blots was the same as for human samples unless otherwise stated. Blots were washed 3 times with TBS-Tween or TBS-Tween containing 1% dry milk. Next, blots were incubated with an HRP-conjugated secondary antibody diluted in TBS-Tween (TBS-Tween containing 1% dry milk for C4BP) for 45 minutes at room temperature. Blots were washed as above, plus an additional wash of 1-4 hours or overnight (C4BP) and treated with ECL. Film was exposed to the blot for 10 seconds-18 hours and developed. In the case of some proteins (C6, human Factor B, Factor I), samples were denatured but not reduced for the SDS PAGE gel, because reduced IgG was similar in size to the reduced complement protein. Page 10 of 46 10 RT-PCR Lung or liver tissue was removed from untreated mice or from mice or rats treated with LPS or saline. Tissue was homogenized in lysis buffer (Biorad AurumTM Total RNA Mini Kit) using the FastPrepTM FP120 with zirconium oxide grinding media (1.251.6mm, Glenmills, Clifton, NJ). RNA was collected using the manufacturer’s protocol for the above Biorad kit, including DNase treatment. RNA was eluted from the Biorad columns in 30 µl elution buffer. The concentration of the RNA was determined using an OD 260/280 ratio and the quality of the RNA was evaluated by analysis on a 1% agaroseformaldehyde gel. RNA was used in a reverse transcriptase reaction to synthesize cDNA. Total RNA (7-8 µg) was used to synthesize 40 µl of cDNA with the Invitrogen (Carlsbad, CA) SuperScript First-Strand Synthesis System for RT-PCR kit, per manufacturer’s directions. Random hexamers were used at 30 ng primer/µg RNA. In order to control for genomic DNA contamination, parallel samples were prepared without the addition of reverse transcriptase. When cDNA was synthesized from RNA isolated from LPS and saline treated mice, equal µg of RNA were added to each reaction. PCR for each complement component was performed using either the BioRad iTaqTM DNA Polymerase kit (used 10x RXN buffer with 1.6 mM MgCl2, 0.2 mM dNTPs, 0.34 µM of each primer, and 0.08U iTaq/µl) or the BioRad iQTM SYBR Green Supermix (use 2x supermix with 0.15 µM of each primer). This basic thermocyle was followed using the annealing temperature and primers listed in Table 2: 94°C-5 minutes and 30 cycles of 94°C-1 Page 11 of 46 11 minute, anneal-1.5 minutes, 72°C-1 minute. PCR products were then separated on a 1% agarose, TAE gel containing ethidium bromide. Quantitative real time RT-PCR was performed using the BioRad MyiQ Single Color Real-Time PCR Detection System to detect amplification of the PCR products and the accompanying Optical System Software (Version 1.0) was used to analyze data. Samples were prepared with BioRad iQTM SYBR Green Supermix containing 0.2 µM of each primer. In each case, reactions were prepared for both the complement gene of interest and the negative control gene ( -actin for mice and -glucuronidase (GUSB) for rats). Mock cDNA synthesis reactions (prepared with no reverse transcriptase, described above) were also added to PCR reactions run in parallel, to control for genomic contamination. Parallel samples were prepared containing serial dilutions of liver cDNA to use as a standard curve. Cycle threshold (Ct) values for each sample were calculated using the MyiQ program and melt curves were also examined to insure analysis of a single PCR product. Standard curves were constructed using the Ct values and relative starting quantities of message for the liver control samples. These curves were used to calculate relative starting quantities of message for each gene of interest and for -actin (mouse) or -glucuronidase (GUSB, rat) in each lung sample. Data are presented as the fold change in RNA message in LPS-treated animals, compared to saline-treated animals. Collection of mouse and rat BAL and concentration of rat BAL After euthanasia, the chest cavity of each mouse was opened, the trachea was exposed and a cannula was inserted. One mL of DPBS was injected through the cannula to wash the lungs. Cells were removed from this BAL fluid by centrifugation at 233 g for 10 Page 12 of 46 12 minutes. For Western blots, aliquots of the BAL collected at various time points after exposure to LPS were stored at -80°C. After euthanasia, the rat lungs were removed and washed with 10-12 mL DPBS (for Western blots) or 50 mL DPBS with 0.2 mM EGTA (for functional assays), by passing the fluid through a cannula inserted into the trachea. Cells were removed by centrifugation, and BAL was stored for Western blots, as above. For functional assays, small debris was removed from the BAL by passing the supernatant over a membrane (Pall Life Sciences (East Hills, NY)-GH Polypro, 90 mm, 0.45 µm). Because the process of lavaging the lung unavoidably dilutes the proteins of the alveolar lining, it is necessary to concentrate the lavage in order to measure complement function. A protocol previously used for human BAL (Watford, et al. 2000) was modified. All steps were performed at 4°C to prevent complement degradation. First BAL from 6 rats was pooled and concentrated approximately 30-fold using a tangential filter pump system (Cole Parmer Masterflex (Vernon Hills, IL) with Millipore (Billerica, MA) Pellicon XL filterPXC010C50). Concentrated BAL was then further concentrated using a centrifugal spin concentrator (Apollo (San Diego, CA) – 3502.2) for a final concentration of approximately 75-fold. Protein concentrations for each preparation were determined using a BCATM Protein Assay Kit (Pierce). The initial and final volumes of saline and LPS BAL were kept the same so that relative comparisons could be made for equal volumes of BAL. The final protein concentrations of the saline and LPS BAL were approximately 3 mg/mL and 5.4 mg/mL, respectively. Page 13 of 46 13 C3b deposition assay A modification of a previously described method for detecting deposition of C3b onto the surface of bacteria was used (Watford, et al. 2000). Group B streptococcus (GBS) were grown and prepared as previously described (Watford, et al. 2000). Bacteria (5x107 bacteria per condition) were incubated for 2 hours at 37°C in buffer (GVBS++, EDTAGVBS++, or EGTA- GVBS++) containing either normal rat serum or concentrated BAL from LPS or saline-only treated rats. Prior to incubation with bacteria, CaCl2 and MgCl2 were added to concentrated BAL to inactivate the EGTA in the lavage buffer (final concentration 0.550 mM CaCl2, 0.500 mM MgCl2). Bacteria were centrifuged and resuspended in 100 µl DPBS and incubated for 20 minutes at 37°C. Bacteria were again centrifuged, resuspended in DPBS containing either FITC-conjugated goat-anti-rat C3 (MP Biomedicals, Aurora, OH) or FITC-conjugated Chrom Pure goat IgG as a negative control (Jackson Immuno Research, West Grove, PA), and incubated on ice for 45 minutes. Finally bacteria were washed twice in 1 mL DPBS and resuspended in 1% formaldehyde in DPBS. Associated fluorescence was measured by Fluorescence Associated Cell Sorting (FACS). Statistics For data shown in Figures 5 and 6, statistical significance was determined using paired two-tailed Student’s t-tests. Error bars represent standard error of the means. Page 14 of 46 14 Results Complement proteins are present in the lung. Previous studies from our laboratory reported that human lung washings (bronchoalveolar lavage (BAL)) contain complement components and complement activity (Watford, et al. 2000). While some complement proteins (C3 and C4) are present at similar levels in BAL and serum samples, others (C1q and Factor B) are much less abundant in BAL samples compared to serum. Classical pathway activity in BAL was present but reduced compared to classical pathway in the serum. Alternative pathway activity was not detected in the BAL. Based on these findings, we hypothesized that regulation of complement activity in the lung may differ from that in the serum at least partly as a consequence of the different relative amounts of complement proteins in the lung and serum. In order to characterize further lung content of complement components, Western blots were performed on human BAL from three different individuals and normal human serum (NHS). The process of lavaging the lung unavoidably dilutes the proteins of the airway lining fluid. To make relative comparisons possible, NHS was diluted to the same final protein concentration as the BAL (a 1:700 dilution of NHS). Western blots showed detectable levels of C2, C5, C6 in the BAL (Figure 1). In order to determine the relative amounts of each protein, densitometric analyses were performed on these Western blots (Table 3). Although relative levels varied between individuals, on average C2 was present at approximately the same level in BAL and serum. In contrast, C5 and C6 were present in BAL at lower concentrations than in serum (41% and 32%, respectively). Page 15 of 46 15 In the serum, complement activation is regulated by a group of proteins known as complement regulatory proteins. These proteins could play an important role in the lung, a delicate organ quite vulnerable to damage caused by excessive or uncontrolled complement activation with extensive local inflammation. Little is know about levels of complement regulatory proteins in the lung. Western blots were performed with human BAL and serum in order to detect two complement regulatory proteins: 1) Factor I, which inactivates C3b and C4b, and 2) C4-binding protein (C4BP), which regulates the classical C3 convertase. Factor I was detected in BAL (Figure 1) at levels slightly less than in serum (Table 3). C4BP was virtually undetectable in BAL (Figure 1). One sample (BAL-2) had a very faint band for this protein. The presence of Factor I in BAL suggests that complement activation in the lung is regulated, at least in part, by complement regulatory proteins. However, virtually undetectable levels of C4BP in the BAL are consistent with the possibility this regulatory system is different in lung than in the serum. Interestingly, C2 (Figure 1), C4 and C3 (previously reported (Watford, et al. 2000)), which are all downstream of C1 in the classical pathway, are present at similar relative levels in the BAL and serum. However, C1q levels in BAL are lower than in serum (Watford, et al. 2000). In fact, the relative levels of both C1q and Factor B, the important initiating components in the classical and alternative pathways, are low in BAL from the normal lungs compared to serum (Watford, et al. 2000). Therefore, we hypothesized that levels of C1q and Factor B are regulated in the lung in order to control complement activation and prevent lung damage. Page 16 of 46 16 Levels of complement proteins change during lung injury. In order to determine whether lung complement levels are altered during acute inflammation, a well-characterized model of lipopolysaccharide (LPS) – induced lung injury was used (Ulich, et al. 1991). LPS is a cell wall component of gram-negative bacteria and is a potent mediator of inflammation (Brigham and Meyrick 1986). Instillation of LPS into the lung leads to the production of inflammatory proteins, an influx of immune cells (in particular neutrophils) into the alveolar space, and an increase in the permeability of the alveolar epithelium leading to pulmonary edema. BAL samples were obtained from human volunteers before and at various times after exposure to aerosolized corn dust extract, containing a known quantity of LPS (Deetz, et al. 1997). Western blots were performed on these samples to determine the levels of complement proteins. Equal volumes of BAL fluid were loaded on the gel for the different time points before and after exposure. Prior to LPS exposure (Pre expo), C1q and Factor B were barely detectable in the BAL, but there was a dramatic increase in the amounts of both proteins in the BAL as early as 4 hours after exposure (Figure 2). The levels of both proteins began to decrease by 24 hours and were again barely detectable by 168 hours (7 days). To test whether this increase in BAL protein was specific to C1q and Factor B, Western blots were performed for C4, C2, C6 and C5 with the same BAL samples. Levels of each of these complement components were also increased after LPS exposure (Figure 2). A Western blot for C3 showed a less dramatic increase after LPS exposure. Even though all of the complement components tested increased in the BAL after LPS exposure, in some cases the time specific changes in the Page 17 of 46 17 levels of the different complement components varied. For example, C1q is dramatically increased at 4 hours after exposure and began to decline by 48 hours, whereas C4 appears not to decline until 96 hours. Similar experiments were performed using mouse and rat models of LPS-induced lung inflammation. LPS suspended in sterile saline was directly instilled into the lungs of these animals, and BAL was collected by lavaging the whole lung at various time points after instillation. As above, equal volumes of BAL from treated or untreated animals were separated on an SDS-Page gel and Western blots were performed. As with human samples, levels of each complement protein tested (Factor B, C3 and C5) increased in the mouse and rat BAL after exposure to LPS (Figure 3). However, the timing of the response to LPS seemed to differ in the human and animal models. Whereas, the response to LPS in the human BAL seemed to occur quite rapidly, peaking in most cases just four hours after exposure, the rodent responses were more delayed. In the mouse model, increased complement protein levels did not appear to change until about 18 hours after exposure and did not peak until 24 hours after exposure (Figure 3A). In the rat model, complement protein levels seemed to increase by 4 hours after exposure, but did not peak until 12 hours after exposure (Figure 3B). The difference in the timing of the LPS response in the human and animal models may be explained by the difference in the way the LPS was administered. The human exposure was at a lower dose than the animals, but the use of aerosolized LPS, rather than direct instillation of LPS in solution, may have resulted in a higher effective dose reaching the alveolar spaces. Additional, inherent differences between the species may have also contributed to the differences in response time. Page 18 of 46 18 Additionally, Western blots were performed on the human BAL samples for Surfactant Protein A (SP-A) (Figure 2). SP-A has several important roles in the innate immune system of the lung, and it has been shown to inhibit complement activation in vitro (Watford, et al. 2001). After exposure to LPS, levels of SP-A in the BAL were also increased. SP-A is an abundant component of the alveolar lining and is present in the serum at very low levels. While the increases in complement proteins in the BAL could be due to proteins entering the alveolar space from the blood stream or to a change in local complement expression, the increase in SP-A levels after LPS exposure are unlikely to be due to leakage from the serum. In rat lung, levels of RNA and protein for SP-A and SP-D were previously shown to increase after exposure to LPS (McIntosh, et al. 1996). Considered together, these data suggest that at least some LPS-induced changes in BAL protein levels are due to changes in local synthesis. Complement is synthesized in the lung and expression is regulated during lung injury. To determine which complement components were synthesized by the lung in vivo, RTPCR was used. RNA was collected from either mouse liver or mouse lung and cDNA was synthesized. To demonstrate that the PCR products were from cDNA and not genomic DNA contamination, control reactions with no reverse transcriptase (No RT) were included. By RT-PCR, C1q-B, C1r, C1s, C2, C4, C3, C5, C6, Factor B, and Factor H were detected in both lung and liver (Figure 4). Interestingly, little or no C9 or C4BP message was detected in the lung, even though both were easily detected in the liver (Figure 4). Page 19 of 46 19 In order to determine if complement is locally regulated during acute lung injury, quantitative real time RT-PCR was used to assess relative levels of mRNA for several complement components. C1q, Factor B, C5, C3, and -actin or -glucuronidase (GUSB) (as a negative controls for mouse and rat, respectively) genes were amplified from lung tissue collected from mice or rats treated with either LPS dissolved in saline or only saline (as vehicle control). In mice, eighteen hours after LPS exposure, there was a 14-fold increase in the expression of Factor B, as compared to the saline control (an 8-fold increase when corrected for -actin) (Figure 5A). At eighteen hours after treatment, there was no significant difference in C1qB expression between the saline and LPS-treated mouse lungs as compared to -actin control. There was about a two-fold increase in the average relative starting quantity of both -actin and C1q after LPS-treatment, probably as a result of differences in the amount of starting total RNA (Figure 5A). There was also no significant change in C1q expression 6 hours after LPS treatment (data not shown). Additionally, there was no significant change in C3 expression after LPS treatment, compared to -actin control (Figure 5A). Interestingly, there was a 3.4 fold decrease in the amount of C5 expression 18 hours after LPS treatment, as compared to saline control (a 6-fold decrease when corrected for -actin) (Figure 5B). In additional experiments, increases in the cytokines TNF-alpha, MCP-1 and MIP-2 were detected after LPS exposure (data not shown)– all of which are expected to rise during lung injury. Significant changes were also observed in complement gene expression in rat lung after exposure to LPS. Eighteen hours after exposure to LPS, there was a 3.2 fold increase in Factor B expression, as compared to the saline control (a 2.3 fold increase Page 20 of 46 20 when corrected for GUSB) (Figure 5C). At an earlier time point, 12 hours after LPS exposure, there was a slightly greater increase in Factor B expression – 4.2 fold (2.6 fold when corrected for GUSB) (data not shown). Eighteen hours after exposure to LPS, there was a 2.9 fold decrease in C5 expression, as compared to the saline control (a 3.9 fold decrease when corrected for GUSB) (Figure 5C). As in the mouse, expression of C1q did not appear to be significantly altered after LPS exposure. C3b opsonization of bacteria is greater in BAL from LPS-treated rats than BAL from normal rats. Our data demonstrate that the composition of the lung’s complement system changes after LPS-induced inflammation. To determine how these changes might affect complement function, in vitro activation assays were performed. We hypothesized that the increase in complement proteins in the alveolar space would lead to an increase in the ability of the lung to use complement to fight invading pathogens. One mechanism by which complement may contribute to pathogen clearance is through opsonization of bacteria with C3b. We hypothesized that the C3b opsonization in the lung would increase after LPS treatment. To test this hypothesis, a rat model of LPS lung injury was required in order to obtain sufficient quantities BAL. As for the Western blot and RT-PCR analyses, rats were treated using direct intratracheal instillation of LPS or saline. Eighteen hours later, BAL was collected and pooled from 6 LPS-treated and 6 saline-treated rats. BAL was concentrated to a volume closer to that of the alveolar lining, since the process of lavage dilutes the alveolar protein. This process was previously shown to be necessary in order Page 21 of 46 21 to detect complement activity in human BAL (Watford, et al. 2000). The concentrated BAL samples (or normal rat serum (NRS) as a control) were incubated with Group B streptococcus to allow C3b to be deposited on the surface of the bacteria. NRS and saline BAL were added to the bacteria at the same total protein concentration; equal volumes of saline and LPS BAL were added to the bacteria. Bacteria were washed and then stained with fluorescently labeled antibody specific for rat C3 or labeled normal goat IgG at same total protein concentration (as a negative control). Bacteria were analyzed using flow cytometry. Six independent experiments were performed using 3 different sets of pooled, concentrated BAL. As expected, there was a significant increase in the amount of C3b deposited on bacteria after incubation with NRS in GVBS++ buffer, which allows activation of complement through the classical and alternative pathways (Figure 6). This increase was also dose-dependant (data not shown). In all cases, control IgG binding was very low and did not change upon addition of NRS or BAL (data not shown). C3b binding was inhibited when bacteria were incubated with NRS in the presence of EDTA (which inhibits both the classical and alternative pathways) and EGTA (which inhibits only the classical pathway). Therefore, the C3b binding measured with NRS is complement dependent and is probably acting through the classical pathway. When bacteria were incubated with concentrated rat BAL in GVBS++, significantly more C3b was detected in the LPS-BAL samples than in the saline controlBAL samples (Figure 6). Therefore, it can be concluded that LPS-treatment increases the ability of BAL to deposit C3b onto bacteria, leading to the hypothesis that bacteria that reach the lung after LPS injury may be more readily opsonized and cleared. C3b binding Page 22 of 46 22 from rat BAL was inhibited by EDTA, which suggests that it is complement dependant (Figure 6). Most of the binding was also inhibited by EGTA, which suggests that C3b deposition is occurring primarily through the classical pathway in the BAL. Page 23 of 46 23 Discussion The purpose of this study was to compare the complement system in the lung and serum and to determine if lung complement levels and function change in response to lung injury. Data presented support an active role for complement in the lung and contribute to the idea that the immune system in the lung is uniquely organized to both optimize clearance of pathogens and modulate inflammation. Additionally, these data demonstrate that the complement system in the lung changes during inflammation through changes in local complement expression and presumably through movement of complement proteins from the bloodstream into the alveolar space. We first compared the levels of complement protein in human bronchioalveolar lavage (BAL) to those in serum. In order to make these relative comparisons possible, serum was diluted and serum and BAL samples were loaded onto SDS-Page gels at the same total protein concentration before Western blot analysis. Western blots of normal human BAL showed that complement proteins C2, C5 and C6 were present in the lung at similar (C2) or lower (C5 and C6) relative levels compared to normal human serum (Figure 1, Table 3). Additional Western blots were performed to detect complement regulatory proteins, Factor I and C4BP. Levels of Factor I in the BAL were slightly less than levels in the serum. However, C4BP could be detected in serum but was virtually undetectable in BAL. Analysis of relative levels of complement proteins in normal human BAL and serum correlates with previous measurements of complement activity in the BAL. Previous work showed that levels of C1q in normal human BAL were low compared to serum but that levels of C4 and C3 were comparable in BAL and serum (Watford, et al. Page 24 of 46 24 2000). We now show that C2 is also present at comparable levels in normal human BAL and serum. Therefore all of the classical complement proteins necessary for C3b deposition are present in the normal lung, but the first component is present only at low levels. These data are consistent with previous experiments demonstrating that classical pathway activity was present in concentrated normal human BAL, albeit at reduced levels compared to serum activity. Previous work also showed that levels of Factor B in the lung were low compared to serum, and correspondingly that alternative pathway activity was undetectable in the BAL from normal lung (Watford, et al. 2000). In order to determine how the lung complement system changes during lung injury, samples of human, mouse and rat BAL were obtained from individuals before and at various time points after exposure to LPS. At each time point, the lung was lavaged with the same volume of fluid. Because an increase in protein within the alveolar space is a physiological consequence of treatment with LPS, the BAL samples at different time points contained different total protein concentrations. Therefore, in order to compare levels of complement components present in the airspace at each time point, equal volumes of BAL for each sample was loaded onto SDS-Page gels before Western blot analysis. Western blots showed a marked, transient increase in several complement proteins in human, mouse and rat BAL. Additionally, levels of Surfactant Protein-A (SPA), a lung specific immune molecule that has been shown to interact with complement, increased in human BAL after LPS exposure. In order to determine whether increases in BAL complement levels were due to changes in local complement synthesis, experiments were first performed to determine which complement components are expressed by lung tissue. RT-PCR was performed on Page 25 of 46 25 mouse lung tissue for a panel of complement genes. C1q-B, C1r, C1s, C2, C4, C3, C5, C6, Factor B, and Factor H were all readily detectable, but C9 and C4BP were not. The absence of detectable C4BP message was particularly interesting, because this was the only complement protein tested that was virtually undetectable in human BAL. Quantitative real-time PCR analysis of mouse and rat lung tissue demonstrated a significant increase in Factor B expression and a significant decrease in C5 expression after animals were exposed to LPS. However, expression of C1q (mouse and rat) and C3 (mouse) were not significantly changed after LPS exposure. The increases in lung complement protein levels after LPS exposure correlate to changes in lung complement function. After exposure to LPS, concentrated rat BAL contained significantly more complement activity, as measured by the ability to deposit C3b onto bacteria. This increase could be due directly to an increase in C3 levels and/or an increase in other complement components, which could increase complement activity. C3 titer experiments demonstrated elevated levels of functional C3 protein in concentrated rat BAL from LPS-treated animals in two out of three independent experiments (unpublished observations) and levels of C3 protein increased in human, mouse and rat BAL after exposure to LPS. Because C1q is at low initial levels in normal BAL, C1 may be a limiting factor in lung complement activity in the unchallenged lung. Therefore, the increase in C1q protein in human BAL after LPS exposure most likely contributes to increased C3b opsonization of bacteria. However, levels of C2, C4 (human), and Factor B (human, mouse and rat) proteins are also increased after LPS exposure and may contribute to the increased activity. The majority of the activity measured in the concentrated BAL from LPS-treated rats is likely to be through the Page 26 of 46 26 classical pathway, because most of the activity was inhibited by both EDTA and EGTA. However, the inhibition by EGTA for these samples was somewhat variable and in some cases there were two populations of bacteria within one sample – one where C3b deposition was inhibited by EGTA and one where C3b deposition was mostly unchanged by the addition of EGTA. These data show that the inhibition by EGTA is not complete and suggest that some of the activity detected in the BAL from LPS-treated animals may be through the alternative pathway. This finding is consistent with our observation that there is an increase in both local Factor B expression and Factor B protein levels in the BAL after LPS exposure. Based upon our and other’s data, we speculate that the complement system is regulated not only to maximize opsonization and clearance of pathogens as they enter the lung, but also to limit harmful inflammation along the delicate lung epithelium. Previous data have linked C5a and to a lesser extent the membrane attack complex (MAC) with lung inflammation. In the lung, complement fragment C5a leads to an influx of neutrophils and protein into the alveolar space, a hallmark of many types of lung inflammation (Desai, et al. 1979;Henson, et al. 1979;Larsen, et al. 1980;Mulligan, et al. 1996). Additionally, fibrin formation and hemorrhage within the lung as well as damage to the alveolar epithelium have been noted in animal lungs treated with C5 cleavage products (Desai, et al. 1979;Larsen, et al. 1980). Either C5a or proteins of the complement membrane attack complex (C5b-9, MAC) combined with IgG immune complexes can lead to greatly enhanced production of CXC and CC chemokines by alveolar macrophages, and MAC can also lead to enhanced production of MCP-1 by cultured endothelial cells (Czermak, et al. 1998). Additionally, C5aR null mice and mice Page 27 of 46 27 treated with an anti-C5 antibody have reduced lung damage in two different models of lung injury (Bozic, et al. 1996;Buras, et al. 2004;Czermak, et al. 1999) . However, it is also important to note that C5a may also have a protective role in the lung, since C5aR null mice die from Pseudomonas aeruginosa lung infections that wild type mice can easily overcome (Hopken, et al. 1996). Restricting levels of C5 and MAC is a possible mechanism by which the lung minimizes inflammation in response to normal levels of foreign particles. We have demonstrated that the level of C5 protein in normal human BAL is approximately 41% of the level in normal human serum (at the same total protein level). Additionally, C6 in BAL is only about 32% of serum levels. These low levels may be important for controlling complement activity in the normal lung. However, during an acute inflammatory episode, which is modeled by the LPS exposure, levels of C5 and C6 proteins rise in the lung, which may contribute to lung damage that occurs with this type of injury. Even though we measured an increase in C5 protein in the BAL of all three species tested, in the mouse and rat models, we measured a significant decrease in C5 expression by lung tissue from LPS-injured animals. The increase in C5 protein within the BAL is most likely due to an influx of serum proteins into the injured lung and not to a change in local expression. Despite the apparent influx of serum C5, the decreased local expression of C5 may serve a protective mechanism to help minimize C5 mediated inflammation in the lung. We cannot rule out the possibility that decreased local expression of C5 does not contribute to a physiologically significant effect in the face of acute LPS-induced inflammation. However, the ability of the lung to decrease C5 expression suggests that there is a local mechanism to regulate complement activity that Page 28 of 46 28 could be even more important in a different or less severe type of lung injury. It is also interesting to note that C9 was one of only two complement genes for which there was no detectable message in the lung tissue. Lack of C9 expression could be another way complement mediated inflammation is controlled in the normal lung, by minimizing formation of the membrane attack complex. Although the majority of complement components are expressed by lung tissue, the changes in expression of complement in response to LPS-induced lung injury seem to be quite specific. We observed changes in expression of Factor B and C5, but not C1q, C3, Factor I, Factor H, Mannose Binding Lectin A, C6, C1r or C1s (unpublished observations). Changes in expression may be limited to specific complement components, but these changes are not limited to only the LPS model of acute lung injury. Changes in local complement expression have also been reported to be associated with mouse models of human diseases including Listeria monocytogenes meningitis, glomerulonephritis, and lupus (Passwell, et al. 1988;Stahel and Barnum 1997;Stahel, et al. 1997;Welch, et al. 2000). Because many changes take place in the lung during LPS-induced injury, it is not clear if the changes in Factor B and C5 expression are a direct effect of LPS on lung cells. These changes could be an indirect response to cytokines, to other inflammatory mediators or possibly even to complement proteins leaking into the lung from the serum. INF- , IL-6 type cytokines, IL-1 type cytokines, hormones, LPS, and C5a have all been reported to effect complement expression both in vivo and by cultured cells (Falus, et al. 1987;Falus, et al. 1990;Lake, et al. 1994;Lappin and Whaley 1991;Rothman, et al. 1989;Schlaf, et al. 2004;Strunk, et al. 1985;Zach, et al. 1992). Detailed analyses of Page 29 of 46 29 promoters and enhancers for Factor B and C3 have demonstrated the intricate control by individual cytokines through specific regulatory sequences (Huang, et al. 2001;Huang, et al. 2002;Kawamura, et al. 1992;Wilson, et al. 1990). Important future studies will be to define the mechanism by which Factor B and C5 expression are modulated during LPSinduced lung injury. Acute lung inflammation is a critical health issue in a variety of human diseases. Because complement is intricately involved in the inflammatory response and serves as an important first line of defense in removing infectious organisms, understanding how this system is regulated in the lung has clear clinical implications. Here, by using both human and rodent models of LPS-induced lung inflammation, we detail changes that are occurring in the complement system of the lung both at the protein and mRNA level. We also present evidence to support the idea that the complement system functions differently in the lung than in the serum. The relative amounts of the complement proteins and the presence of complement regulatory proteins are very important determinants of complement function in the serum. The relative amounts of complement proteins are different in the lung than in the serum and C4BP, a key complement regulatory protein, is virtually undetectable in the normal lung. Maintaining specific levels of each complement protein and complement regulatory protein, regulating complement expression in response to inflammatory stimuli and using SP-A to regulate complement activity all appear to be mechanisms used in the lung to help insure a balance between the helpful and harmful activities of complement. Page 30 of 46 30 Acknowledgements We would like to thank Kathy Evans for performing Western blots for SP-A and for general experimental support. We would also like to acknowledge Dr. John Whitesides, Patrice McDermott, and Danielle King from the Human Vaccine Institute facility for the FACS analysis. Grants This work was supported by a National Institute of Health-National Heart, Lung and Blood Institute Grant RO1HL-51134 (J.R. Wright and M.M. Frank). Contact Information Address for reprint requests and other correspondence: J. R. Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center, Durham, NC 27710 (e-mail: [email protected]) Page 31 of 46 31 Figure Legends Figure1: Complement proteins are present in BAL at different levels compared to serum. Normal human BAL from three different individuals, normal human serum diluted to the same total protein concentration (NHS 1:700), purified complement proteins (C2, C5, C6, Factor I, and C4BP), and human IgG and HSA (as controls) were analyzed by Western blot using antibodies against complement proteins C2, C5, and C6, Factor I or C4BP. Figure 2: Levels of complement proteins in human BAL change after exposure to LPS. Human volunteers were exposed to aerosolized LPS at a dose of 0.4 µg/kg body mass. Lungs were lavaged prior to exposure (Pre expo) and at various time points after exposure (4, 24, 48, 96, and 168 hours). BAL samples, NHS, purified complement proteins, IgG, and HSA were analyzed by Western blot using antibodies against C1q, Factor B, C2, C4, C5, C6, C3 and Surfactant Protein A (SP-A). Equal volumes of BAL were loaded for each time point. All Western blots were performed with samples from at least two individuals. Blots shown are representative data. *individual number 1, #individual number 2, **individual number 3. Figure 3: Levels of complement proteins in mouse and rat BAL change after exposure to LPS. Male mice or rats were treated with LPS via direct intratracheal instillation at a dose of 100 µg LPS/kg mouse body mass. BAL was collected from untreated animals and from animals at various time points after exposure. BAL samples, dilute mouse or rat serum (NMS or NRS, respectively) and mouse or rat IgG (as a control) were analyzed by Western blot using antibodies against C3, C5, and Factor B. Equal volumes of BAL Page 32 of 46 32 were loaded for each time point. All Western blots were performed with samples from at least two animals at each time point. Blots shown are representative data. Figure 4: Complement protein mRNA is detectable in the mouse lung. RNA was collected from whole male mouse lung or liver and cDNA was synthesized. RT-PCR was performed with primers specific for a variety of complement genes. No reverse transcriptase (No RT) controls serve as a control for genomic contamination. Water was added to some reactions instead of cDNA as a no template control. Lane 1-liver cDNA, lane 2-lung cDNA, lane 3-lung no RT control, lane 4-water only. All RT-PCR reactions were performed with samples from at least two animals. Data shown are representative. Figure 5: The lung regulates expression of specific complement components. Male mice (A and B) or rats (C) were intratracheally instilled with LPS (at a dose of 100 µg/kg body mass) or saline (as a control) and sacrificed 18 hours later. RNA was collected from lung tissue and cDNA was synthesized. Real-time quantitative RT-PCR was performed using the BioRad Sybr green system to detect amplification of each PCR product. Factor B, C1q C3, and C5 were amplified and in each case -actin (for mice) or -glucuronidase (GUSB, for rats) was amplified at the same time as a control for differences in how much total cDNA was added. Also standard curves were constructed to determine relative starting quantities of cDNA for each unknown sample. Comparisons were then made between relative starting quantities in LPS and saline control samples and are presented as a fold change for each gene. Fold changes in expression for C5, Factor B, C1q or C3 can then be compared to fold changes in the Page 33 of 46 33 control gene. Parts A and B: *significantly different from -actin control (student t-test, p<0.05). N=4 for Factor B, C5, and -actin. N=3 for C1q and C3. Part C: *significantly different from GUSB control (student t-test, p<0.05). N=4 for Factor B, C5, and GUSB. Figure 6: BAL from LPS treated rats supports greater C3b deposition onto bacteria than does BAL from saline treated rats. Freshly harvested Group B streptococcus were washed and then incubated with NRS, BAL from male saline-treated rats (Sal BAL) or BAL from male LPS-treated rats at 37ºC, for 1 hour. Next bacteria were washed for 20 min at 37°C, stained with FITC labeled goat-anti-rat C3 or FITC- labeled normal goat IgG as a control, washed again and fixed. Incubations with NRS or BAL were performed in GVBS++ (which allows complement activation though alternative and classical pathways), in EDTA buffer (which inhibits all complement activation) or EGTA buffer (which inhibits only the classical pathway). The same amount of total protein was added for NRS and Sal BAL samples. The same volume was added for Sal BAL and LPS BAL. C3b deposition is shown as the median fluorescence detected when FACS analysis was performed on bacteria. *significantly different from bacteria only control, #significantly different from saline BAL (student t-test, p<0.05), N=6 individual experiments, with three different sets of pooled BAL samples. Page 34 of 46 34 References 1. Ackerman SK, Friend PS, Hoidal JR, and Douglas SD. Production of C2 by human alveolar macrophages. Immunology 35: 369-72, 1978. 2. Alper C, Raum D, Awdeh Z, Petersen B, Taylor P, and Starzl T. Studies of hepatic synthesis in vivo of plasma proteins, including orosomucoid, transferrin, alpha 1antitrypsin, C8, and Factor B. Clin Immunol Immunopathol 16: 84-89, 1980. 3. Bossi F, Fischetti F, Pellis V, Bulla R, Ferrero E, Mollnes TE, Regoli D, and Tedesco F. Platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex. J Immunol 173: 6921-7, 2004. 4. Bozic CR, Lu B, Hopken UE, Gerard C, and Gerard NP. Neurogenic amplification of immune complex inflammation. Science 273: 1722-5, 1996. 5. Brigham KL, and Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis 133: 913-27, 1986. 6. Brown JS, Hussell T, Gilliland SM, Holden DW, Paton JC, Ehrenstein MR, Walport MJ, and Botto M. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice. Proc Natl Acad Sci U S A 99: 16969-74, 2002. 7. Buras JA, Rice L, Orlow D, Pavlides S, Reenstra WR, Ceonzo K, and Stahl GL. Inhibition of C5 or absence of C6 protects from sepsis mortality. Immunobiology 209: 629-35, 2004. 8. Cole FS, Matthews WJ, Jr., Rossing TH, Gash DJ, Lichtenberg NA, and Pennington JE. Complement biosynthesis by human bronchoalveolar macrophages. Clin Immunol Immunopathol 27: 153-9, 1983. 9. Colten H, and Garnier G. Regulation of Complement Protein Gene Expression. In: The Human Complement System in Health and Disease, edited by Volanakis J and M Frank. New York, NY: Marcel Dekker, Inc., 1998. 10. Czermak BJ, Lentsch AB, Bless NM, Schmal H, Friedl HP, and Ward PA. Role of complement in in vitro and in vivo lung inflammatory reactions. J Leukoc Biol 64: 408, 1998. 11. Czermak BJ, Breckwoldt M, Ravage ZB, Huber-Lang M, Schmal H, Bless NM, Friedl HP, and Ward PA. Mechanisms of enhanced lung injury during sepsis. Am J Pathol 154: 1057-65, 1999. 12. Deetz DC, Jagielo PJ, Quinn TJ, Thorne PS, Bleuer SA, and Schwartz DA. The kinetics of grain dust-induced inflammation of the lower respiratory tract. Am J Respir Crit Care Med 155: 254-9, 1997. 13. Desai U, Kreutzer DL, Showell H, Arroyave CV, and Ward PA. Acute inflammatory pulmonary reactions induced by chemotactic factors. Am J Pathol 96: 7183, 1979. 14. Falus A, Beuscher HU, Auerbach HS, and Colten HR. Constitutive and IL 1regulated murine complement gene expression is strain and tissue specific. J Immunol 138: 856-60, 1987. 15. Falus A, Rokita H, Walcz E, Brozik M, Hidvegi T, and Meretey K. Hormonal regulation of complement biosynthesis in human cell lines-- II. Upregulation of the Page 35 of 46 35 biosynthesis of complement components C3, factor B and C1 inhibitor by interleukin-6 and interleukin-1 in human hepatoma cell line. Mol Immunol 27: 197-201, 1990. 16. Figueroa JE, and Densen P. Infectious diseases associated with complement deficiencies. Clin Microbiol Rev 4: 359-95, 1991. 17. Gross GN, Rehm SR, and Pierce AK. The effect of complement depletion on lung clearance of bacteria. J. Clin. Invest. 373-378, 1978. 18. Guo RF, and Ward PA. Role of C5a in inflammatory responses. Annu Rev Immunol 23: 821-52, 2005. 19. Henson PM, McCarthy K, Larsen GL, Webster RO, Giclas PC, Dreisin RB, King TE, and Shaw JO. Complement fragments, alveolar macrophages, and alveolitis. Am J Pathol 97: 93-110, 1979. 20. Hopken UE, Lu B, Gerard NP, and Gerard C. The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 383: 86-9, 1996. 21. Huang Y, Krein PM, and Winston BW. Characterization of IFN-gamma regulation of the complement factor B gene in macrophages. Eur J Immunol 31: 3676-86, 2001. 22. Huang Y, Krein PM, Muruve DA, and Winston BW. Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages. J Immunol 169: 2627-35, 2002. 23. Kawamura N, Singer L, Wetsel RA, and Colten HR. Cis- and trans-acting elements required for constitutive and cytokine-regulated expression of the mouse complement C3 gene. Biochem J 283 ( Pt 3): 705-12, 1992. 24. Lake FR, Noble PW, Henson PM, and Riches DW. Functional switching of macrophage responses to tumor necrosis factor-alpha (TNF alpha) by interferons. Implications for the pleiotropic activities of TNF alpha. J Clin Invest 93: 1661-9, 1994. 25. Langlois P, and Gawryl M. Complement Activation Occurs through Both Classical and Alternative Pathways Prior to Onset and Resolution of Adult Respiratory Distress Syndrome. Clin Immunol Immunopathol 47: 152-163, 1988. 26. Langlois P, and Gawryl M. Accentuated Formation of the Terminal C5b-9 Complement Complex in Patient Plasma Precedes Development of the Adult Respiratory Distress Syndrome. Am Rev Respir Dis 138: 368-375, 1988. 27. Lappin DF, and Whaley K. Modulation of complement gene expression by glucocorticoids. Biochem J 280 ( Pt 1): 117-23, 1991. 28. Larsen GL, McCarthy K, Webster RO, Henson J, and Henson PM. A differential effect of C5a and C5a des Arg in the induction of pulmonary inflammation. Am J Pathol 100: 179-92, 1980. 29. Liszewski M, and Atkinson J. Regulatory Proteins of Complement. In: The Human Complement System in Health and Disease, edited by Volanakis J and M Frank. New York, NY: Marcel Dekker, Inc., 1998. 30. McIntosh JC, Swyers AH, Fisher JH, and Wright JR. Surfactant proteins A and D increase in response to intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol 15: 509-19, 1996. 31. Mulligan MS, Schmid E, Beck-Schimmer B, Till GO, Friedl HP, Brauer RB, Hugli TE, Miyasaka M, Warner RL, Johnson KJ, and Ward PA. Requirement and role of C5a in acute lung inflammatory injury in rats. J Clin Invest 98: 503-12, 1996. Page 36 of 46 36 32. Passwell J, Schreiner GF, Nonaka M, Beuscher HU, and Colten HR. Local extrahepatic expression of complement genes C3, factor B, C2, and C4 is increased in murine lupus nephritis. J Clin Invest 82: 1676-84, 1988. 33. Reynolds HY, and Newball HH. Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage. J. Lab. Clin. Med. 84: 559-573, 1974. 34. Robertson J, Caldwell JR, Castle JR, and Waldman RH. Evidence for the presence of components of the alternative (properdin) pathway of complement activation in respiratory secretions. J Immunol 117: 900-3, 1976. 35. Rothman BL, Merrow M, Despins A, Kennedy T, and Kreutzer DL. Effect of lipopolysaccharide on C3 and C5 production by human lung cells. J Immunol 143: 196202, 1989. 36. Schlaf G, Nitzki F, Heine I, Hardeland R, Schieferdecker HL, and Gotze O. C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells. Eur J Immunol 34: 3257-66, 2004. 37. Schutte M, DiCamelli R, Murphy P, Sadove M, and Gewurz H. Effects of anesthesia, surgery and inflammation upon host defense mechanisms. I. Effects upon the complement system. Int Arch Allergy Appl Immunol 48: 706-20, 1975. 38. Stahel PF, and Barnum SR. Bacterial meningitis: complement gene expression in the central nervous system. Immunopharmacology 38: 65-72, 1997. 39. Stahel PF, Frei K, Eugster HP, Fontana A, Hummel KM, Wetsel RA, Ames RS, and Barnum SR. TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis. J Immunol 159: 861-9, 1997. 40. Strunk RC, Whitehead AS, and Cole FS. Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide. J Clin Invest 76: 985-90, 1985. 41. Strunk RC, Eidlen DM, and Mason RJ. Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J Clin Invest 81: 1419-26, 1988. 42. Ulich TR, Watson LR, Yin SM, Guo KZ, Wang P, Thang H, and del Castillo J. The intratracheal administration of endotoxin and cytokines. I. Characterization of LPSinduced IL-1 and TNF mRNA expression and the LPS-, IL-1-, and TNF-induced inflammatory infiltrate. Am J Pathol 138: 1485-96, 1991. 43. Vinci G, Lynch NJ, Duponchel C, Lebastard TM, Milon G, Stover C, Schwaeble W, and Tosi M. In vivo biosynthesis of endogenous and of human C1 inhibitor in transgenic mice: tissue distribution and colocalization of their expression. J Immunol 169: 5948-54, 2002. 44. Volanakis JE. Transcriptional regulation of complement genes. Annu Rev Immunol 13: 277-305, 1995. 45. Wagner E, Jiang H, and Frank M. Complement and kinins: mediators of inflammation. In: Clinical Diagnosis and Management by Laboratory Methods, edited by Henry J. Philadelphia, PA: W.B. Saunders Company, 2001. 46. Watford WT, Ghio AJ, and Wright JR. Complement-mediated host defense in the lung. Am J Physiol Lung Cell Mol Physiol 279: L790-8, 2000. 47. Watford WT, Wright JR, Hester CG, Jiang H, and Frank MM. Surfactant protein A regulates complement activation. J Immunol 167: 6593-600, 2001. Page 37 of 46 37 48. Watford WT, Smithers MB, Frank MM, and Wright JR. Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 283: L1011-22, 2002. 49. Welch TR, Frenzke M, and Witte D. Evidence of a role for local complement expression in a murine model of progressive glomerulonephritis. Pediatr Res 48: 200-5, 2000. 50. Wilson DR, Juan TS, Wilde MD, Fey GH, and Darlington GJ. A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6. Mol Cell Biol 10: 6181-91, 1990. 51. Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 5: 58-68, 2005. 52. Zach TL, Hill LD, Herrman VA, Leuschen MP, and Hostetter MK. Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line. J Immunol 148: 3964-9, 1992. Page 38 of 46 Page 39 of 46 Page 40 of 46 Page 41 of 46 Page 42 of 46 Page 43 of 46 Page 44 of 46 Page 45 of 46 Page 46 of 46