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Am J Physiol Lung Cell Mol Physiol 292: L748-L759, 2007. First published October 27, 2006; doi:10.1152/ajplung.00127.2006
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Complement levels and activity in the normal and LPS-injured lung

Molly S. Bolger,1 DeAndre S. Ross,1 Haixiang Jiang,2 Michael M. Frank,2 Andrew J. Ghio,3 David A. Schwartz,4 and Jo Rae Wright1

Departments of 1Cell Biology and 2Pediatrics, Duke University Medical Center, Durham, 3Human Studies Division, Environmental Protection Agency, Chapel Hill, and 4the National Institute of Environmental Health Sciences and National Toxicology Program, Durham, North Carolina

Submitted 4 April 2006 ; accepted in final form 23 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

inflammation; bronchoalveolar lavage; lipopolysaccharide; C3b deposition; complement expression

DESPITE 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 (51).

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 (3).

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 activator, C5a, is formed. The terminal complement proteins C6, C7, C8, and C9 form the membrane attack complex (MAC) that can directly lyse bacteria.

The major site of complement synthesis is the liver, which secretes complement proteins into the circulation (2), although other tissues have been shown to produce complement proteins (reviewed in Ref. 9), 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 (1, 8, 41). 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 (33, 34, 46).

Complement activity has also been observed in the lung. Classical pathway activity was measured in human BAL using standard in vitro activation assays (46). 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 (16). Additionally, mice infected intranasally with Streptococcus pneumonia are more likely to die if they do not express C1q (6), and mice treated with cobra venom factor to deplete their complement system are less able to clear S. pneumonia or Pseudomonas aeruginosa from their lungs (17). Furthermore, in acute respiratory distress syndrome (ARDS), complement activation products measured in serum can be predictive of disease outcome (25, 26). Although complement has a role in lung host defense, its activity in the normal lung is reduced compared with serum even at same total protein concentrations (17–40% of serum activity; Ref. 46), which may be due in part to complement inhibition by surfactant protein A (SP-A), a lung-specific immune molecule (47).

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 Ref. 18). 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 (29). C1 inhibitor, which inhibits classical pathway activation through interaction with the C1 complex, has been detected in human BAL and is expressed in mouse lung (43, 46). 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 (37). In vitro, cytokines, hormones, and lipopolysaccharide (LPS) are able to modulate complement expression (14, 15, 24, 27, 35, 40, 52, and reviewed in Ref. 44).

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Buffers. Buffers were as follows: isotonic veronal-buffered saline (VBS) with metals and gelatin (GVBS+/+), prepared as previously described (45, 48); EDTA-GVBS–/–, made without MgCl2 or CaCl2 and containing 0.01 M EDTA; EGTA-GVBS+/+, containing 5 mM MgCl2 and 8 mM EGTA; Dulbecco's phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO); and Tris-buffered saline (TBS; 20 mM Tris base, 137 mM NaCl, 3.8 mM HCl, pH 7.6).

Human LPS exposure and BAL. Normal human BAL was obtained from healthy human volunteers as previously described (46). 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 (12). 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 min and then on ice for 30 min. Clotted blood and cells were removed from serum by centrifugation at 2,095 g for 10 min at 4°C. Serum was stored at –20°C.

Animal models. Pathogen-free, male, C57/BL6 mice (Jackson Labs, Bar Harbor, ME) were anesthetized by injection of ketamine and xylazine and a brief inhalation of isoflurane. An incision was made to expose the trachea, and 50 µl of LPS (Escherichia coli 026:B6, Sigma-Aldrich) dissolved in sterile saline or only saline (as a vehicle control) was injected into the trachea using a 0.5-ml 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, rats were euthanized by intraperitoneal injection of pentobarbital and transection of the renal artery. The work with rats and mice was approved by the Duke University Institutional Animal Care and Use Committee. The approved protocols under which the work was performed were A0930603 (alveolar factors in the uptake of surfactant proteins) and A1670605 (roles of surfactant proteins in pulmonary host defense).

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, 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 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, Keene, NH). Blots were blocked with TBS-Tween [0.1% Tween 20 (Sigma-Aldrich) in TBS] containing 5% dry milk for 1 h at room temperature. Blots were washed three 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 h at room temperature (C4, C5 for human samples, factor I, and SP-A), 2 h 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 three times with TBS-Tween or TBS-Tween containing 1% dry milk. Next, blots were incubated with a horseradish peroxidase-conjugated secondary antibody diluted in TBS-Tween (TBS-Tween containing 1% dry milk for C4BP) for 45 min at room temperature. Blots were washed as above, plus an additional wash of 1–4 h or overnight (C4BP) and treated with enhanced chemiluminescence. Film was exposed to the blot for 10 s to 18 h 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.


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  		Table 1. Antibodies used for Western blots

 
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 (Bio-Rad Aurum Total RNA Mini Kit) using the FastPrep FP120 with zirconium oxide grinding media (1.25–1.6 mm; Glen Mills, Clifton, NJ). RNA was collected using the manufacturer's protocol for the above Bio-Rad kit, including DNase treatment. RNA was eluted from the Bio-Rad columns in 30-µl elution buffer. The concentration of the RNA was determined using a 260:280 optical density ratio, and the quality of the RNA was evaluated by analysis on a 1% agarose-formaldehyde 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. 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 micrograms of RNA were added to each reaction. PCR for each complement component was performed using either the Bio-Rad iTaq DNA Polymerase kit [10x reaction buffer with 1.6 mM MgCl2, 0.2 mM deoxynucleotide triphosphates (dNTPs), 0.34 µM of each primer, and 0.08 U/µl iTaq] or the Bio-Rad iQ SYBR Green Supermix (2x Supermix with 0.15 µM of each primer). This basic thermocycle was followed using the annealing temperature and primers listed in Table 2: 94°C, 5 min; 30 cycles of: 94°C, 1 min; anneal, 1.5 min; 72°C, 1 min. PCR products were then separated on a 1% agarose TAE (40 mM Tris-acetate and 1 mM EDTA) gel containing ethidium bromide.


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  		Table 2. Primers used for RT-PCR

 
Quantitative real-time RT-PCR was performed using the Bio-Rad MyiQ Single Color Real-Time PCR Detection System to detect amplification of the PCR products, and the accompanying Optical System Software v1.0 was used to analyze data. Samples were prepared with Bio-Rad iQ 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 [beta-actin for mice and beta-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 ensure 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 beta-actin (mouse) or GUSB (rat) in each lung sample. Data are presented as the fold change in RNA message in LPS-treated animals, compared with 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 milliliter 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 min. 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 of DPBS (for Western blots) or 50 ml of 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 (90 mm, 0.45 µm; GH Polypro; Pall Life Sciences, East Hills, NY). Because the process of lavaging the lung unavoidably dilutes the proteins of the alveolar lining, it is necessary to concentrate the lavage to measure complement function. A protocol previously used for human BAL (46) was modified. All steps were performed at 4°C to prevent complement degradation. First, BAL from six rats was pooled and concentrated ~30-fold using a tangential filter pump system (Cole-Parmer Masterflex, Vernon Hills, IL) with Pellicon XL filter PXC010C50 (Millipore, Billerica, MA). Concentrated BAL was then further concentrated using a centrifugal spin concentrator (3502.2; Apollo, San Diego, CA) for a final concentration of ~75-fold. Protein concentrations for each preparation were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). 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 ~3 mg/ml and 5.4 mg/ml, respectively.

C3b deposition assay. A modification of a previously described method for detecting deposition of C3b onto the surface of bacteria was used (46). Group B streptococcus (GBS) were grown and prepared as previously described (46). Bacteria (5 x 107 bacteria per condition) were incubated for 2 h at 37°C in buffer (GVBS+/+, EDTA-GVBS+/+, or EGTA-GVBS+/+) containing either normal rat serum (NRS) or concentrated BAL from LPS- or saline-only-treated rats. Before 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 of DPBS and incubated for 20 min 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 min. Finally, bacteria were washed twice in 1 ml of DPBS and resuspended in 1% formaldehyde in DPBS. Associated fluorescence was measured by fluorescence-activated cell sorting (FACS).

Statistics. For data shown in GoGoGoGoFigs. 5 and 6, statistical significance was determined using paired two-tailed Student's t-tests. Error bars represent standard error of the means.


Figure 1
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  		Fig. 1. Complement proteins are present in bronchoalveolar lavage (BAL) at different levels compared with serum. Normal human BAL from 3 different individuals, normal human serum (NHS) diluted to the same total protein concentration (1:700), purified complement proteins [C2, C5, C6, factor I, and C4 binding protein (C4BP)], and human IgG and human serum albumin (HSA) (as controls) were analyzed by Western blot using antibodies against complement proteins C2, C5, and C6, factor I, or C4BP.

 

Figure 2
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  		Fig. 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 before exposure (Pre expo) and at various time points after exposure (4, 24, 48, 96, and 168 h). BAL samples, NHS, purified complement proteins, IgG, and HSA were analyzed by Western blot using antibodies against C1q, factor B (Fact 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 2 individuals. Blots shown are representative data. *Individual no. 1; #individual no. 2; **individual no. 3.

 

Figure 3
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  		Fig. 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 (A) or rat (B) serum [normal mouse serum (NMS) or normal rat serum (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 were loaded for each time point. All Western blots were performed with samples from at least 2 animals at each time point. Blots shown are representative data.

 

Figure 4
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  		Fig. 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 2 animals. Data shown are representative.

 

Figure 5
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  		Fig. 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 killed 18 h later. RNA was collected from lung tissue, and cDNA was synthesized. Real-time quantitative RT-PCR was performed using the Bio-Rad SYBR Green system to detect amplification of each PCR product. Factor B, C1q, C3, and C5 were amplified, and, in each case, beta-actin (for mice) or beta-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 with fold changes in the control gene. A and B: *significantly different from beta-actin control (Student's t-test, P < 0.05); n = 4 for factor B, C5, and beta-actin, n = 3 for C1q and C3. C: *significantly different from GUSB control (Student's t-test, P < 0.05); n = 4 for factor B, C5, and GUSB.

 

Figure 6
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  		Fig. 6. BAL from LPS-treated rats supports greater C3b deposition onto bacteria than does BAL from saline-treated rats (Saline BAL). Freshly harvested Group B streptococcus (GBS) were washed and then incubated with NRS, BAL from male saline-treated rats, or BAL from male LPS-treated rats at 37°C for 1 h. 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 isotonic veronal-buffered saline (VBS) with metals and gelatin (GVBS+/+, which allows complement activation through alternative and classical pathways), 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 Saline BAL samples. The same volume was added for Saline BAL and LPS BAL. C3b deposition is shown as the median fluorescence detected when fluorescence-activated cell sorting (FACS) analysis was performed on bacteria. *Significantly different from bacteria-only control; #significantly different from Saline BAL (Student's t-test, P < 0.05); n = 6 individual experiments, with 3 different sets of pooled BAL samples.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Complement proteins are present in the lung. Previous studies from our laboratory reported that human lung washings (BAL) contain complement components and complement activity (46). Whereas 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 with serum. Classical pathway activity in BAL was present but reduced compared with 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.

To characterize further lung content of complement components, Western blots were performed on human BAL from three different individuals and on 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, and C6 in the BAL (Fig. 1). 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).


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  		Table 3. Comparison of complement protein levels in normal human BAL and normal human serum

 
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 to detect two complement regulatory proteins: 1) factor I, which inactivates C3b and C4b; and 2) C4BP, which regulates the classical C3 convertase. Factor I was detected in BAL (Fig. 1) at levels slightly less than in serum (Table 3). C4BP was virtually undetectable in BAL (Fig. 1). One sample (BAL-2, the BAL from individual no. 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 (Fig. 1), and C4 and C3 [previously reported (46)], 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 (46). 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 with serum (46). Therefore, we hypothesized that levels of C1q and factor B are regulated in the lung to control complement activation and prevent lung damage.

Levels of complement proteins change during lung injury. To determine whether lung complement levels are altered during acute inflammation, a well-characterized model of LPS-induced lung injury was used (42). LPS is a cell wall component of Gram-negative bacteria and is a potent mediator of inflammation (5). 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 (12). 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. Before 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 h after exposure (Fig. 2). The levels of both proteins began to decrease by 24 h and were again barely detectable by 168 h (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 (Fig. 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 levels of the different complement components varied. For example, C1q is dramatically increased at 4 h after exposure and began to decline by 48 h, whereas C4 appears not to decline until 96 h.

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 (Fig. 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 4 h after exposure, the rodent responses were more delayed. In the mouse model, increased complement protein levels did not appear to change until about 18 h after exposure and did not peak until 24 h after exposure (Fig. 3A). In the rat model, complement protein levels seemed to increase by 4 h after exposure but did not peak until 12 h after exposure (Fig. 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. Additionally, inherent differences between the species may have also contributed to the differences in response time.

Additionally, Western blots were performed on the human BAL samples for SP-A (Fig. 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 (47). 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. Whereas 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 (30). 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, RT-PCR 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 (Fig. 4). Interestingly, little or no C9 or C4BP message was detected in the lung, even though both were easily detected in the liver (Fig. 4).

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 beta-actin or GUSB (as a negative control for mouse or 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, 18 h after LPS exposure, there was a 14-fold increase in the expression of factor B compared with the saline control (an 8-fold increase when corrected for beta-actin) (Fig. 5A). At 18 h after treatment, there was no significant difference in C1q-B expression between the saline- and LPS-treated mouse lungs compared with beta-actin control. There was an approximately twofold increase in the average relative starting quantity of both beta-actin and C1q after LPS treatment, probably as a result of differences in the amount of starting total RNA (Fig. 5A). There was also no significant change in C1q expression 6 h after LPS treatment (data not shown). Additionally, there was no significant change in C3 expression after LPS treatment compared with beta-actin control (Fig. 5A). Interestingly, there was a 3.4-fold decrease in the amount of C5 expression 18 h after LPS treatment compared with saline control (a 6-fold decrease when corrected for beta-actin) (Fig. 5B). In additional experiments, increases in the cytokines TNF-{alpha}, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (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 compared with the saline control (a 2.3-fold increase when corrected for GUSB) (Fig. 5C). At an earlier time point, 12 h 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, compared with the saline control (a 3.9-fold decrease when corrected for GUSB) (Fig. 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 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 to obtain sufficient quantities of 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 six LPS-treated and six 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 to detect complement activity in human BAL (46). The concentrated BAL samples (or NRS as a control) were incubated with GBS 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 three 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 (Fig. 6). This increase was also dose-dependant (data not shown). In all cases, control IgG binding was very low and did not change after the 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 control BAL samples (Fig. 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 from rat BAL was inhibited by EDTA, which suggests that it is complement dependent (Fig. 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.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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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 BAL to those in serum. 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 with NHS (Fig. 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 with serum but that levels of C4 and C3 were comparable in BAL and serum (46). 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 with serum activity. Previous work also showed that levels of factor B in the lung were low compared with serum and, correspondingly, that alternative pathway activity was undetectable in the BAL from normal lung (46).

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, to compare levels of complement components present in the air space at each time point, equal volumes of BAL for each sample were 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 SP-A, a lung-specific immune molecule that has been shown to interact with complement, increased in human BAL after LPS exposure.

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 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 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 on our and others' 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 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 (13, 19, 28, 31). 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 (13, 28). Either C5a or proteins of the complement MAC (C5b-9) 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 (10). Additionally, C5aR null mice and mice treated with an anti-C5 antibody have reduced lung damage in two different models of lung injury (4, 7, 11). However, it is also important to note that C5a may also have a protective role in the lung, since C5aR null mice die from P. aeruginosa lung infections that wild-type mice can easily overcome (20).

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 ~41% of the level in NHS (at the same total protein level). Additionally, C6 in BAL is only ~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 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 MAC.

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 (32, 38, 39, 49).

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. IFN-{gamma}, 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 (14, 15, 24, 27, 35, 36, 40, 52). Detailed analyses of promoters and enhancers for factor B and C3 have demonstrated the intricate control by individual cytokines through specific regulatory sequences (21, 22, 23, 50). Important future studies will be to define the mechanism by which factor B and C5 expression are modulated during LPS-induced 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 at both 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 ensure a balance between the helpful and harmful activities of complement.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a National Heart, Lung, and Blood Institute Grant RO1-HL-51134 (to J. R. Wright and M. M. Frank).


   ACKNOWLEDGMENTS
 
We thank Kathy Evans for performing Western blots for SP-A and for general experimental support. We also acknowledge Dr. John Whitesides, Patrice McDermott, and Danielle King from the Human Vaccine Institute facility for the FACS analysis.


   FOOTNOTES
 

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: j.wright{at}cellbio.duke.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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