Generation of C5a by Phagocytic Cells
2002; Elsevier BV; Volume: 161; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64461-6
ISSN1525-2191
AutoresMarkus Huber‐Lang, Ellen M. Younkin, J. Vidya Sarma, Niels C. Riedemann, Stephanie McGuire, Kristina T. Lu, Robin Kunkel, John G. Younger, Firas S. Zetoune, Peter A. Ward,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system. The complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system. The complement system generating the complement activation products, C3a, C5a, and C5b-9 and the cellular defense system involving macrophages and neutrophils are known to form the first line of defense (innate immunity) against microorganisms and other tissue-damaging factors.1Ishii Y Kobayashi J Kitamura S Chemotactic factor generation and cell accumulation in acute lung injury induced by endotracheal acid instillation.Prostaglandins Leukot Essent Fatty Acids. 1989; 37: 65-70Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 2Ward PA Role of complement in lung inflammatory injury.Am J Pathol. 1996; 149: 1081-1086PubMed Google Scholar During acute lung inflammation, leukocytes are recruited from the vascular space into interstitial and distal airway compartments by complement activation products, especially C5a3Sibille Y Reynolds HY Macrophages and polymorphonuclear neutrophils in lung defense and injury.Am Rev Respir Med. 1990; 141: 471-501Crossref PubMed Scopus (923) Google Scholar, 4Mulligan MS Schmid E Beck-Schimmer B Till GO Friedl HP Brauer RB Hugli TE Miyasaka M Warner RL Johnson KJ Ward PA Requirement and role of C5a in acute lung injury in rats.J Clin Invest. 1996; 98: 503-512Crossref PubMed Scopus (175) Google Scholar, 5Solomkin JS Cotta LA Satoh PS Hurst JM Nelson RD Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man.Surgery. 1984; 97: 668-678Google Scholar and various chemotactic cytokines.2Ward PA Role of complement in lung inflammatory injury.Am J Pathol. 1996; 149: 1081-1086PubMed Google Scholar, 6Kazmierowski JA Gallin JI Reynolds HY Mechanism for the inflammatory response in primate lungs. Demonstration and partial characterization of an alveolar macrophage-derived chemotactic factor with preferential activity for polymorphonuclear leukocytes.J Clin Invest. 1977; 59: 273-283Crossref PubMed Scopus (115) Google Scholar There is also evidence that C5a and C5b-9 enhance lung macrophage generation of cytokines and chemokines.7Czermak BJ Sarma V Bless NM Schmal H Friedl HP Ward PA In vitro and in vivo dependency of chemokine generation on C5a and TNFα.J Immunol. 1999; 162: 2321-2325PubMed Google Scholar Systemic complement activation by intravenous infusion of purified cobra venom factor has been shown to cause pulmonary capillary injury and neutrophil accumulation in lungs, leading to acute lung injury.8Till GO Morganroth ML Kunkel R Ward PA Activation of C5 by cobra venom factor is required in neutrophil-mediated lung injury in the rat.Am J Pathol. 1987; 129: 44-53PubMed Google Scholar Although the pathways of complement activation in plasma (alternate, classical, and lectin-binding) are well established, there is less definitive evidence about generation of complement components and complement activation products within the extravascular compartment.7Czermak BJ Sarma V Bless NM Schmal H Friedl HP Ward PA In vitro and in vivo dependency of chemokine generation on C5a and TNFα.J Immunol. 1999; 162: 2321-2325PubMed Google Scholar, 9Hetland G Johnson E Royset P Eskeland T Human alveolar macrophages and monocytes generate the functional classical pathway of complement in vitro.Acta Pathol Microbiol Immunol Scand. 1987; 95: 117-122Google Scholar In bronchoalveolar lavage (BAL) fluids, C5 fragments with C5a-like properties have been detected during acute6Kazmierowski JA Gallin JI Reynolds HY Mechanism for the inflammatory response in primate lungs. Demonstration and partial characterization of an alveolar macrophage-derived chemotactic factor with preferential activity for polymorphonuclear leukocytes.J Clin Invest. 1977; 59: 273-283Crossref PubMed Scopus (115) Google Scholar, 10Henson PM McCarthy K Larsen GL Webster RO Giclas PC Dreisin RB King TE Shaw JO Complement fragments, alveolar macrophages, and alveolitis.Am J Pathol. 1979; 97: 93-110PubMed Google Scholar and chronic lung inflammation.11Groneck P Oppermann M Speer CP Levels of complement anaphylatoxin C5a in pulmonary effluent fluids of infants at risk of chronic lung disease and effects of dexamethasone treatment.Pediatr Res. 1993; 34: 586-590Crossref PubMed Scopus (26) Google Scholar In these BAL fluids, a higher level of hemolytic C5 activity was found when compared to levels found in serum, suggesting that complement components may be formed in extravascular sites.10Henson PM McCarthy K Larsen GL Webster RO Giclas PC Dreisin RB King TE Shaw JO Complement fragments, alveolar macrophages, and alveolitis.Am J Pathol. 1979; 97: 93-110PubMed Google Scholar An extravascular cellular source of complement seems to be macrophages, which are ubiquitous in most tissues and are known to generate a variety of complement proteins, including many of the components required for activation of the alternative pathway.12Ooi YM Harris DE Edelson PJ Colten HR Post-translational control of complement (C5) production by resident and stimulated mouse macrophages.J Immunol. 1980; 124: 2077-2081PubMed Google Scholar, 13Hetland G Johnson E Aasebo U Human alveolar macrophages synthesize the functional alternative pathway of complement and active C5 and C9 in vitro.Scand J Immunol. 1986; 24: 603-608Crossref PubMed Scopus (26) Google Scholar Some in vitro studies have also suggested that noncomplement-derived convertases, namely, bacterially derived arginine-specific cysteine protease14Wingrove JA DiScipio RG Chen Z Potempa J Travis J Hugli TE Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from porphyromonas (bacteroides) gingivalis.J Biol Chem. 1992; 276: 18902-18907Google Scholar and several serine proteases (eg, trypsin and elastase) have the ability to cleave complement components, such as C3 and C5, to produce biologically active anaphylatoxins.15Wiggins RC Giclas PC Henson PM Chemotactic activity generated from the fifth component of complement by plasma kallikrein of the rabbit.J Exp Med. 1981; 153: 1391-1404Crossref PubMed Scopus (72) Google Scholar, 16Ward PA Newman LJ Neutrophil chemotactic factor from human C'5.J Immunol. 1969; 102: 93-99PubMed Google Scholar Thus, C3a and C5a, which are powerful phlogistic peptides, can be generated by complement convertases as well as complement-independent convertases. It has been shown that the co-presence of C5a or C5b-9, bacterial lipopolysaccharide (LPS), or immune complexes cause enhanced production and release of chemotactic cytokines by alveolar macrophages (AMs).7Czermak BJ Sarma V Bless NM Schmal H Friedl HP Ward PA In vitro and in vivo dependency of chemokine generation on C5a and TNFα.J Immunol. 1999; 162: 2321-2325PubMed Google Scholar When C5a or C5b-9 were given into the airways of rats undergoing lung deposition of IgG immune complexes, there was enhanced pulmonary neutrophil accumulation and intensified inflammatory lung injury.7Czermak BJ Sarma V Bless NM Schmal H Friedl HP Ward PA In vitro and in vivo dependency of chemokine generation on C5a and TNFα.J Immunol. 1999; 162: 2321-2325PubMed Google Scholar These data suggest that C5 activation products generated within lung in the presence of a co-stimulus can lead to the recruitment of neutrophils into the alveolar space. Relatively little is known about the extravascular generation of C5 activation products, the C5-cleaving enzyme(s) involved, and the biological functions of such products. In the current studies we have demonstrated that activated rat AMs and activated human neutrophils [but not rat alveolar epithelial cells (AECs) or human peripheral blood mononuclear cells (PBMCs)] can cleave human C5 to generate product(s) that in Western blots align with C5a immunoprecipitated from activated human serum. This C5a was chemotactically active for neutrophils and its functional activity could be blocked by antibody (Ab) to human C5a. Further, serine protease inhibitors [soybean trypsin inhibitor (SBTI) and secretory leukocyte protease inhibitor (SLPI)] were found to block the cleavage of C5 by activated macrophages. These studies imply that C5a can be directly generated by activated phagocytic cells in the presence of C5, extending the potential sources of the anaphylatoxin C5a- and C5-cleaving enzymes beyond proteins present in the plasma. Unless otherwise specified, chemicals and reagents and recombinant human C5a were purchased from Sigma Chemical Co. (St. Louis, MO). Purified human C5 was obtained from Quidel, Inc. (Mountain View, CA). Recombinant human SLPI and human tissue inhibitor of matrix metalloproteases inhibitor-2 (TIMP-2) were kindly provided by Drs. Thomas R. Ulich and Clifford D. Wright (Amgen, Inc., Thousand Oaks, CA). Based on the recently published potent in vitro and in vivo effects of different anti-C5a Ab preparations,17Huber-Lang MS Sarma JV McGuire SR Lu KT Guo RF Padgaonkar VA Younkin EM Laudes IJ Riedemann NC Younger JG Ward PA Protective effects of anti-C5a peptide antibodies in experimental sepsis.EMBO J. 2001; 15: 568-570Google Scholar polyclonal anti-C5a antibodies to the carboxyl-terminal peptide region of the rat C5a molecule (with the sequence CTIADKIRKESHHKGMLLGR, corresponding to amino acid residues 58 to 77) and to the carboxyl-terminal peptide domain of human C5a (CVVASQLRANISHKDMQLGR, corresponding to residues 55 to 74) were used in the present study. Peptide synthesis, Ab production, and affinity purification was done by Research Genetics, Inc. (Huntsville, AL). The anti-rat C5a polyclonal Ab does not cross-react with recombinant human C5a (data not shown). However, the anti-human C5a polyclonal Ab does cross-react with recombinant rat C5a but does not pick up human C5 on a Western blot (data not shown). We are unable to determine the crossreactivity of these antibodies with rat C5 because of the nonavailability of rat C5. Anti-human C5 Ab was obtained from Quidel. Rat AMs were isolated by repeatedly lavaging lungs of anesthetized, healthy animals (Long Evans rats; Harlan, Inc., Indianapolis, IN). After centrifugation of BAL fluids, cells were resuspended in Dulbecco's modified Eagle's medium (DMEM), pH 7.4 (Whittaker Bioproducts, Walkersville, MD). AMs (1 to 10 × 106 cells) were stimulated with phorbol ester (PMA) (100 ng/ml), while 10 mmol/L of ethylenediaminetetraacetic acid (EDTA) was added to nonstimulated AMs to avoid contact-mediated activation. To both groups, human C5, at concentrations ranging from 1 to 120 μg, was added and the cells incubated for the indicated time periods at 37°C. After an incubation period of 4 hours (or at other indicated time intervals), supernatant fluids were collected and remaining cell debris removed by a centrifugation step at 4°C. The supernatant fluids were then used for detection of C5-cleavage products as described below. AECs were isolated as primary cultures from specific pathogen-free male Long Evans rats (Harlan). The isolation was performed using a modified version of the elastase method. After anesthesia with ketamine (1 g/kg body weight), rats were bled out by cutting the abdominal aorta. An intratracheal catheter was then placed. After flushing of the pulmonary artery with 10 ml of Dulbecco's phosphate-buffered saline (DPBS), the lungs were carefully removed together with the heart and subjected to BAL for 8 to 10 times with 10 ml of DPBS. This resulted in the removal of the majority of AMs. Next, the lungs were placed in a waterbath (37°C) for 25 minutes and 40 ml of DPBS containing ∼90 U of Elastase (Worthington, Lakewood, NJ) was slowly infused into the airways. The heart and any remaining tissue was removed and the lungs were minced for 3 minutes with scissors after adding 1000 U of deoxyribonuclease I (Sigma Chemical Co.). Enzyme activity was stopped by the addition of 5 ml of ultra low IgG fetal bovine serum (Life Technologies Inc., Rockville, MD). The suspension containing the AECs was incubated at room temperature and gently stirred for 20 minutes. The suspension was then filtered three times through a mesh (500 mm, 175 mm, 105 mm) (Spectrum Laboratories, Inc., Rancho Dominguez, CA) and suspended with 30 ml of DMEM. The cells were centrifuged at 1500 rpm for 10 minutes and then resuspended in 45 ml of DMEM. Next the cells were plated onto 100-mm Petri dishes precoated with 30 mg/ml of rabbit IgG, for 1 hour at 37°C to remove remaining AMs. Cells were than carefully collected by washing over the Petri dishes three to four times. After centrifugation, the cell pellet was carefully resuspended in 10 to 20 ml of DMEM containing 1% penicillin streptomycin mix, 1% l-glutamine (200 mmol/L), and 1% nonessential amino acids (10 mmol/L) purchased from Life Technologies, Inc. (Grand Island, NJ) and 10% heat-inactivated fetal bovine serum. Finally, cells were plated onto various tissue culture plates as needed, and cultured for 2 days before stimulation. All nonadherent cells were removed by washing the tissue culture dishes with DMEM at days 1 and 2 after isolation. Residual macrophages were <2% of total cells. Human blood neutrophils and PBMCs, were isolated as described below and elsewhere.18Foreback JL Remick DG Crockett-Torabi E Ward PA Cytokine responses of human blood monocytes stimulated with Ig's.Inflammation. 1997; 21: 501-517Crossref PubMed Scopus (26) Google Scholar Supernatant fluids from the indicated cell suspensions, which were unstimulated or stimulated with PMA and to which human C5 (in the amounts indicated) was added, were electrophoresed under reducing conditions on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Schleicher & Schuell, Keene, NH) using a semidry electrophoresis apparatus (LkB Multiphor II; Pharmacia Biotech, Uppsala, Sweden). The blot was incubated with 5% fat-free milk in DPBS for 1 hour at room temperature and then probed with the indicated rabbit anti-human C5a IgG (at a final concentration of 3 μg/ml) overnight at 4°C. After washing, an alkaline phosphatase-conjugated goat anti-rabbit IgG Ab (Jackson ImmunoResearch, Inc., West Grove, PA) was used as a secondary Ab (1:1000) at room temperature for 1 hour, followed by an additional washing step (with DPBS containing 0.1% Tween 20), and alkaline phosphatase substrate color development (Bio-Rad Laboratories, Hercules, CA). When goat anti-human C5 IgG was used as the primary Ab, the secondary Ab was rabbit anti-goat IgG that was conjugated to alkaline phosphatase. In some experiments C5a was generated in normal human serum by the addition of 10 mg of oyster glycogen/ml serum, followed by incubation at 37°C for 30 minutes. The serum was then diluted 1:5 with DPBS and C5a immunoprecipitated with protein A Sepharose beads and anti-human C5a rabbit IgG (Calbiochem, La Jolla, CA). Rat AMs and human neutrophils were obtained in the usual way and were suspended in DMEM at 10 × 106 cells/ml. The cells were pretreated with cycloheximide or actinomycin D (20 μg/ml and 200 ng/ml, respectively) for 20 minutes at 37°C. After treatment, cells were incubated with the indicated amounts of C5 and PMA, as described above. The cells were then further incubated for 4 hours at 37°C. The cells were then pelleted and the supernatants analyzed by Western blotting. Cells were also incubated alone or with C5 and PMA treatment in the absence of exposure to actinomycin D or cycloheximide. Some of the AM supernatant fluids samples were electrophoretically separated on a 15% polyacrylamide gel electrophoresis and the gels were silver-stained (Silver Stain Plus; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. Briefly, after electrophoresis, gels were placed in a fixative enhancer solution for 20 minutes, followed by two washing steps in deionized distilled water. The final silver complex solution was added for 20 minutes in the presence of the image development reagent and the reaction stopped in 5% acetic acid solution. The gels were then washed again, dried, and photo-documented. The ability of protease inhibitors to interfere with the CH50 activity in whole human and rat serum was assessed by measurement of the hemolytic activity of fresh human and rat serum in the presence or absence of protease inhibitors. Briefly, sensitized sheep red blood cells (Colorado Serum Company, Denver, CO) were exposed at 37°C for 60 minutes to various dilutions of serum samples in triethanolamine-buffered saline (TBS), pH 7.35 (268 mmol/L NaCl, 20 mmol/L triethanolamine, 150 μmol/L CaCl2, and 500 μmol/L MgCl2) in presence or absence of SLPI or SBTI (each at 100 μg/ml). The complement reaction was stopped by ice-cold TBS (with 0.05% gelatin) followed by a centrifugation step (2500 × g, 5 minutes). Absorption values of the supernatant fluids were determined at 541 nm and the hemolytic activity of human and rat serum in the presence or absence of the indicated protease inhibitors was determined.19Amsterdam EA Stahl GL Pa HL Rendig SV Fletcher MP Longhur JC Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs.Am J Physiol. 1995; 268: H448-H457PubMed Google Scholar Whole blood of healthy human volunteers was drawn from the antecubital vein into syringes containing the anticoagulant, anticoagulate citrate dextrose (ACD) (Baxter Health Care, Deerfield, IL), in a volume ratio of 1:10 (ACD:blood). Neutrophils were isolated using Ficoll-Paque gradient centrifugation (Pharmacia Biotech AB) followed by a dextran sedimentation step. After hypotonic lysis of residual red blood cells, neutrophils were resuspended in Hanks' balanced salt solution and fluorescein-labeled with BCECF (2′, 7′-bis [2-carboxyethyl]-5-[and 6]-carboxy-fluorescein acetoxymethyl ester) (Molecular Probes, Inc., Eugene, OR) for 30 minutes at 37°C. After a washing step with DPBS, labeled neutrophils (5 × 106 cells/ml) were loaded into the upper chamber of a 96-well mini chamber (NeuroProbe, Inc., Gaithersburg, MD), separated by a polycarbonate filter with a porosity of 3 μm (NeuroProbe). The lower chambers were loaded with human C5a (0.1 to 1000 ng/ml) or different dilutions of supernatant fluids of the AM preparations in the presence or absence of anti-human C5a and/or anti-rat C5a antibodies (each at 10 μg/ml). Neutrophils were then incubated for 30 minutes at 37°C. The number of cells migrating through the polycarbonate membrane to the lower surface was determined by cytofluorometry (Cytofluor II; Per Septive Biosystems, Inc., Framingham, MA). For each measurement, samples were done at least in quadruplicates. All values were expressed as mean ± SEM. Data sets of chemotaxis assays and CH50 assays were analyzed with one-way analysis of variance; differences in the mean values among the experimental groups were then compared using the Tukey multiple comparison test. Results were considered statistically significant when P was 2 × 107 cells were incubated with C5 and PMA (data not shown). Primary cultures of rat AECs (type II cells) ( 97% cells that were cytokeratin-positive) as well as freshly isolated rat AMs (≥97% macrophages) were incubated with PMA (100 ng/ml) and C5 (90 μg) for 4 hours at 37°C under the various conditions are shown in Figure 2. The typical pattern for recombinant human C5a was found, with bands positioned slower or faster than the 14.3-kDa marker (Figure 2, lanes 1 and 11). AECs (106) activated with PMA failed to produce any evidence of cleavage of C5 (Figure 2A, lane 2). AECs in the absence of PMA also showed no C5a product (Figure 2, lane 3). In the same experiment, activated AMs (107) showed a cleavage product that was detected by anti-C5a and aligned with the 14.3-kDa marker (Figure 2, lane 5). To assess the dose responses of PMA-stimulated AMs to generate C5a as a function of macrophage numbers, 1, 5, and 10 × 106 AMs were incubated with PMA and C5 under the conditions described above. The results (Figure 2, lanes 7, 8, and 9) indicated a C5a product in each case. Accordingly, when equivalent numbers (106) of activated rat AMs (Figure 2, lane 7) and AECs (Figure 2, lane 2) were compared, only the former cells were capable of generating C5a, suggesting that C5a generation by various cells may be a selective cell response. Two phagocytic cell types (human PMNs and rat AMs) with a robust ability to generate C5a from human C5 (as determined in Figure 1, Figure 2) were evaluated for the extent of C5 cleavage as a function of time using Ab to whole C5. The results are shown in Figure 3. The α and β chains of otherwise unmanipulated C5 could be readily detected as the predominant proteins in the expected positions (Figure 3, lane 5). Not surprisingly, the anti-human C5 Ab had a poor ability to detect human C5a (Figure 3, lane 4). Incubation of C5 with PMNs for 30 minutes at 37°C in the presence of PMA led to extensive hydrolysis of both the α and β chains of human C5, resulting in many hydrolysis products (Figure 3, lane 3). By 3 hours, virtually no detectable C5 cleavage products remained (Figure 3, lane 2). The co-presence of EDTA, as might be expected, reduced the extent of hydrolysis of C5 by activated PMNs at 3 hours, with detectable α and β chains of C5 still present (Figure 3, lane 1). When C5 was incubated for 30 minutes or 3 hours at 37°C with rat AMs stimulated with PMA, the α and β chains of C5 could still be detected and extensive hydrolysis products were not found (Figure 3, lanes 7 and 8). Supernatant fluids were evaluated from rat AMs and human PMNs (10 × 106 cells) that had been stimulated with 100 ng/ml of PMA in the presence of 90 μg of C5 at 37°C as a function of time (Figure 4). Samples were subjected to electrophoresis and the products detected by Western blot analysis using anti-human C5a Ab. A C5 cleavage product that aligned with the 14.3-kDa marker and was reactive with anti-human C5a Ab was found, with increasing intensity as a function of time of incubation, faintly detectable at 1 hour and appearing to reach a maximum between 2 and 4 hours in activated macrophages (Figure 4A). Similarly, in human PMNs, a C5a cleavage product was detectable as early as 30 minutes and appeared to peak at 2 hours. No other cleavage products from C5 were found when anti-C5 or anti-C5a Ab was used (Figure 1, Figure 2, Figure 3). In macrophages the intensity of the cleavage product as detected in Western blots increased when increasing doses of C5 was used (Figure 4C). Thus, incubation of C5 with activated rat AMs and PMNs consistently yielded a C5 cleavage product that aligned with the 14.3-kDa marker. No evidence of this cleavage product from C5 was found when human C5 was omitted from or when C5 (90 μg) was present in the absence of cells (Figure 4C). By Western blot analysis, recombinant human C5a showed the main band near the 6.5-kDa marker and a less intense band between the 14.3- and 21.5-kDa markers (Figure 5A, lanes 1 and 2). The C5 cleavage product generated by activated rat AMs is consistent with glycosylated C5a (Figure 5, lane 3). C5a immunoprecipitated from glycogen-activated human serum also aligned near the 14.3-kDa position (Figure 5, lane 4). No band could be detected in nonactivated human serum (data not shown). As shown in Figure 5B, the C5 cleavage product generated by PMA-activated AMs in the presence of C5 (Figure 5, lane 7) was not found in the supernatant fluids that contained 10 mmol/L of EDTA (Figure 5, lane 6). To further characterize the C5 cleavage products, the anti-C5a Ab was preabsorbed with recombinant human C5a, resulting in the disappearance of the ability to detect the expected cleavage product (Figure 5, lane 8). The presence of a cleavage product was further confirmed by silver staining. The band produced by activated AMs in the presence of C5 (Figure 5, lane 10) aligned with the 14.3-kDa marker, this band being barely detectable if EDTA had also been present in the mixture of C5 and rat AMs (Figure 5, lane 9). These data indicate that the C5 cleavage product produced by activated AMs aligns with the 14.3-kDa marker and is likely C5a. Because Western blot analysis indicated the presence of a product reactive with Ab to human C5a in supernatant fluids from activated rat AMs incubated with human C5 (described above), we performed functional analysis on these fluids by measuring the chemoattractant activity for human blood neutrophils and the ability of anti-human C5a to block this biological activity. The data are shown in Figure 6 in which cytofluorometric analysis of neutrophils that had migrated through the micropore filters was used. The chemotactic response of human neutrophils to 0.1 μmol/L of fMLP is shown in the second (from left) white bar in Figure 6. The migratory response was very low in the presence of Hanks' balanced salt solution alone (Figure 6, first white bar) or in supernatant fluids to which either EDTA (Figure 6, cross-hatched bar) or PMA (Figure 6, gray bar) had been added to rat AMs (107) in the absence of C5. In contrast, supernatant fluids from PMA-stimulated rat AMs in the presence of human C5 showed a very robust migratory response (Figure 6, first black bar), which was similar to the neutrophil chemotactic response to 100 nmol/L fMLP (Figure 6, second white bar). Addition of anti-rat C5a Ab (10 μg/ml) to the supernatant fluids from activated AMs incubated with 90 μg of C5 showed only
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