Both free radicals and complement activation can injure tissue. Our study determined whether free radicals alter complement production by the myocardium. Isolated hearts from New Zealand White rabbits were perfused on a Langendorff apparatus and exposed to xanthine (X; 100 μM) plus xanthine oxidase (XO; 8 mU/ml) (X/XO). The free radical-generating system significantly (P < 0.05) increased C1q and also increased C1r, C3, C8, and C9 transcription compared with controls. Immunohistological examination revealed augmented membrane attack complex deposition on X/XO-treated tissue. X/XO-treated hearts also exhibited significant (P < 0.05) increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in left-ventricular developed pressure.N-(2-mercaptopropionyl)-glycine (3 mM), in conjunction with the superoxide dismutase mimetic SC-52608 (100 μM), significantly (P < 0.05) reduced the upregulation of C1q, C1r, C3, C8, and C9 mRNA expression elicited by X/XO. The antioxidants also ameliorated the deterioration in function caused by X/XO. Local complement activation may represent a mechanism by which free radicals mediate tissue injury.
- gene expression
- membrane attack complex
free radicals are involved in advancing the tissue destruction associated with the inflammatory process (18). Lipid peroxidation represents a direct manifestation of the deleterious effects of free radicals on cell membranes, and initiation of certain intracellular signaling cascades by free radicals may propagate tissue injury (1,35). These oxidants also damage cellular proteins and nucleic acids (2). Reperfusion of the ischemic heart generates free radicals that are speculated to mediate a portion of the irreversible tissue injury subsequent to ischemia-reperfusion (18, 34). Many studies have demonstrated the efficacy of antioxidants in ameliorating ischemia-reperfusion injury, affirming the significance of free radicals in promoting tissue damage in this setting (18).
In vivo, free radicals can arise from extracellular and intracellular sources. Reperfusion of the ischemic myocardium delivers neutrophils to the area at risk that then infiltrate the tissue and release cytotoxic reactive oxygen species, such as superoxide anion. Studies examining the effects of extracellular free radical-generating systems on isolated hearts have demonstrated the direct, detrimental consequences of these species on ultrastructure and function (19,32). Free radicals also have been detected in the reperfused isolated heart, indicating that organ-derived oxidants participate in promoting reperfusion injury (34). Reactive oxygen species can originate intracellularly from enzyme-substrate interactions and organelles such as mitochondria (18,29).
Complement activation constitutes another destructive facet of inflammation, such as that which occurs during ischemia and reperfusion (13). A variety of entities activate complement, including antibodies, membranes of microorganisms, and free radicals (6). Complement causes tissue injury via the direct lytic activity of the membrane attack complex (MAC) and augments the inflammatory response (13). The complement components C3a and C5a function as chemoattractants, recruiting potentially harmful leukocytes into the area at risk (13). We previously determined that the rabbit isolated heart can locally produce complement, and that ischemia and reperfusion significantly increase myocardial complement expression (30). The modulation in tissue complement expression in response to an ischemic attack suggests a role for local complement generation in reperfusion injury.
Oxygen radicals have been shown to stimulate the transcription of a variety of genes as well as activate the ubiquitous transcription factor nuclear factor (NF)-κB (1,23). Reactive oxygen species also initiate several phosphorylation cascades that affect gene expression (1). Although it is known that free radicals activate the complement system, the effect of free radicals on complement transcription remains unexplained.
The present study investigates the effect of free radicals on tissue complement expression by the rabbit isolated heart. We demonstrate that reactive oxygen species induce complement production in the myocardium, and that antioxidants attenuate this effect. Our results suggest that local activation of complement may represent a mechanism by which free radicals advance the pathogenesis of ischemia-reperfusion injury and other inflammatory-mediated disorders.
MATERIALS AND METHODS
Guidelines for animal research.
The procedures used in this study were in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Health Care, and the animal care use program conforms to the standards in the National Research Council's Guide for the Care and Use of Laboratory Animals.
Langendorff perfused heart.
Male New Zealand White rabbits (1.8–2.2 kg) were rendered unconscious by cervical dislocation. Hearts were removed, mounted, and perfused on a Langendorff apparatus with modified, oxygenated Krebs-Henseleit (K-H) buffer (pH 7.44, 37°C) through the aorta at a constant flow (20–24 ml/min). The perfusion medium was composed of (in mM) 117 NaCl, 4.0 KCl, 1.2 MgCl2 · 6H2O, 1.1 KH2PO4, 5.0 glucose, 25.0 NaHCO3, and 2.6 CaCl2 · 2H2O. The buffer passed through a membranous “lung” composed of Silastic medical-grade tubing (Dow Corning, Midland, MI). The membrane lung was gassed continuously with a mixture of 95% O2-5% CO2. The hearts were paced through the right atrium with electrodes attached to a laboratory stimulator (Grass SD-5, Quincy, MA). A left ventricular drain, thermistor probe, and latex balloon were placed via the left atrium and secured with a purse string suture at the atrial appendage. The latex balloon was expanded with water to achieve a left ventricular end-diastolic pressure of 5 mmHg. Isolated hearts were stabilized under normoxic conditions for a 10- to 15-min equilibration period before starting the protocol. The preparation has been described in detail previously (30).
Effluents collected from the Langendorff apparatus (buffer alone), isolated heart, xanthine solution, and aliquots of xanthine oxidase were tested for endotoxin with the Limulus assay (Sigma, St. Louis, MO). In addition, aliquots of xanthine oxidase were tested in a hemolytic assay for complement activation activity. The hemolytic assay was performed as described previously (25). Endotoxin is known to activate complement. The Limulus test was negative for endotoxin, and the hemolytic assay was negative for complement activation by xanthine oxidase.
Three experimental groups were studied. Group 1 (control) consisted of hearts perfused for 50 min with K-H buffer; group 2 hearts were perfused for 40 min, with buffer containing a free radical-generating system containing 100 μM xanthine and 8 U/l xanthine oxidase dissolved in K-H buffer; and group 3 hearts were pretreated with the hydroxyl radical scavengerN-(2-mercaptopropionyl)-glycine (MPG; 3 mM) and the superoxide dismutase (SOD) mimetic SC-52608 (100 μM) for 10 min before perfusion for 40 min with 100 μM xanthine and 8 U/l xanthine oxidase. The antioxidants were present for the duration of the protocol.
Unless specified otherwise, all chemicals were purchased from Sigma Chemical and dissolved readily in K-H buffer. Xanthine was sonicated in 1 ml of 0.9% saline before addition to the buffer. SC-52608 was supplied by Monsanto (St. Louis, MO).
Choice of specific primers.
The DNA sequences of rabbit C3, C8, C9, and cyclophilin were obtained from the GenBank data base (accession numbers M32434, U20055, L26980, and YO0052, respectively). Cyclophilin mRNA was chosen as the internal standard because it is expressed at a relatively constant level in virtually all tissues. We previously reported on the primer sequences used to detect mRNAs for C3, C9, and cyclophilin (30). The primer sequences chosen for amplifying C8 were as follows: forward 5′-TAAAAGACCGACACAAAAGGGACAC-3′ and reverse 5′-ATGAAGACCAGCGAGACCAGCAACT-3′. Primers for human C1q and C1r (31) were used to determine whether rabbit cDNA products could be obtained. Total RNA from rabbit heart was reverse transcribed, and the cDNAs were amplified with the human C1q and C1r primers by methods previously described in detail (30,31). Single products close to the expected sizes were obtained on polyacrylamide gels. These products were then subcloned into PGEM-T plasmid vector (Promega) for sequencing. The sequences were determined by the cycle-sequencing method with the use of T7 and sp6 sequencing primers on an autosequencer (NAPS unit, Univ. of British Columbia). The sequenced rabbit clone for C1q was 361 bp long compared with 358 for the comparable human cDNA product. There was 82.6% homology overall, with 100% homology in the primer region. Primers for rabbit C1q were as follows: forward 5′-CCCAGGGATAAAAGGAGAGAAAGG-3′ and reverse 5′-GGCGTGGTAGGTGAAGTAGTAGAG-3′. The assigned Genbank accession number is AF089083. The sequenced rabbit clone for C1r was 218 bp long compared with 216 for the human product. There was 85.3% homology overall, with only a 1-bp difference in the primer region. The assigned GenBank accession number is AF108768. New primers for rabbit C1r were designed: forward 5′-GCCTCCCTGACAACGATACCTTCTA-3′ and reverse 5′-TGTCCTGCTTTAGAGATGGGTGTCC-3′.
RNA preparation and RT-PCR.
Total RNA from ∼500 mg of each tissue sample was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (5). The extracted RNA was quantified by scanning spectrophotometry. The absorbance 260/280 ratio of all preparations was >1.8. The RNA was then reverse transcribed, and specific cDNAs were amplified by the PCR technique as previously described in detail (30,31).
In preliminary studies, we found that the amount of PCR product increased exponentially from 20 to 29 cycles for cyclophilin and from 25 to 37 cycles for the complement cDNAs. A plateau phase was reached after 29 and 37 cycles, respectively, because of the plateau effect (15). Accordingly, each cDNA sample was treated by the PCR procedure with the cyclophilin product being amplified for 27 cycles and the complement products for 35 cycles. Each PCR reaction product was electrophoresed through a 6% polyacrylamide gel, and the product was visualized by incubation for 10 min in a solution containing 10 ng/ml ethidium bromide. Resulting gel bands were imaged using a GDS 6700 image analyzer (Ultra Violet, Uplands, CA). The relative intensities of the bands, expressed as optical density (OD) units, were quantitatively analyzed with the use of National Institutes of Health Image software 1.61. Each complement mRNA amplification was run in parallel with a cyclophilin mRNA amplification to provide an internal standard. Direct OD values were analyzed as were values relative to cyclophilin. Polaroid photographs of the gels were taken.
Restriction digest analysis.
The PCR products were purified by the ethanol precipitation procedure (31). Unique restriction sites and restriction enzymes were selected with the use of the DNA Strider computer program. The restriction enzymes chosen were as follows: Sac I for C1q,Sau3A I for C1r, Hinc II for C3, Mse I for C8, and BamH I for C9. The restriction digestion reaction was carried out for 2 h at 37°C. The digested PCR products were analyzed by electrophoresis on a 6% nondenaturing polyacrylamide gel.
At the completion of the experiments, hearts were removed from the Langendorff apparatus, cut into transverse sections, and frozen in liquid nitrogen. The apex and atrial tissue were discarded. Sections were embedded in optimum cutting temperature (OCT) compound-embedding medium (Miles, Ekhart, IN), cut at 3 μm, and placed on poly-l-lysine-coated slides. After being rinsed with PBS, sections were incubated with 4% paraformaldehyde in PBS at room temperature. Heart sections were rinsed with PBS and incubated with 1% BSA for 15 min to minimize nonspecific staining. After being rinsed with PBS, sections were incubated with a polyclonal chicken anti-rabbit MAC antibody at a 1:50 dilution at room temperature for 1 h. The anti-rabbit MAC antibody was developed in conjunction with Lampire Biological Laboratories (Pipersville, PA) with the use of rabbit C5b-9 antigen supplied by Dr. S. Bhakdi (Institute of Medical Microbiology and Hygiene Johannes Gutenberg Univ., Mainz, Germany). Sections were then rinsed with PBS and incubated at room temperature for 1 h with a donkey anti-chicken FITC-conjugated antibody (Accurate Antibodies, Westbury, NY) at a 1:50 dilution. After a final rinse with PBS, sections were mounted with Gel/Mount (Biomeda, Foster City, CA) and protected with a coverslip. Controls included sections in which the primary antibody was omitted.
Data were expressed as means ± SE. Differences between control and experimental groups were checked for statistical significance (P < 0.05) by the Student's t-test for unpaired observations. Dunnet's t-tests with Holm's (12) correction for multiple comparisons were used for determining significant differences. Data were analyzed in both the raw form and after correction for levels of the housekeeping gene cyclophilin. The corrections were minimal, and similar Pvalues were obtained in each case.
Forty minutes of perfusion with the free radical-generating system, xanthine/xanthine oxidase (X/XO), resulted in significant (P < 0.05) increases in coronary perfusion pressure (CPP) (Fig. 1) and left ventricular end-diastolic pressure (LVEDP) (Fig.2) compared with control (expressed as percentage of baseline values). The hydroxyl radical scavenger, MPG, and the SOD mimetic, SC-52608, attenuated the increases in CPP and LVEDP observed in the X/XO-treated hearts (Figs. 1 and 2). MPG/SC-52608 alone produced a significant (P < 0.05) but transient decrease in CPP after 10 and 20 min of perfusion (data not shown); however, CPP did not significantly differ from controls after 40 min of perfusion with MPG/SC-52608/X/XO. Exposure to X/XO also caused a significant (P < 0.05) decrease in left ventricular developed pressure (LVDP) (Fig. 3). MPG/SC-52608 significantly (P < 0.05) preserved LVDP compared with hearts perfused only with X/XO (Fig. 3). This is expressed as percentage of LVDP after 10-min exposure to MPG/SC-52608 immediately before addition of X/XO, because perfusion with MPG/SC-52608 alone caused a significant decrease in LVDP (data not shown).
RT-PCR amplification from total RNA extracts was used to establish the presence and relative values of the mRNAs for C1q, C1r, C3, C8, and C9 in all heart samples. These are the only complement genes for which the sequences have been determined for rabbit and are therefore the only ones that could be amplified. The primers chosen to amplify each cDNA yielded a single product corresponding to the anticipated size based on the known sequences (data not shown). The C1q primers generated a product of 361 bp, which gave the expected fragments of 214 and 147 bp when treated with the restriction enzyme Sac I. The C1r primers generated a product of 218 bp, which was cleaved bySau3A I to yield the predicted digestion fragments of 65 and 153 bp. The C3 primer generated a product of 298 bp, and treatment withHinc II yielded fragments of 253 and 45 bp. The C8 primers yielded a product of 441 bp, and treatment with Mse I gave fragments of 160 and 281 bp. The C9 primers generated a product of 202 bp, and treatment with BamH I resulted in fragments of 125 and 67 bp. The cyclophilin primers yielded a product corresponding to the calculated size of 206 bp. These results establish that unique reaction products were being amplified that correspond to each complement mRNA being analyzed.
The relative intensities of all gel bands were determined as described in materials and methods, and the quantitative values were expressed as relative OD units. Figure 4illustrates the comparative levels of C1q, C1r, C3, C8, and C9 mRNA for all three groups. X/XO treatment increased mRNA levels for each complement component compared with buffer-perfused hearts. However, after multiple comparison statistical analysis, significance at theP < 0.05 level was reached only for C1q, where an 18.3-fold increase over control levels was observed (Table1, group 1 vs. group 2). The antioxidants MPG and SC-52608 significantly (P < 0.05) attenuated the transcription of C1q, C1r, C3, C8, and C9 mRNA compared with hearts exposed to X/XO alone (Table1, group 2 vs. group 3). Treatment with MPG and SC-52608 also reduced C1r, C3, and C8 mRNA expression below control levels, although significance at the P < 0.05 level was not observed (Table 1, group 1 vs. group 3).
Tissue samples from each group were stained for the presence of the MAC. Hearts perfused with X/XO exhibited an intense red stain compared with controls, suggesting that free radicals induce MAC assembly in myocytes (Fig. 5). It cannot be excluded that resident leukocytes are the cells exhibiting MAC immunofluorescence. However, hematoxylin and eosin staining of isolated heart tissue samples from other experiments performed in our laboratory demonstrates an almost complete absence of leukocytes in the preparation (unpublished observations). The red fluorescence of the MPG/SC-52608-treated hearts was considerably weaker than that of the X/XO-perfused hearts, indicating that the antioxidants attenuated MAC formation in hearts exposed to free radicals. These results support the mRNA data. The MAC immunofluorescence also indicates that the rabbit myocardium is capable of synthesizing all the complement components needed to form the MAC.
The present study demonstrates that free radicals increase C1q, C1r, C3, C8, and C9 mRNA and MAC production by the isolated myocardium. The antioxidants MPG and SC-52608 inhibit free radical-stimulated complement expression, confirming the role of these reactive species in our model. Cytotoxic oxygen species are produced during the inflammatory response, which comprises a key part of many pathological conditions including ischemia-reperfusion injury (34). We propose that local activation of complement by free radicals may occur during oxidative stress and may contribute to the tissue injury associated with this event.
The metabolism of xanthine by xanthine oxidase generates reactive oxygen species, most notably the superoxide anion (O2 −) and hydrogen peroxide (H2O2) (18). Both entities cause cellular damage and propagate the generation of free radicals via the Fenton and Haber-Weiss reactions (11, 24). Exposure to xanthine and xanthine oxidase is used frequently for evaluating the effects of free radicals on cell structure and function (18, 32). Perfusion of isolated hearts with the enzyme system results in tissue edema, disorganization of myofilaments, and mitochondrial swelling in myocytes (32). Treatment with X/XO also alters the hemodynamic function of the myocardium, decreasing contractility and coronary flow as well as inducing mitochondrial ultrastructure (18,28). In agreement with previous studies, we observed deterioration of myocardial hemodynamic function in the X/XO-treated hearts. The combination of MPG and SC-52608 significantly preserved the hemodynamic function of hearts exposed to X/XO, suggesting that free radicals mediate the decline in function noted in this model. It is not clear from this study whether activation of myocardial complement also contributed to impaired contractility and increased CPP. Previous work showing that human plasma severely damages rabbit isolated hearts through the activation of complement indicates that complement components directly alter myocardial function (14).
MPG and SC-52608 primarily scavenge hydroxyl and superoxide anions, respectively (16, 20). We elected to use both antioxidants to effectively quench the superoxide generated by the metabolism of xanthine by xanthine oxidase, as well as the hydroxyl radicals that may be produced by the interaction of hydrogen peroxide and iron. Although exogenous iron was not supplied in this system, trace amounts of iron present in the buffer appeared to effectively catalyze the reaction, as evidenced by the antioxidant-sensitive changes in function and complement production. Myoglobin also may have served as an important intracellular source of iron, because oxygen radicals and hydrogen peroxide can cross cell membranes. SC-52608 is a low-molecular-weight superoxide dismutase mimetic (16). MPG is a sulfhydryl compound mainly recognized as a hydroxyl radical scavenger, but it has also been shown to scavenge superoxide (20). In addition, MPG inhibits complement activation (17); however, its effects in this study appear to be at the transcriptional level. MPG and SC-52608 are both lipid soluble and have been shown to attenuate ischemia-reperfusion injury, presumably through their antioxidant capabilities (3,16, 20). The data demonstrate that these agents effectively inhibit complement expression stimulated by exposure to free radicals. Future studies examining the effects of these antioxidants on complement production in an ischemia-reperfusion model are warranted and would prove informative in determining the role of free radicals under these conditions.
MPG and SC-52608 significantly inhibited complement mRNA synthesis of C1q, C1r, C3, C8, and C9 compared with X/XO-perfused hearts. The antioxidants also attenuated C1r, C3, and C8 mRNA expression below control levels, indicating that perfusion of isolated hearts with buffer alone slightly stimulates complement production. The differential sensitivity to reactive oxygen species of the complement components measured suggests that several distinct mechanisms may govern the transcription of each complement gene. The intracellular signaling events and transcriptional regulators involved in expression of complement genes remain largely unknown.
Activation of the complement system mediates a significant portion of the tissue damage that occurs during ischemia-reperfusion (13). Previously, it was assumed that plasma delivered all complement components to the site of injury. However, recent investigations have demonstrated that myocardial tissue upregulates complement expression in response to ischemia-reperfusion, suggesting a role for local complement activation in this setting (30). Complement injures tissue directly by lysing cells through formation of the MAC (13). The complement cleavage products C3a and C5a also act as chemoattractants for leukocytes, thereby augmenting the inflammatory response in the reperfused area at risk (13). Both the classical and alternative pathways of complement activation may participate in reperfusion injury (22). Free radicals have been shown to activate the alternative pathway (6). Although the classical pathway is primarily initiated by antibodies, upregulation of C1q and C1r in our study suggests that oxidative stress may also activate the classical pathway in the isolated heart.
Our laboratory has found that preconditioning, the phenomenon whereby brief episodes of ischemia before a prolonged ischemic event reduce reperfusion injury, attenuates myocardial complement production stimulated by ischemia-reperfusion (26). Preconditioning has also been shown to protect against free radical-mediated injury, possibly by stimulating endogenous antioxidant defenses (27, 33). Oxygen radicals initiate the transcription of a variety of genes as well as activate the transcription factor NF-κB, which regulates several inflammatory genes (1, 23). In addition, it has been suggested that NF-κB mediates C3 expression in human epithelial cells (21).
Under physiological conditions, free radicals can originate from a variety of sources. Neutrophils destroy invading and native cells via the production of oxidants and their cytotoxic metabolites (18). Because C3a and C5a are chemoattractants, increased local complement production may recruit these cell types into the area at risk and maintain the presence of free radical-donating inflammatory cells throughout the reperfusion period. Yasojima et al. (30) demonstrated that complement activation in infarcted human myocardium continues long after the initial ischemic insult. Mitochondria may also leak oxidants during respiration, and intracellular enzymes, such as xanthine oxidase, produce free radicals during catalysis (18, 30). Aside from the lipid peroxidation and the intracellular calcium overload associated with oxidative stress, our study suggests that tissue-derived complement may also play a role in the mechanism of free radical-induced cytotoxicity.
Although the present study clearly demonstrates that antioxidants attenuate myocardial complement generation, the possibility that endotoxin may have contributed to this effect cannot be excluded. Because the xanthine oxidase used in our experiments was purified from a microorganism, it is possible that trace amounts of endotoxin were present in the system even though measures were taken to minimize endotoxin contamination. However, Limulus tests performed on effluents taken from the Langendorff apparatus (buffer alone), isolated heart, xanthine solution, and aliquots of the xanthine oxidase used in the experiments were negative for endotoxin contamination. Furthermore, addition of xanthine oxidase to rabbit plasma failed to activate rabbit complement in a hemolytic assay. Endotoxin has been reported to stimulate the production of cytokines such as tumor necrosis factor (TNF)-α (7), and cytokines have been shown to increase complement generation in vitro (8, 10). However, because complement production was upregulated in 1 h of our study, it is unlikely that endotoxin induction of TNF-α (or any other cytokine) production followed by cytokine-induced complement upregulation successively occurred in this brief amount of time. On the other hand, the extreme reactivity of free radicals could elicit this response within this time window. Endotoxin is also known to cause tissue injury via oxidative stress (4, 9). If this is the case in the present investigation, it does not change the conclusion that free radicals increase complement gene and protein expression.
In conclusion, we demonstrate that free radicals stimulate complement production in the myocardium. Because free radicals and complement activation participate in a variety of inflammatory processes, these two events may promote one another and advance tissue injury.
We are grateful to Dr. S. Bhakdi for the kind donation of the rabbit C5b-9 antigen, which made the MAC immunofluorescence staining possible. We also thank Monsanto for furnishing the SC-52608.
This study was supported by the Cardiovascular Research Fund, Univ. of Michigan, and donations from individual British Columbians.
Address for reprint requests and other correspondence: B. R. Lucchesi, Dept. of Pharmacology, Univ. of Michigan, 1150 West Medical Center Drive, A220C MSRB III, Ann Arbor, MI 48109-0632 (E-mail address:).
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