J. PHILIP McCOY, PhD, KENT J. JOHNSON, MD, and. JAMES VARANI, PhD ... berg and Michael.2" The kidneys from 40 rats were collected, and the capsules ...
Protein Degradation Following Hydrogen Peroxide
SUZANNE E. G. FLIGIEL, MD, EUISUNG C. LEE, J. PHILIP McCOY, PhD, KENT J. JOHNSON, MD, and JAMES VARANI, PhD
Treatment With
From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
Pretreatment of hemoglobin with 50-5000 nmol hydrogen peroxide (H202) increased its susceptibility to proteolysis by a number of purified enzymes, including trypsin, chymotrypsin, elastase, and plasmin, and by the neutral protease of rat peritoneal leukocytes. Pretreatment of the protein substrate with catalaseinactivated H202 had no effect. Separation of the proteolytic fragments by G-75 Sephadex gel filtration indicated no apparent differences in the size distribution of the fragments produced by treatment with the H202/proteolytic enzyme combination as compared with enzyme treatment alone. A partially purified preparation of rat glomerular basement membrane was
also treated with proteolytic enzyme alone or in combination with H202. As with the hemoglobin, pretreatment of the glomerular basement membrane with H202 increased its susceptibility to subsequent proteolytic attack. In addition, treatment of a basement membrane glycoprotein, fibronectin, with H202 also increased its sensitivity to subsequent proteolysis. These results suggest that in addition to their other proinflammatory activities, oxygen-derived metabolites may contribute to tissue destruction by altering the susceptibility of proteins to hydrolytic enzymes. (Am J Pathol 1984, 115:418-425)
SEVERAL RECENT studies have shown the ability of oxygen metabolites to produce cell and tissue injury."1-1 How oxygen-mediated injury occurs is not known with certainty, and several potential mechanisms exist. It has been shown, for example, that oxygen free radicals can participate in the generation of a potent chemotactic factor in plasma.12 It has also been shown that oxygen metabolites can inactivate the major proteinase inhibitor of plasma."-3"4 Both of these effects could be expected to potentiate the tissue destructive activities of the inflammatory systems. Other studies have suggested that various species of oxygen free radicals can have a direct, destructive effect on tissue components, including collagen, proteoglycans, and glycosaminoglycans (hyaluronic acid).15-19 In this report we describe an additional (potential) role for oxygen metabolites. We describe studies which suggest that, under control conditions at least, pretreatment of proteins with H202 can increase their susceptibility to degradation by proteolytic enzymes.
crystallized from bovine pancreas; Sigma Chemical Company, St. Louis, Mo), chymotrypsin (Type II, three times crystallized from bovine pancreas; Sigma), elastase (Type I, two times crystallized from hog pancreas; Sigma), and plasmin (from porcine blood; Sigma). In addition to these enzymes, whole cell extracts of glycogen-elicited, rat peritoneal neutrophils were used as an additional enzyme source. The procedures used to obtain the rat peritoneal cells and prepare the extract as well as a description of the enzyme(s) characteristics were described in a recent report.20 H202 was obtained as a 3007 solution and catalase was obtained as a purified powder from bovine liver with 'v15,000 units of activity per milligram of protein. Both were from Sigma Chemical Company. Substrates for proteolysis were acid-denatured bovine hemoglobin, fibronectin, and a partially purified preparation of rat glomerular basement membrane. The bovine hemoglobin and human plasma fibronectin were obtained from Sigma Chemical
Materials and Methods
Supported in part by NHLBI Clinical Investigator Award 1 K08 HL00889. Accepted for publication January 16, 1984. Address reprint requests to James Varani, PhD, Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI 48109.
Reagents Four purified proteolytic enzymes were used in this study. They include trypsin (Type III, two times
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HYDRC)GEN PEROXDE AND PROIEN DEGRADATION
Company. The glomerular basement membrane was prepared in our laboratory as described below.
Glomerular Basement Membrne Preparation Rat glomerular basement membranes were isolated by the use of a modification of the procedure of Westberg and Michael.2" The kidneys from 40 rats were collected, and the capsules were removed. After midsagittal sectioning, the renal cortex was dissected away from the medulla. These cortical sections were then minced with a razor blade and gently pushed through a number 80 copper mesh (Fisher Scientific Company, Detroit, Mich). The tissue was then suspended in sterile saline and washed once more through a number 80 mesh followed by a number 270 mesh. The intact glomeruli were retained on this mesh, and they were gently removed by the use of a rubber policeman and saline washes. The glomeruli were examined at this point by light microscopy and were found to be mostly free of tubules and fibrous tissue. For the preparation of the isolated glomerular basement membranes the glomeruli were sonicated five times by the use of a model W-225R Sonicator (Heat Systems-Ultrasonics, Inc., Plainview, NY) at 30 seconds each at a setting of 2.5. The preparation was then centrifuged twice at 120g for 10 minutes, and the resulting pellet was checked for purity by the use of electron microscopy as shown in Figure 1. Measurement of H202 Quantitation of the H202 used in these experiments was done by the technique of Thurman et al,22 which measures the formation of a ferrithiocyanate complex. Measurement of Protease Activity and Protocol for Determining Effects of Substrate Pretreatment With H202 The hemoglobin and rat glomerular basement membrane were available in quantities of up to several milligrams. Therefore, we used a direct procedure to measure proteolytic breakdown of these substrates. The procedure was a modification of that originally described by Anson.' For this assay, each substrate was prepared as a 1 mg/ml solution in 0.05 M sodium phosphate buffer (pH 7.2), and 0.5 ml of substrate was mixed with varying concentrations of each protease (in 0.2 ml) to be tested. After incubation at 37 C for varying lengths of time, the reaction was terminated and the protein precipitated by the addition of 0.250 ml of 407o trichloroacetic acid (TCA). The
419
precipitate was allowed to settle for 10 minutes and then pelleted by centrifugation for 20 minutes at 2000 RPM. The supernatant was carefully aspirated. The amount of TCA-soluble fragments present in the supernatant was determined by the use of the Folin method.24 Bovine serum albumin was used as the standard with each assay. Although this assay procedure is not as sensitive as newer procedures which make use of radiolabeled or chromogenic substrates, the products measured are the solubilized protein fragments rather than an indirect tag such as a radioactive marker which is presumed to reflect the amount of protein fragments present. While this distinction may not be important when a purified proteolytic enzyme is examined in isolation, it becomes critical when a combination of reactants, including strong oxidizing agents and proteases, are examined together. To determine whether pretreatment of protein substrates with H202 could alter their susceptibility to subsequent proteolysis, we used the following protocol. The protein substrate was incubated for 1 hour with 50-5000 nmol of H202 in 0.1 ml. The controls consisted of the substrate incubated with the buffer alone or with inactivated H202. Inactivation of the H202 was accomplished by incubating the H202 with 20 pg of catalase (in 0.1 ml) for 15 minutes at 37 C. Preliminary studies indicated that this amount of catalase was 10 times greater than the amount needed to inactivate essentially all of the H202 used in the experiments. After incubation of the protein substrate with the H202, the reaction tubes in which active H202 was used were then treated with 20 pg of catalase for 15 minutes at 37 C to inactivate the residual H202 activity. Following this, all of the treated and control reaction tubes were mixed with varying concentrations of the proteases (in 0.2 ml) and incubated at 37 C for up to 2 hours. At the end of the incubation period, the reactions were terminated by the addition of 407o TCA. The reaction tubes were then handled as described above for the estimation of TCA-soluble protein fragments. We subjected native hemoglobin and trypsintreated hemoglobin to molecular sieving gel filtration to determine whether the different treatment protocols affected the size distribution of the protein fragments produced. The preparation of the hemoglobin and the treatments of the hemoglobin were conducted as described above. The column consisted of Sephadex G-75 fine (Pharmacia Fine Chemicals) prepared according to manufacturer's instructions and poured into a BioRad Standard Econo-column (0.7 cm x 30 cm). The running buffer consisted of 0.01 M phosphate-buffered saline, pH 7.2. Routinely, 0.3 ml of sample was added to the column, and 13-drop frac-
420
FLIGIEL ET AL
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Figure 1 -Electron micrograph of the partially purified rat glomerular basement membrane. (x 6000)
tions were collected. To calibrate the column we used blue dextran (mol wt >> 80,000), a-chymotrypsin (mol wt 25,100), soybean trypsin inhibitor (mol wt 22,700) ribonuclease A (mol wt 12,700), and phenol red (mol wt 354). With the purified basement membrane component fibronectin, we used a biologic assay to measure its activity and determine the effects of various treatment protocols on this molecule. The biologic assay used is based on the ability of this glycoprotein to facilitate cell attachment in the absence of serum. A line of tumor cells which is routinely maintained in our lab-
oratory was used as the indicator cells for this assay. The cells were maintained in culture under standard conditions as described previously.25 For assay, they were harvested by trypsinization, washed four times in serum-free RPMI-1640 medium, and plated in plastic culture dishes at 2.5 x 105 cells per dish. The culture medium consisted of 2 ml of RPMI-1640 medipm containing 200,ug of bovine serum albumin. Without additional supplements, the cells rapidly adhere to the dish. However, they are unable to spread and begin to detach after 2-4 hours. By 24 hours, only a very small percentage of cells remain attached.
421
HYDROGEN PEROXIDE AND PROTEIN DEGRADATION
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Table 1 - H202 Enhancement of Hemoglobin Degradation* Hemoglobin fragments produced (ug)t H202 (± SEM) Pretreatment Enzyme 1 ± 1 Nonet None
c
I.. 0
o
Ea
;jg trypsin
10
0
U.
0
10
20
30
40
SO
60
1 8g trypsin
TIME (minutes)
Figure 2-Time course of trypsin-mediated degradation of untreated hemoglobin and H202-treated hemoglobin. The protocol used to assess the H202-protease interaction was described in Materials and Methods. In each reaction, the hemoglobin was treated for 1 hour with the H202. The substrate was then incubated with 10 mg trypsin for varying periods of time (-0, no H202 and no trypsin; *--, no H202 and 10 pg of trypsin; 0-0, 50 nmol H202 and 10 mg trypsin; [:-E, 500 nmol H202 and 10 mg trypsin; A-A, 5000 nmol H202 and 10 ig trypsin).
10 Ag chymotrypsin
1
pg chymotrypsin
1
Wg
pg
elastase
elastase
100 ;g plasmin
measure the effects of protease treatment and H202/
protease treatments on this molecule, the following protocol was carried out. The fibronectin was added to the culture dishes and exposed to varying concen-
trations of the H202 solution (or buffer alone) in 0.1 ml for 2 hours at 37 C. Following this, the residual H202 was inactivated by the addition of 20 .g of catalase. The protein substrate was then treated with the proteolytic enzymes for 2 hours at 37 C. The desired amount of each protease was added directly to the dishes in a volume of 0.1 ml. Residual protease activity was eliminated by the addition of excess soybean or lima bean trypsin inhibitor at the termination of the incubation. The cells were then added to the dishes and incubated for 18 hours at 37 C and 5%o CO2. Following this, the nonattached cells were removed and discarded. The dishes were washed one time, and the attached cells were removed by trypsinization. The cells were counted with an electronic particle counter, and the percentage of adhering cells was determined. Results Potentiation of Hemoglobin Degradation by H202
In the initial studies we examined the ability of four different proteolytic enzymes to degrade the acid-
500S0nmol 500nmol 50 nmol None 5000 nmol 500 nmol 50 nmol None
5000nmol 500nmol 50 nmol
10
If fibronectin is added to the dishes, the cells rapidly attach and spread. After 24 hours, virtually all of the cells that have attached remain adherent and spread. Dose-response curves for fibronectin indicate detectable activity as low as 2 ig per dish and maximal attachment at concentrations of 10-20 pg per dish. For our experiments we chose to use 10 pg per dish. To
5000 nmol 500 nmol 50 nmol None
10
;g
plasmin
Leukocyte
extract§
(1:4) Leukocyte extract (1: 10)
None 5000 nmol 500nmol 50 nmol None 5000 nmol 500nmol 50 nmol None 5OO0 nmol 500 nmol 50 nmol None 5O0 nmol 500nmol 50 nmol None 5000 nmol 500nmol 50 nmol None 5000 nmol 500nmol 50nmol None 5000 nmol 500nmol 50nmol
1±1 0±1 1 0 36 ± 2
60±2 42±2 43 3 20 ± 2 40 ± 3 38 ± 1 30 ± 2 48± 15
42±5 17 ± 2 26 8
20±6 59 ± 8 100 ± 10 78± 11 26 ± 2 52 4 34 8 25 ± 1 43 2
25±3 35
8
8 ± 1 11 1
8±1 23 ± 2 24 ± 4
36±8
35±6
7 ± 1 12 ± 2
6±2 13±2 The protocol used for assessment of protease and H202lprotease
effects was decribed in Materials and Methods. t The values are the averages of three independent experiments. The values represent the amount of Folin-reactive material present in the supernatants after TCA precipitation. They were obtained by direct comparison with a bovine serum albumin standard curve. t The values shown under "none" are averages of four different groups (ie, buffer alone and three different concentrations of catalase-inactivated H202). There were no significant differences among these four groups. § The undiluted leukocyte extract was prepared to contain approximately 10 mg protein/ml, and the amounts used per reaction were 100-250 9g.
denatured hemoglobin either before or after treatment of the substrate with H202. These results are shown in Figure 2 and Table 1. In Figure 2, the time course for trypsin-mediated hydrolysis can be seen. For these studies, a single concentration of tryp-
422
FLIGIEL ET AL
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A
14
0
la
5 10 15 20 25 30 35 40
0
B 0
20[ 15
0 0
10
0
I-
5
'a
A
I0
5 10 15 20 25 30 35 4C
C 20 15
10 5
XI . 0
.
.i
5 10 15 20 25 303540
Fraction Number Figure 3-Sephadex G-75 gel filtration chromatography of intact hemoglobin, trypsin-treated hemoglobin, and H202/trypsin-treated hemoglobin. For these experiments hemoglobin was prepared at a concentration of 2 mg/ml in 0.01 M phosphate-buffered saline. Intact hemoglobin is shown in the top panel: 0.5 ml of the hemoglobin was incubated with 0.03 ml of buffer, and 0.3 ml of this mixture was applied to the column. Trypsin-treated hemoglobin is shown in the middle panel: 0.5 ml of the hemoglobin with incubated for 2 hours at 37 C with 0.03 ml trypsin containing 10 l*g protein. After incubation, 0.3 ml was applied to the column. In the bottom panel is shown the H202/trypsin-treated hemoglobin: 0.5 ml of the hemoglobin solution was incubated for 1 hour at 37 C with H202 (5000 nmol in 0.01 ml), for 15 minutes with 2 jig catalase in 0.01 ml, and for 2 hours at 37 C with 0.01 ml trypsin containing 10 mg protein. After incubation, 0.3 ml was applied to the column. The panels present the results of a single experiment. The experiment was run two times with nearly
identical results. Markers used to calibrate the column included blue dextran (mol wt >80,000); a-chymotrypsin (mol wt 25,100); soybean trypsin inhibitor (mol wt 22,700); ribonuclease A (mol wt 12,700); and phenol red (mol wt 354). The peak of blue dextran eluted in Fraction 11, the a-chymotrypsin and soybean trypsin inhibitor eluted in Fraction 17, the ribonuclease A eluted in Fraction 19, and the phenol red eluted in Fraction 31.
sin (10 jg/reaction) was used. At all times examined, there was more breakdown of the H202-treated hemoglobin than the untreated substrate. Table 1 shows the results obtained at a single time point with varying concentrations of the H202 and with varying concentrations of the different enzymes. It can be seen from this table that there was no significant production of acid-soluble protein fragments by H202 treatment alone (ie, in the absence of subsequent protease treatment) but that H202 treatment increased the suscepti-
bility of the hemoglobin substrate to hydrolysis by each of the four purified proteolytic enzymes. H202 concentrations ranging from 50 to 5000 nmol per reaction were effective. In addition to facilitating hydrolysis by the purified enzymes, H202 pretreatment also enhanced hydrolysis by the leukocyte extract (Table 1). Although the overall effects were less dramatic with the whole cell extract than with the purified enzymes, this may be due to the large amount of contaminating protein in the whole cell extracts. The data shown in Figure 2 and Table 1 were obtained by measuring the total amount of protein fragments remaining in solution after TCA precipitation. In order to obtain further information regarding the size distribution of the fragments produced under the various conditions used, samples of untreated hemoglobin, trypsin-treated hemoglobin, and H202/trypsin-treated hemoglobin were separated on a molecular sieving column. The results shown in Figure 3 indicate no significant difference in the molecular weight distribution patterns of the fragments produced by trypsin treatment alone and by H202/trypsin treatment. Based on the molecular weight standards, it appears that the molecular weights of the major proteolytic fragments are between 400 and 12,000 daltons. Although there were no detectable differences in the size distribution, the amount of protein recovered in the low molecular weight peak was increased after H202/trypsin treatment relative to treatment with trypsin alone. Potentiation of Basement Membrane Degradation by H202 Table 2 shows that H202 enhancement of proteolysis is not limited to hemoglobin. A partially purified glomerular basement membrane preparation was used in the studies shown here. It can be seen that treatment of the substrate with 500-5000 nmol H202 increased its suceptibility to hydrolysis by trypsin. Very interestingly, we observed some increase in the formation of TCA-soluble, Folin-reactive fragments in the glomerular basement membrane treated with high concentrations of H202 alone. The significance of this is not known. It should be noted that Greenwald et al"6 reported a direct solubilization of collagen in the presence of xanthine oxidase/ hypoxanthine. Effects of H202 Treatment on the Functional Activity of Fibronectin We next examined the effects of protease treatment and H202/protease treatment on the ability of fibro-
HYDROGEN PEROXIDE AND PROTEIN DEGRADATION
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Table 2- H202 Enhancement of Basement Membrane Degradation* Basement membrane fragments produced (pg) H202 (± SEM)t Pretreatment Enzyme None
10 jig trypsin 1
pg trypsin
Nonet 5000 nmol 500 nmol None 5000 nmol 500 nmol None 5000 nmol
2± 1 8±3 0 10 ± 2 34 ± 5 23 ± 4 8±2 36 ± 7
* The protocol used to assess protease and H202iprotease effects was decribed in Materials and Methods. t The values are the averages of three or four independent experiments. The values represent the amount of Folin-reactive material present in the supematant after TCA precipitation. They were obtained by direct comparison with a bovine serum albumin standard curve. t The values shown under "none' are averages of values obtained in the presence of buffer alone and the values obtained in the presence of catalase-inactivated H202.
nectin to serve as cell attachment factor. For these studies we used 10 *g of fibronectin per reaction and carried out the degradation protocol and cell attachment assay as described in the Materials and Methods section. Ten micrograms was chosen after preliminary dose-response studies showed that this amount was the lowest dose that induced maximal responses with our indicator cells. Fibronectin proved to be very susceptible to proteolytic enzymes (Table 3), and proteolytic treatment prevented fibronectin from serving as a cell attachment factor. H202 treatment of fibronectin increased its subsequent sensitivity to the proteases. This was most dramatic when the fibronectin was treated with the H202 and then subsequently treated with chymotrypsin (Table 3).
that other tissue components, including cartilage proteoglycan and collagen, were also affected by the oxygen metabolites."5-" The molecular basis for these effects was not established, although it was shown that smaller disaccharide units were produced from the hyaluronatel' and that the radiolabel was released into solution fromr prelabeled collagen.16 It was also shown in these studies that while the intact hyaluronate was resistant to carbohydrases, the oxidized product could be further broken down by enzymes, including P-glucuronidase and (3-N-acetylglucosaminidase.1l Thus, not only was there a direct effect of the oxygen free radicals on the substrate, but, in addition, there appeared to be a synergistic interaction between the oxygen metabolites and the hydrolases. In this report we provide evidence suggesting that oxygen metabolites (in this case, H202) can act with various proteolytic enzymes to facilitate the degradation of proteins. Several proteases were used in this study, including trypsin, chymotrypsin, elastase, and plasmin, as well as the proteases present in rat penitoneal leukocyte extracts. With each of these enzymes, hydrolysis of the protein substrate was in-
Table 3-H202 Enhancement of Fibronectin Degradation* % Tumor cells that adhered (±+ SEM)t H202 Pretreatment Enzyme 100 Nonet None 95 + 3 5000 nmol 98 + 4 500 nmol 98 + 5 50 nmol 10
pg
trypsin
0.1 pg chymotrypsin
Discussion Oxygen free radicals either generated directly by enzyme substrate reactions or released from activated leukocyttes are capable of causing the death of several kinds of cells" in vitro and are thought to contribute to the tissue injury characteristic of inflammatory conditions.' Recent studies have suggested a number of potentially important, proinflammatory mechanisms to account for these effects,""12 although some studies have suggested an antiinflammatory role as well.26"2, The idea that oxygen metabolites have a direct tissue-destructive effect was first suggested by McCord,"8 who showed that a purified hyaluronate solution as well as bovine synovial fluid could be depolymerized by 0; generated from xanthine and xanthine oxidase. Additional studies confirmed these findings and showed
423
0.01 pig chymotrypsin
14 ± 2 10 ± 1 13 ± 2
None 5000 nmol 500 nmol 50 nmol None 5000 nmol 500 nmol 50 nmol
81 ± 3 58 ± 8 64 ± 6 68 ± 3
None 5000 nmol 500 nmol 50 nmol
99 ± 2 89 ± 1 91 ± 3 88 + 1
14 ± 2
* The protocol used to assess protease and H202lprotease effects was decribed in Materials and Methods. We used the cell attachment assay to determine the biologic activity of the fibronectin. t The values are the averages of three independent experiments. In each experiment, approximately 20% of the cells attached to the dishes in the absence of fibronectin. In the presence of 10 Ag of fibronectin (the amount used per dish in each experiment), approximately 90% of the cells attached. The percentage of cells which attached in the presence of untreated fibronectin (ie. in the absence of H202 and proteolytic enzymes but with catalase and the protease inhibitors present) was arbitrarily assigned a value of 100%. The other values were then calculated based on this. t The values shown under -none" are average of four different treatment groups (ie, buffer alone and three different concentrations of catalase-inactivated H202). There were no significant differences among these four groups.
424
FLIGIEL ET AL
creased by pretreatment of the substrate with H202. Although the H202 was provided from an artificial source, the amounts used (as little as 50 nmol/reaction) were small enough to be present in vivo at sites of inflammation.28-32 H202 is one of the central oxygen metabolites produced by stimulated leukocytes. In addition to playing a direct role in tissue destruction, it is also a key intermediate in the generation of other species of oxygen free radicals such as hydroxyl radical (OH-).30 Most of our studies were carried out with hemoglobin as the substrate. However, the results obtained in this study were not limited to proteins containing H202-susceptible prosthetic groups such as heme. Proteolysis of a partially purified glomerular basement membrane preparation was also facilitated by pretreatment of the substrate with H202. In addition, a basement membrane glycoprotein, fibronectin, was also found to be susceptible to H202 treatment. When this glycoprotein was sequentially treated with H202 and proteolytic enzymes, its ability to function as a cell attachment factor was inhibited. Treatment of fibronectin with the proteolytic enzymes alone also reduced its ability to support cell attachment but not to the same extent as when the H202 and proteases were both used. The mechanism(s) by which H202 treatment of the protein substrates facilitates their subsequent hydrolysis is not known. It may be that in the case of hemoglobin, H202 combines with the heme and in doing so alters the exposure of the globin molecule to the proteases. Likewise, with the glomerular basement membrane and with fibronectin, combination of the oxygen metabolite with H202-sensitive portions of the molecules may alter the protein structure in such a way as to further expose protease-sensitive regions of the molecules. Additional studies will have to be done to determine whether this is, in fact, correct. Additional studies will also have to be done before the in vivo significance of these results can be determined. However, the finding that components of basement membrane are made increasingly susceptible to proteolytic attack following treatment with H202 (in amounts available at sites of inflammation) may provide an additional explanation for the role of oxygen metabolites in tissue injury.
References 1. Sachs T, Moldow CF, Craddock PR, Bowers JK, Jacob HS: Oxygen radical mediated endothelial cell damage by complement-stimulated granulocytes: An in vitro
AJP * June 1984
model of immune vascular damage. J Clin Invest 1978, 61:1161-1167 2. Simon RH, Scoggin CH, Patterson D: Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals. J Biol Chem 1981, 256:7181-7186 3. Clark RA, Klebanoff SJ, Einstein AB, Fefer A: Peroxidase-H202-halide system: Cytotoxic effect on mammalian tumor cells. Blood 1975, 45:161-170 4. Baehner RL, Boxer LA, Allen JM, Davis J: Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood 1977, 50:327-335 5. Clark RA, Klebanoff SJ: Myeloperoxidase-mediated platelet release reaction. J Clin Invest 1979, 63:177-183 6. Smith DC, Klebanoff SJ: A uterine fluid-mediated sperm-inhibitory system. Biol Reprod 1970, 3:229-235 7. Johnson KJ, Fantone JC, Kaplan J, Ward PA: In vivo damage of rat lungs by oxygen metabolites. J Clin Invest 1981, 67:983-993 8. Johnson KJ, Ward PA: Role of oxygen metabolites in immune complex injury of lung. J Immunol 1981, 126: 2365-2369 9. Oyanagui Y: Participation of superoxide anions at the prostaglandins phase of carrageenin foot edema. Biochem Pharmacol 1976, 25:1465-1472 10. Marten WJ, Gadek JE, Hunninghake GW, Crystal RG: Oxidant injury of lung parenchymal cells. J Clin Invest 1981, 68:1277-1288 11. McCormick JR, Harkin MM, Johnson KJ, Ward PA: The effect of superoxide dismutase on pulmonary and dermal inflammation. Am J Pathol 1981, 102:55-61 12. Petrone WF, English DK, Wong K, McCord JM: Free radicals and inflammation: The superoxide dependent activation of a neutrophil chemotactic factor in plasma. Proc Nat Acad Sci USA 1980, 77:1159-1163 13. Corp H, Janoff A: In vitro suppression of serum elastase-inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes. J Clin Invest 1979, 63:793-797 14. Matheson NR, Wong PS, Travis J: Enzymatic inactivation of human alpha-l-proteinase inhibitor by neutrophil myeloperoxidase. Biochem Biophys Res Commun 1979, 88:402-409 15. Greenwald RA, Moy WW: Inhibition of collagen gelation by action of the superoxide radical. Arthritis Rheum 1979, 22:251-259 16. Greenwald RA, Moy WW, Lazarus D: Degradation of cartilage proteoglycans and collagen by superoxide radical (Abstr). Arthritis Rheum 1976, 19:799 17. Greenwald RA, Moy WW: Effect of oxygen-derived free radicals on hyaluronic acid. Arthritis Rheum 1980, 23:455-463 18. McCord JM: Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science 1974, 185:529-531 19. Puig-Parellada P, Planas JM: Synovial fluid degradation induced by free radicals. In vitro action of several free radical scavengers and anti-inflammatory drugs. Biochem Pharmacol 1978, 27:535-537 20. Varani J, Ward D, Johnson KJ: Nonspecific protease and elastase activities in rat leukocytes. Inflammation 1982, 6:177-187 21. Westberg NG, Michael AF: Human glomerular basement membrane. Preparation and composition. Biochemistry 1970, 9:3837-3846 22. Thurman RG, Ley HG, Scholz R: Hepatic microsomal ethanol oxidation: Hydrogen peroxide formation and the role of catalase. Euro J Biochem 1972, 25:420-430 23. Anson ML: The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 1940, 23:79-89
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24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-275 25. Varani J, Lovett EJ, Wicha M, Malinoff H, McCoy JP: Cell surface a-D-galactopyranosyl end group: Use as markers in the isolation of munne tumor cell lines with different cancer-causing potentials. J Nat Cancer Inst 1983, 71:1281-1286 26. Clark RA, Szot S, Venkatasubramanian K, Schiffman E: Chemotactic factor inactivation by myeloperoxidase-mediated oxidation of methionine. J Immunol 1980, 124:2020-2026 27. Henderson WR, J6rg A, Klebanoff SJ: Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4. J Immunol 1982, 128:2609-2613 28. Senn HJ, Jungi WF: Neutrophil Migration in Health and Disease, Neutrophil Physiology and Pathology.
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Edited by JR Humbert, PA Miescher, ER Jaffe. New York, Grune and Stratton, 1975, pp 25-43 Ward PA, Duque RE, Sulavik MC, Johnson KJ: In vitro and in vivo stimulation of rat neutrophils and alveolar macrophages by immune complexes: Production of 0 and H20A Am J Pathol 1983, 110:297-309 Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1982, 107:397-418 Dahinden CA, Fehr J, Hugli TE: Role of cell surface contact in the kinetics of superoxide production by granulocytes. J Clin Invest 1983, 72:113-121 Lehmeyer JE, Snyderman R, Johnston RB Jr: Stimulation of neutrophil oxidative metabolism by chemotactic peptides: Influence of calcium ion concentration and cytochalasin B and comparison with stimulation by phorbol myristate acetate. Blood 1979, 54:35-45
