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LIGAND-DEPENDENT RELEASE OF ACTIVE NEUTROPHIL COLLAGENASE W. WINN CHATHAM, LOUIS W. HECK, and WARREN D. BLACKBURN, JR. The amount of active neutrophil (PMN) collagenase released extracellularly is dependent on the PMNactivating ligand. Neutrophils stimulated with soluble ligands, including FMLP, platelet-activating factor, or heat-aggregated IgG, released very little active collagenase, in contrast to cells stimulated with opsonized zymosan or surface-bound IgG. However, opsonized zymosan and surface-bound IgG did not differ appreciably from soluble ligands in effecting PMN production of superoxide, release of the specific granule component lactoferrin, or total (latent plus active) collagenase release, which suggests that there is more efficient collagenase activation during PMN stimulation with surface-bound ligands. These results suggest a role for surface (cartilage)-bound IgG in the release and activation of human neutrophil collagenase in the joints of patients with rheumatoid arthritis.
The activation of human neutrophils (PMN) through ligand binding to membrane receptors is associated with the extracellular release of a variety of constituents that may lead to tissue injury at sites of inflammation. Among the lysosomal proteases reFrom the Division of Clinical Immunology and Rheumatology, Birmingham Veterans Administration Hospital and The University of Alabama at Birmingham. Supported by a Veterans Administration Research Associate Career Development award, a Veterans Administration Merit Review award, and NIH grant AR-20614. W. Winn Chatham, MD: Associate in Medicine; Louis W. Heck, MD: Associate Professor of Medicine; Warren D. Blackburn, Jr., MD: Assistant Professor of Medicine. Address reprint requests to W. Winn Chatham, MD, Research Service (15l-F), Birmingham Veterans Administration Hospital, 700 19th Street South, Birmingham, AL 35233. Submitted for publication May 4, 1989; accepted in revised form September 15, 1989. Arthritis and Rheumatism, Vol. 33, No. 2 (February 1990)
leased during PMN activation is human neutrophil collagenase (HNC), which is stored in the specific granule in a latent form (1). Once activated, H N C is capable of cleaving collagen types I, 11, and 111 (2), which are found in cartilage (type II), vascular tissue (type 111), and periarticular structures such as bone, ligaments, and tendons (type I). Collagenase activity has been detected in some rheumatoid synovial fluids (3), but the cellular origins of this activity have been difficult to determine. Fibroblasts and macrophages in the synovial pannus are likely sources of collagenase, but whether or how the collagenases secreted by these cells contribute to cartilage erosion is uncertain: Likewise, PMN are frequently seen in abundance in r k u matoid synovial fluid, but how HNC is activafed in vivo and its role in mediating cartilage destruction remain speculative. Activation of latent HNC has been demonstrated during PMN stimulation in vitro with phorbol myristate acetate (PMA) and zymosan, and appears to be dependent on the generation of hypochlorous acid (HOCI) from H,O, and chloride ions (4). Other neutrophilactivating ligands, including platelet-activating factor (PAF) (3,immune complexes (both soluble [6] and surface-bound [7]), and activated complement components (8), are of considerable relevance since they are readily detectable in the inflamed joints of patients with rheumatoid arthritis (9-11). PMN binding of these ligands triggers cellular activating pathways that appear to be bypassed by PMA, a direct activator of protein kinase C. As such, cellular responses required for the release of activated HNC may proceed differently during PMN stimulation in inflamed tissue than with PMA. To determine whether and to what extent the release and activation of HNC occurs during neutrophil stimulation by
NEUTROPHIL COLLAGENASE RELEASE
aggregates of IgG and surface-bound IgG.
MATERIALS AND METHODS Reagents and materials. Native type I collagen used in the reconstituted fibril assay was obtained from fresh rat tail tendons by solubilization in acetic acid and selective salt precipitation (12). A portion of the solubilized collagen was labeled with 3H-acetic anhydride (New England Nuclear, Boston, MA) (13), yielding a specific activity of 570,000 counts per minutehg collagen, for use in the collagenase assay described by Johnson-Wint (14). Soluble pepsintreated type I (from human placenta) and type I1 (from bovine cartilage) collagens were used to confirm the presence of specific collagenase activity by examination of cleavage products on sodium dodecyl sulfate gels (15). Ficoll-Hypaque for neutrophil isolation was obtained from Pharmacia (Piscataway, NJ). PMA, FMLP, PAF, L-methionine, sodium azide, femcytochrome c, salyrganic acid (mersalyl), DFP, and zymosan were obtained from Sigma (St. Louis, MO). Clostridial collagenase was obtained from Millipore (Freehold, NJ). IgG for use as a surfacebound PMN-activating ligand and for preparation of heataggregated IgG was prepared from Cohn fraction I1 as previously described (16,17). Serum-treated zymosan was prepared by incubating 5 mg zymosan with 1.0 ml pooled human sera from normal donors for 1 hour at 37°C; this was followed by 3 washes of the treated zymosan with Hanks' balanced salt solution (HBSS). Cell preparation. Venous blood from normal donors was drawn into heparinized syringes (20 units/ml). Following dextran sedimentation, the leukocyte-rich plasma was centrifuged, and the cell pellet was washed with phosphate buffered saline (PBS) containing 0.2 mM EDTA. After 2 washes, the cells were resuspended in PBS, layered onto Ficoll-Hypaque gradients, and centrifuged at 750g for 30 minutes. Following hypotonic lysis of residual red cells in the PMN pellet, the neutrophils were resuspended in HBSS to a concentration of 5 x 106 PMN/ml. Differential counts on the cell suspensions consistently revealed >97% PMN. Viability as assessed by trypan blue exclusion was consistently greater than 95%. Activation of neutrophils. For activation of PMN with PMA, FMLP, PAF, heat-aggregated IgG, and zymosan (serum-treated and untreated), PMN suspended in HBSS (5 x 10" cells/ml) were placed into polypropylene tubes (Sarstedt, Newton, NC) precoated with 0.1% bovine serum albumin (BSA). The respective ligands were added and dispersed into the suspended cells by gentle agitation; this was followed by incubation of the mixtures for 45 minutes at 37°C. For activation of PMN with surface-bound IgC, roundbottom microtiter wells (Dynatech, Alexandria, VA) were incubated overnight with 100 pl/well of 7 s IgG at a concentration of 100 tLglml (10 pg/well). The wells were then washed with PBS and blocked with 0.1% BSA in HBSS. Cell
229
microtiter wells containing no IgG. Following incubation, the cell mixtures activated with each ligand were centrifuged (750g) for 5 minutes at 4°C. The supernatants were removed and treated with DFP (final concentration IOP3M) to inactivate serine proteases, and were then evaluated for granule constituents and collagenase activity as described below. Measurement of superoxide generation. In parallel experiments, superoxide production was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c (18). Determination of collagenase activity. Collagenase activity in the DFP-treated supernatants was determined by incubating, in triplicate, 200-pl aliquots of supernatant with 'H-labeled, reconstituted type I collagen fibrils in 7-mm flat-bottom tissue culture wells (Linbro; Flow Laboratories, McLean, VA). The reconstituted fibril in each well contained 75 pg of a mixture of 3H-labeled and unlabeled collagen with an activity of 10,OOO cpm. To determine the total radioactivity potentially released from the fibrils in each experiment, the reconstituted fibrils were also incubated with a mixture of clostridial collagenase (250 pg/ml HBSS). To maximize the sensitivity and specificity of the assay, incubations were done in triplicate for 18 hours at 37°C (see Results). At the end of the incubation period, the supernatants were aspirated from each well, and the radioactivity was determined by counting in a liquid scintillation counter. Greater than 99% of the radioactivity applied to the well was recovered when wells were incubated with bacterial collagenase. The mean cpm released by fibrils incubated with buffer (HBSS) alone was subtracted from the cpm measured in each supernatant. The resulting triplicate values for each PMN supernatant were averaged and divided by the mean cpm released by the bacterial collagenase to determine the percent fibril lysis produced by each PMN supernatant. The total collagen released during the 18-hour incubation was then calculated and divided by the incubation time to yield values for the collagenase activity (ng degradedhinute) in each supernatant (HNC-ACT). In parallel experiments, release of total collagenase (HNC-TOT) into the PMN supernatants was determined by activating latent collagenase in the supernatants with 1.0 mM mersalyl (15) prior to the addition of the supernatants to the radiolabeled collagen fibrils. To avoid underestimation of HNC-TOT due to inhibition of protease activity by oxidative metabolites generated during PMN activation, the supernatants used for these determinations were derived from PMN activated in the presence of 1.0 mM sodium azide (an inhibitor of myeloperoxidase). Incubations and calculations of collagenase activity in the mersalyl-treated supernatants were done as described above. &granulation. Lactoferrin (a specific granule constituent) was measured in the cell-free supernatants by radioimmunoassay , as previously described (17).
CHATHAM ET AL
230
Statistical analysis. Significant differences in PMN responses to the different activating ligands were determined using the t-distribution.
251
I
T
20
RESULTS Collagenase assay for HNC activity. As demonstrated in Figure 1, incubation of the PMN supernatants with the 3H-labeled, reconstituted fibrils for 18 hours maximized the sensitivity of the assay for collagenase activity. Limited nonspecific proteolysis occurred during incubation with trypsin, but virtually all of the proteolytic activity in the DFP-treated PMN supernatant was mediated through a metalloprotease, as demonstrated by the near complete inhibition of fibril lysis with the addition of EDTA. Furthermore, specific collagenase activity in the PMN supernatants was confirmed by demonstration of the characteristic %-Y4 cleavage of the a-chains by incubation of the PMN supernatants with pepsin-treated type I and type I1 collagen. The extent of the specific a-chain cleavage noted on Coomassie blue-stained sodium dodecyl sulfate gels correlated with the increased release of radioactivity in the radioassay employed (data not shown).
5
n
z x i 0
4
3
2
3
6 9 12 Hour8 Incubation 37OC
15
18
Figure 1. Lysis of 3H-labeled reconstituted type I collagen fibrils by neutrophil (PMN) supernatants. PMN (5 x 10"/ml Hanks' balanced salt solution [HBSS]) were incubated with phorbol myristate acetate (500 ng/ml) for 45 minutes. Cell-free supernatants were collected, and I .0 mM DFP and I .0 m M mersalyl were added. The supernatants were not treated (0)or were treated with 1.0 mM EDTA (A) followed by incubation with radiolabeled collagen fibrils for various times, as described in Materials and Methods. W = radioactivity released by fibrils into HBSS alone; 0 = lysis of fibril by 0.005% trypsin in HBSS. Each point represents the mean of 3 observations. The standard deviation of each point was
LIGAND-DEPENDENT RELEASE OF ACTIVE NEUTROPHIL COLLAGENASE W. WINN CHATHAM, LOUIS W. HECK, and WARREN D. BLACKBURN, JR. The amount of active neutrophil (PMN) collagenase released extracellularly is dependent on the PMNactivating ligand. Neutrophils stimulated with soluble ligands, including FMLP, platelet-activating factor, or heat-aggregated IgG, released very little active collagenase, in contrast to cells stimulated with opsonized zymosan or surface-bound IgG. However, opsonized zymosan and surface-bound IgG did not differ appreciably from soluble ligands in effecting PMN production of superoxide, release of the specific granule component lactoferrin, or total (latent plus active) collagenase release, which suggests that there is more efficient collagenase activation during PMN stimulation with surface-bound ligands. These results suggest a role for surface (cartilage)-bound IgG in the release and activation of human neutrophil collagenase in the joints of patients with rheumatoid arthritis.
The activation of human neutrophils (PMN) through ligand binding to membrane receptors is associated with the extracellular release of a variety of constituents that may lead to tissue injury at sites of inflammation. Among the lysosomal proteases reFrom the Division of Clinical Immunology and Rheumatology, Birmingham Veterans Administration Hospital and The University of Alabama at Birmingham. Supported by a Veterans Administration Research Associate Career Development award, a Veterans Administration Merit Review award, and NIH grant AR-20614. W. Winn Chatham, MD: Associate in Medicine; Louis W. Heck, MD: Associate Professor of Medicine; Warren D. Blackburn, Jr., MD: Assistant Professor of Medicine. Address reprint requests to W. Winn Chatham, MD, Research Service (15l-F), Birmingham Veterans Administration Hospital, 700 19th Street South, Birmingham, AL 35233. Submitted for publication May 4, 1989; accepted in revised form September 15, 1989. Arthritis and Rheumatism, Vol. 33, No. 2 (February 1990)
leased during PMN activation is human neutrophil collagenase (HNC), which is stored in the specific granule in a latent form (1). Once activated, H N C is capable of cleaving collagen types I, 11, and 111 (2), which are found in cartilage (type II), vascular tissue (type 111), and periarticular structures such as bone, ligaments, and tendons (type I). Collagenase activity has been detected in some rheumatoid synovial fluids (3), but the cellular origins of this activity have been difficult to determine. Fibroblasts and macrophages in the synovial pannus are likely sources of collagenase, but whether or how the collagenases secreted by these cells contribute to cartilage erosion is uncertain: Likewise, PMN are frequently seen in abundance in r k u matoid synovial fluid, but how HNC is activafed in vivo and its role in mediating cartilage destruction remain speculative. Activation of latent HNC has been demonstrated during PMN stimulation in vitro with phorbol myristate acetate (PMA) and zymosan, and appears to be dependent on the generation of hypochlorous acid (HOCI) from H,O, and chloride ions (4). Other neutrophilactivating ligands, including platelet-activating factor (PAF) (3,immune complexes (both soluble [6] and surface-bound [7]), and activated complement components (8), are of considerable relevance since they are readily detectable in the inflamed joints of patients with rheumatoid arthritis (9-11). PMN binding of these ligands triggers cellular activating pathways that appear to be bypassed by PMA, a direct activator of protein kinase C. As such, cellular responses required for the release of activated HNC may proceed differently during PMN stimulation in inflamed tissue than with PMA. To determine whether and to what extent the release and activation of HNC occurs during neutrophil stimulation by
NEUTROPHIL COLLAGENASE RELEASE
aggregates of IgG and surface-bound IgG.
MATERIALS AND METHODS Reagents and materials. Native type I collagen used in the reconstituted fibril assay was obtained from fresh rat tail tendons by solubilization in acetic acid and selective salt precipitation (12). A portion of the solubilized collagen was labeled with 3H-acetic anhydride (New England Nuclear, Boston, MA) (13), yielding a specific activity of 570,000 counts per minutehg collagen, for use in the collagenase assay described by Johnson-Wint (14). Soluble pepsintreated type I (from human placenta) and type I1 (from bovine cartilage) collagens were used to confirm the presence of specific collagenase activity by examination of cleavage products on sodium dodecyl sulfate gels (15). Ficoll-Hypaque for neutrophil isolation was obtained from Pharmacia (Piscataway, NJ). PMA, FMLP, PAF, L-methionine, sodium azide, femcytochrome c, salyrganic acid (mersalyl), DFP, and zymosan were obtained from Sigma (St. Louis, MO). Clostridial collagenase was obtained from Millipore (Freehold, NJ). IgG for use as a surfacebound PMN-activating ligand and for preparation of heataggregated IgG was prepared from Cohn fraction I1 as previously described (16,17). Serum-treated zymosan was prepared by incubating 5 mg zymosan with 1.0 ml pooled human sera from normal donors for 1 hour at 37°C; this was followed by 3 washes of the treated zymosan with Hanks' balanced salt solution (HBSS). Cell preparation. Venous blood from normal donors was drawn into heparinized syringes (20 units/ml). Following dextran sedimentation, the leukocyte-rich plasma was centrifuged, and the cell pellet was washed with phosphate buffered saline (PBS) containing 0.2 mM EDTA. After 2 washes, the cells were resuspended in PBS, layered onto Ficoll-Hypaque gradients, and centrifuged at 750g for 30 minutes. Following hypotonic lysis of residual red cells in the PMN pellet, the neutrophils were resuspended in HBSS to a concentration of 5 x 106 PMN/ml. Differential counts on the cell suspensions consistently revealed >97% PMN. Viability as assessed by trypan blue exclusion was consistently greater than 95%. Activation of neutrophils. For activation of PMN with PMA, FMLP, PAF, heat-aggregated IgG, and zymosan (serum-treated and untreated), PMN suspended in HBSS (5 x 10" cells/ml) were placed into polypropylene tubes (Sarstedt, Newton, NC) precoated with 0.1% bovine serum albumin (BSA). The respective ligands were added and dispersed into the suspended cells by gentle agitation; this was followed by incubation of the mixtures for 45 minutes at 37°C. For activation of PMN with surface-bound IgC, roundbottom microtiter wells (Dynatech, Alexandria, VA) were incubated overnight with 100 pl/well of 7 s IgG at a concentration of 100 tLglml (10 pg/well). The wells were then washed with PBS and blocked with 0.1% BSA in HBSS. Cell
229
microtiter wells containing no IgG. Following incubation, the cell mixtures activated with each ligand were centrifuged (750g) for 5 minutes at 4°C. The supernatants were removed and treated with DFP (final concentration IOP3M) to inactivate serine proteases, and were then evaluated for granule constituents and collagenase activity as described below. Measurement of superoxide generation. In parallel experiments, superoxide production was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c (18). Determination of collagenase activity. Collagenase activity in the DFP-treated supernatants was determined by incubating, in triplicate, 200-pl aliquots of supernatant with 'H-labeled, reconstituted type I collagen fibrils in 7-mm flat-bottom tissue culture wells (Linbro; Flow Laboratories, McLean, VA). The reconstituted fibril in each well contained 75 pg of a mixture of 3H-labeled and unlabeled collagen with an activity of 10,OOO cpm. To determine the total radioactivity potentially released from the fibrils in each experiment, the reconstituted fibrils were also incubated with a mixture of clostridial collagenase (250 pg/ml HBSS). To maximize the sensitivity and specificity of the assay, incubations were done in triplicate for 18 hours at 37°C (see Results). At the end of the incubation period, the supernatants were aspirated from each well, and the radioactivity was determined by counting in a liquid scintillation counter. Greater than 99% of the radioactivity applied to the well was recovered when wells were incubated with bacterial collagenase. The mean cpm released by fibrils incubated with buffer (HBSS) alone was subtracted from the cpm measured in each supernatant. The resulting triplicate values for each PMN supernatant were averaged and divided by the mean cpm released by the bacterial collagenase to determine the percent fibril lysis produced by each PMN supernatant. The total collagen released during the 18-hour incubation was then calculated and divided by the incubation time to yield values for the collagenase activity (ng degradedhinute) in each supernatant (HNC-ACT). In parallel experiments, release of total collagenase (HNC-TOT) into the PMN supernatants was determined by activating latent collagenase in the supernatants with 1.0 mM mersalyl (15) prior to the addition of the supernatants to the radiolabeled collagen fibrils. To avoid underestimation of HNC-TOT due to inhibition of protease activity by oxidative metabolites generated during PMN activation, the supernatants used for these determinations were derived from PMN activated in the presence of 1.0 mM sodium azide (an inhibitor of myeloperoxidase). Incubations and calculations of collagenase activity in the mersalyl-treated supernatants were done as described above. &granulation. Lactoferrin (a specific granule constituent) was measured in the cell-free supernatants by radioimmunoassay , as previously described (17).
CHATHAM ET AL
230
Statistical analysis. Significant differences in PMN responses to the different activating ligands were determined using the t-distribution.
251
I
T
20
RESULTS Collagenase assay for HNC activity. As demonstrated in Figure 1, incubation of the PMN supernatants with the 3H-labeled, reconstituted fibrils for 18 hours maximized the sensitivity of the assay for collagenase activity. Limited nonspecific proteolysis occurred during incubation with trypsin, but virtually all of the proteolytic activity in the DFP-treated PMN supernatant was mediated through a metalloprotease, as demonstrated by the near complete inhibition of fibril lysis with the addition of EDTA. Furthermore, specific collagenase activity in the PMN supernatants was confirmed by demonstration of the characteristic %-Y4 cleavage of the a-chains by incubation of the PMN supernatants with pepsin-treated type I and type I1 collagen. The extent of the specific a-chain cleavage noted on Coomassie blue-stained sodium dodecyl sulfate gels correlated with the increased release of radioactivity in the radioassay employed (data not shown).
5
n
z x i 0
4
3
2
3
6 9 12 Hour8 Incubation 37OC
15
18
Figure 1. Lysis of 3H-labeled reconstituted type I collagen fibrils by neutrophil (PMN) supernatants. PMN (5 x 10"/ml Hanks' balanced salt solution [HBSS]) were incubated with phorbol myristate acetate (500 ng/ml) for 45 minutes. Cell-free supernatants were collected, and I .0 mM DFP and I .0 m M mersalyl were added. The supernatants were not treated (0)or were treated with 1.0 mM EDTA (A) followed by incubation with radiolabeled collagen fibrils for various times, as described in Materials and Methods. W = radioactivity released by fibrils into HBSS alone; 0 = lysis of fibril by 0.005% trypsin in HBSS. Each point represents the mean of 3 observations. The standard deviation of each point was
