Inflammation, Vol. 16, No. 5, 1992
ENZYMATIC
ACTIVITY
IMMUNOREACTIVITY PHOSPHOLIPASE
AND
OF EXTRACELLULAR
A 2 IN INFLAMMATORY
SYNOVIAL
FLUIDS
W. PRUZANSKI, 1 K. SCOTT, 2 G. SMITH, 2 RAJKOVIC, 2 E. STEFANSKI, 1 and P. VADAS t llnflamr~tion Research Group, University of Toronto Toronto, Ontario, Canada 2Garvan Insti:ute of Medical Research, Darlinghurst, Australia
Abstract--Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant "synovial-type" group II phospholipase A2. This ELISA was specific for synovial-type PLAz and did not detect pancreatic (group I) PLA 2. In all synovial fluids examined, including rheumatoid, osteoarthfitic, Izsoriatic, and gouty fluids, synovial fluid PLA 2 enzyme activity significantly correlated with PLA2 immunoreactivity (P < 0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA2 activity by either putative PLA 2 activating or inhibitory proteins.
INTRODUCTION Phospholipase A2 (PLA:,) has been well characterized as a key enzyme in the arachidonic acid cascade (1). To date, two different extracellular PLA2's have been identified, the pancreatic type (2) and a nonpancreatic "synovial" type (3, 4). Whereas the former belongs to the group ! (5) phospholipases, the latter represents a group II (5) enzyme. In humans, extracellular nonpancreatic PLA2 (EC-PLA2) was originally discovered in and purified from rheumatoid synovial fluid (6, 7). Subsequently, high activity of EC-PLA2 was also found in synovial fluids from inflamed osteoarthritic, gouty, psoriatic, and other joints (8). Activity of EC-PLA 2 in the
451 0360-3997/92/1000-0451506,50/0 9 1992 Plenum Publishing Corporation
452
Pruzanski et aL
serum correlated with disease activity in rheumatoid (9) and in juvenile chronic arthritis (10). Eventually, EC-PLA2 was recognized as a proinflammatory enzyme capable of inducing an inflammatory reaction in joints (11, 12), skin (13), and lungs (14, 15) of experimental animals. It is not clear whether PLA2 activity found in synovial fluid is attributable to a single enzyme or whether several group II isoenzymes may coexist in the same fluid (16). Current standard assays of extracellular PLA2, based on the hydrolysis of radiolabeled phospholipids of various substrates, assess the catalytic activity of the enzyme. It was suggested that PLA2 activity may be biologically up- or down-regulated by various agents. Enhancement of EC-PLA 2 activity (17) and conversely suppression of PLA2 activity (18, 19) have been described. Thus, the enzymatic activity of PLA: in inflammatory sites may depend on the presence of these putative modulators. It was reported that some PLA2 activators per se may be proinflammatory (20) and that inhibitors may have antiinflammatory activity (21). The correlation of PLA2 enzyme activity with PLA a immunoreactivity would provide supportive evidence for the presumption that EC-PLA2 enzyme activity in inflammatory sites reflects changes in PLA2 concentration rather than modulation by activating or inhibitory factors. Such data may be obtained only now with the availability of monoclonal antibodies against synovial fluid type EC-PLA: (22). Herein, we provide evidence that in synovial fluids from inflamed joints enzymatic activity and immunoreactivity of EC-PLA2 correlate very closely. These data suggest that the possible presence of PLA2 modulators does not have a significant impact on PLA2 enzymatic activity in the synovial fluids.
MATERIALS AND METHODS Synovial fluids were obtained from 65 patients including 27 with rheumatoid arthritis, 12 with osteoarthritis, 9 with psoriatic arthritis, and 6 with gout. Synovial fluid PLA 2 activity was quantitated as described (7). Cell-free synovial fluids were stored at - 2 0 ~ until tested. PLAz activity was determined using phospholipid substrate from [14C]oleic acid-labeled E. coil membranes. Reaction mixtures included 10 mg of bovine serum albumin, 2 mM CaC12, 8 • 10s radi'olabeled E. coli, and 0.1 M Tris HC1 buffer, pH 7.5, in a total volume of 1.5 ml. Reaction mixtures were incubated at 37~ for 30 min, and the reaction was terminated by filtration through a 0.45-/zm Millipore filter retaining intact E. coli membranes and allowing the [Inc]oleic acid bound to the albumin carrier, released as a result of PLA2 hydrolysis, to pass through the filter. Assays were performed in duplicate, and values shown represent the mean of two determinations with a SD less than 5% of the mean. All assays were performed in substrate excess, and enzyme activities were corrected for nonenzymatic hydrolysis. Immunoreactivity of synovial fluid PLA2 was assayed using a modification of a capture ELISA technique reported by Green et al. (22). Two murine monoclonal antibodies raised against recombinant human PLA2 were used, a 9C1 capture antibody, and a 4A1 alkaline phosphatase conjugate. Dynatech Immulon microtiter plates (Fisher Scientific Company, Toronto, Canada) were coated
Synoviai Fluid Phospholipase Az
453
with 9C1 antibody at a concent:ration of 2/zg/ml in phosphate-buffered saline for 16 h at 4~ Plates were blocked with 200 #1 of 1% skimmed milk powder and 0.1% bovine serum albumin in phosphate-buffered saline for 3 h at 37~ and washed twice. PLA~ standards (including human recombinant PLA2, purified human pancreatic PLA2, purified porcine pancreatic PLA2, and serum samples) were incubated with immobilized antibody for 2 h at 37~ The wells were rinsed and 100/4 of alkaline phosphatase-conjugated antibody was added for 1 h at 37~ The unbound 4A1 conjugated antibody was removed and a sabstrate (100/zl) of P-nitrophenyl phosphate, 1 mg/ml in carbonate buffer, was added for 30 min at 37~ absorbance was read at 405 nm.
RESULTS
Sixty-five cell-free synovial fluids were tested for enzymatic activity and immunoreactivity of secretory PLA2. Twenty-seven fluids from rheumatoid joints were tested over a wide range of enzymatic activity (Figure 1) (Table 1). There was close correlation (P < 0.001) between enzymatic activity and immunoreactivity in rheumatoid fluids. Identical correlation (P < 0.001) was found in 12 osteoarthritic (Figure 2) and 9 psoriatic synovial fluids (Table 1). In six fluids from gouty joints, the correlation was slightly less marked but significant (P < 0.02). Eleven other fluids included four from patients with ankylosing spondylitis, four from SLE patients, and three from juvenile chronic arthritis. Again, significant correlation between enzymatic activity and immunoreactivity was noted. In the entire group of 65 synovial fluids (Figure 3) PLA2 enzymatic activity ranged from 4161 units/ml to 92614 units/ml and immunoreactivity ranged from 450 ng/ml to 7620 ng/ml (r = 0.838, P < 0.001). 8000--.
6O00
4000 c
I
9
2000
/. 20000
40000
60000
80000
100000
U/ml
Fig. 1. Phospholipase A2 in :~eum~oid synovial fluids. Co~el~ion of immunoreacfivity (ng/ml) to enzyme activity(units/ml):N= 27, r = 0.878, P < 0.001.
454
Pruzanski et al. Table 1. Phospholipase A2 in Synovial Fluids
Phospholipase A2 Correlation" Diagnosis
N
Enzymatic activity range (nnits/ml)
Rheumatoid arthritis Osteoarthritis Psoriatic arthritis Gouty arthritis Othersb Total
27 12 9 6 11 65
6392-91614 4355-23548 6335-38088 4161-37923 2894-37923 4161-92614
Immunoreactivity range (ng/ml)
r
P
770-7620 450-1065 752-3128 1284-4630 499-3748 450-7620
0.878 0.865 0.890 0.894 0.749 0.838
ENZYMATIC
ACTIVITY
IMMUNOREACTIVITY PHOSPHOLIPASE
AND
OF EXTRACELLULAR
A 2 IN INFLAMMATORY
SYNOVIAL
FLUIDS
W. PRUZANSKI, 1 K. SCOTT, 2 G. SMITH, 2 RAJKOVIC, 2 E. STEFANSKI, 1 and P. VADAS t llnflamr~tion Research Group, University of Toronto Toronto, Ontario, Canada 2Garvan Insti:ute of Medical Research, Darlinghurst, Australia
Abstract--Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant "synovial-type" group II phospholipase A2. This ELISA was specific for synovial-type PLAz and did not detect pancreatic (group I) PLA 2. In all synovial fluids examined, including rheumatoid, osteoarthfitic, Izsoriatic, and gouty fluids, synovial fluid PLA 2 enzyme activity significantly correlated with PLA2 immunoreactivity (P < 0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA2 activity by either putative PLA 2 activating or inhibitory proteins.
INTRODUCTION Phospholipase A2 (PLA:,) has been well characterized as a key enzyme in the arachidonic acid cascade (1). To date, two different extracellular PLA2's have been identified, the pancreatic type (2) and a nonpancreatic "synovial" type (3, 4). Whereas the former belongs to the group ! (5) phospholipases, the latter represents a group II (5) enzyme. In humans, extracellular nonpancreatic PLA2 (EC-PLA2) was originally discovered in and purified from rheumatoid synovial fluid (6, 7). Subsequently, high activity of EC-PLA2 was also found in synovial fluids from inflamed osteoarthritic, gouty, psoriatic, and other joints (8). Activity of EC-PLA 2 in the
451 0360-3997/92/1000-0451506,50/0 9 1992 Plenum Publishing Corporation
452
Pruzanski et aL
serum correlated with disease activity in rheumatoid (9) and in juvenile chronic arthritis (10). Eventually, EC-PLA2 was recognized as a proinflammatory enzyme capable of inducing an inflammatory reaction in joints (11, 12), skin (13), and lungs (14, 15) of experimental animals. It is not clear whether PLA2 activity found in synovial fluid is attributable to a single enzyme or whether several group II isoenzymes may coexist in the same fluid (16). Current standard assays of extracellular PLA2, based on the hydrolysis of radiolabeled phospholipids of various substrates, assess the catalytic activity of the enzyme. It was suggested that PLA2 activity may be biologically up- or down-regulated by various agents. Enhancement of EC-PLA 2 activity (17) and conversely suppression of PLA2 activity (18, 19) have been described. Thus, the enzymatic activity of PLA: in inflammatory sites may depend on the presence of these putative modulators. It was reported that some PLA2 activators per se may be proinflammatory (20) and that inhibitors may have antiinflammatory activity (21). The correlation of PLA2 enzyme activity with PLA a immunoreactivity would provide supportive evidence for the presumption that EC-PLA2 enzyme activity in inflammatory sites reflects changes in PLA2 concentration rather than modulation by activating or inhibitory factors. Such data may be obtained only now with the availability of monoclonal antibodies against synovial fluid type EC-PLA: (22). Herein, we provide evidence that in synovial fluids from inflamed joints enzymatic activity and immunoreactivity of EC-PLA2 correlate very closely. These data suggest that the possible presence of PLA2 modulators does not have a significant impact on PLA2 enzymatic activity in the synovial fluids.
MATERIALS AND METHODS Synovial fluids were obtained from 65 patients including 27 with rheumatoid arthritis, 12 with osteoarthritis, 9 with psoriatic arthritis, and 6 with gout. Synovial fluid PLA 2 activity was quantitated as described (7). Cell-free synovial fluids were stored at - 2 0 ~ until tested. PLAz activity was determined using phospholipid substrate from [14C]oleic acid-labeled E. coil membranes. Reaction mixtures included 10 mg of bovine serum albumin, 2 mM CaC12, 8 • 10s radi'olabeled E. coli, and 0.1 M Tris HC1 buffer, pH 7.5, in a total volume of 1.5 ml. Reaction mixtures were incubated at 37~ for 30 min, and the reaction was terminated by filtration through a 0.45-/zm Millipore filter retaining intact E. coli membranes and allowing the [Inc]oleic acid bound to the albumin carrier, released as a result of PLA2 hydrolysis, to pass through the filter. Assays were performed in duplicate, and values shown represent the mean of two determinations with a SD less than 5% of the mean. All assays were performed in substrate excess, and enzyme activities were corrected for nonenzymatic hydrolysis. Immunoreactivity of synovial fluid PLA2 was assayed using a modification of a capture ELISA technique reported by Green et al. (22). Two murine monoclonal antibodies raised against recombinant human PLA2 were used, a 9C1 capture antibody, and a 4A1 alkaline phosphatase conjugate. Dynatech Immulon microtiter plates (Fisher Scientific Company, Toronto, Canada) were coated
Synoviai Fluid Phospholipase Az
453
with 9C1 antibody at a concent:ration of 2/zg/ml in phosphate-buffered saline for 16 h at 4~ Plates were blocked with 200 #1 of 1% skimmed milk powder and 0.1% bovine serum albumin in phosphate-buffered saline for 3 h at 37~ and washed twice. PLA~ standards (including human recombinant PLA2, purified human pancreatic PLA2, purified porcine pancreatic PLA2, and serum samples) were incubated with immobilized antibody for 2 h at 37~ The wells were rinsed and 100/4 of alkaline phosphatase-conjugated antibody was added for 1 h at 37~ The unbound 4A1 conjugated antibody was removed and a sabstrate (100/zl) of P-nitrophenyl phosphate, 1 mg/ml in carbonate buffer, was added for 30 min at 37~ absorbance was read at 405 nm.
RESULTS
Sixty-five cell-free synovial fluids were tested for enzymatic activity and immunoreactivity of secretory PLA2. Twenty-seven fluids from rheumatoid joints were tested over a wide range of enzymatic activity (Figure 1) (Table 1). There was close correlation (P < 0.001) between enzymatic activity and immunoreactivity in rheumatoid fluids. Identical correlation (P < 0.001) was found in 12 osteoarthritic (Figure 2) and 9 psoriatic synovial fluids (Table 1). In six fluids from gouty joints, the correlation was slightly less marked but significant (P < 0.02). Eleven other fluids included four from patients with ankylosing spondylitis, four from SLE patients, and three from juvenile chronic arthritis. Again, significant correlation between enzymatic activity and immunoreactivity was noted. In the entire group of 65 synovial fluids (Figure 3) PLA2 enzymatic activity ranged from 4161 units/ml to 92614 units/ml and immunoreactivity ranged from 450 ng/ml to 7620 ng/ml (r = 0.838, P < 0.001). 8000--.
6O00
4000 c
I
9
2000
/. 20000
40000
60000
80000
100000
U/ml
Fig. 1. Phospholipase A2 in :~eum~oid synovial fluids. Co~el~ion of immunoreacfivity (ng/ml) to enzyme activity(units/ml):N= 27, r = 0.878, P < 0.001.
454
Pruzanski et al. Table 1. Phospholipase A2 in Synovial Fluids
Phospholipase A2 Correlation" Diagnosis
N
Enzymatic activity range (nnits/ml)
Rheumatoid arthritis Osteoarthritis Psoriatic arthritis Gouty arthritis Othersb Total
27 12 9 6 11 65
6392-91614 4355-23548 6335-38088 4161-37923 2894-37923 4161-92614
Immunoreactivity range (ng/ml)
r
P
770-7620 450-1065 752-3128 1284-4630 499-3748 450-7620
0.878 0.865 0.890 0.894 0.749 0.838
