Clin. exp. Immunol. (1990) 79, 195-201
Depressed degranulation response of synovial fluid polymorphonuclear leucocytes from patients with rheumatoid arthritis to IgG aggregates B. DULARAY, P. A. DIEPPE* & C. J. ELSON Department of Pathology, University of Bristol; and *Department of Rheumatology, Bristol Royal Infirmary, Bristol, England
(Acceptedfor publication 21 September 1989)
SUMMARY No difference was found between the degranulation responses to FMLP of synovial fluid (SF) polymorphonuclear leucocytes (PMNL), from patients with rheumatoid arthritis (RA), and either paired blood PMNL or blood PMNL from a healthy donor. In contrast, the response of SF PMNL to heat-aggregated IgG was often reduced compared with autologous blood PMNL. Similarly, SF from some (35%) RA patients stimulated degranulation of PMNL but the response of SF-derived PMNL to autologous stimulatory SF was reduced compared with the response of blood PMNL. The stimulatory activity of the SF was removed by sepharose-protein A. These results were taken to suggest that the activity is due to immunoglobulin aggregates and that SF PMNL (from some RA patients) are tachyphylactic to stimulation by immunoglobulin aggregates as measured by degranulation because they have been stimulated by immunoglobulin aggregates in vivo. In other studies the concentration of myeloperoxidase (MPO) was measured enzymically in RA SF and was found to be present in varying amounts. However, only a weak relationship was found between MPO levels and either PMNL numbers or levels of complement-bearing IgG aggregates in SF. It is considered that the relationship between MPO and immunoglobulin aggregates levels is obscured by the presence of a peroxidase inhibitor in the fluids and/or because only aggregates bound to tissue stimulate degranulation in vivo. Keywords degranulation synovial fluid rheumatoid arthritis polymorphonuclear leucocytes
INTRODUCTION
demonstrated that interleukin-1 (IL-1), a substance known to be present in RA SF (Fontana et al., 1982; Nouri, Panayi & Goodman, 1984), primed PMNL to give enhanced stimulusinduced degranulation responses. These results raised the possibility that SF PMNL may be capable of enhanced degranulation responses and the primary purpose of the current work was to test this idea. In addition, it is known that stimulants of PMNL degranulation, for example immunoglobulin aggregates (Goldstein et al., 1975; Treadway et al., 1979), leucotriene B4 (Klickstein, Shapleigh & Goetzl, 1980) and platelet-activating factor (Dewald & Baggiolini, 1986) occur in RA SF together with PMNL-derived lysosomal enzymes (Hadler, Spitznagel & Quinet, 1979). Consequently it is often assumed that degranulation of PMNL does take place in rheumatoid joints. To examine this assumption we investigated whether RA SF can actually stimulate RA blood and SF PMNL to degranulate, and sought for evidence that such degranulation had occurred in vivo.
Since the discovery of large numbers of polymorphonuclear leucocytes (PMNL) in the joint fluid of patients with rheumatoid arthritis (RA), considerable efforts have been made to determine how these cells are involved in the disease process. Studies in vitro have revealed that several agents can stimulate PMNL to release lysosomal enzymes (degranulation response) and/or free radicals (oxidative response) into the external environment. Although blood PMNL responses to such agents have been studied in detail there have been few corresponding studies using synovial fluid (SF) PMNL. We therefore (Dularay, Elson & Dieppe, 1988) compared the oxidative responses of SF PMNL from RA patients with that of autologous PMNL and PMNL from healthy donors and showed that the former were primed in that they gave enhanced oxidative responses. Moreover, incubating normal blood PMNL in RA SF primed PMNL to give enhanced oxidative responses suggesting that these SF contained a priming factor. Recently (Dularay et al., 1989) we
MATERIALS AND METHODS
Synovialfluids
Correspondences: Dr C. J. Elson, Department of Pathology, University of Bristol, Bristol BS8 ITD, England.
SF were obtained from patients attending the Rheumatology
195
196
B. Dularay, P. A. Dieppe & C. J. Elson
Out Patients Clinic at the Bristol Royal Infirmary. All patients fulfilled the standard criteria for classical or definite rheumatoid arthritis. The patients were receiving a variety of non-steroidal anti-inflammatory and slow-acting anti-rheumatic drugs. In the degranulation studies, 21 patients were investigated (13 women, mean age 55-7 years, range 32-73; and eight men, mean age 53 8 years, range 37-72). SF were obtained by therapeutic aspiration of inflamed knee joints which were free from bacterial infections. Samples were collected in 0 1-volume acid citrate dextrose and centrifuged at 1 840g for 10 min to remove cells. Cell-free SF 70'C. were aliquoted and stored at Of the 93 SF assayed for myeloperoxidase (MPO) activity, 71 SF were from patients with RA (51 women, mean age 57; 20 men, mean age 55), 16 were from patients with osteoarthritis (seven women, mean age 61; and nine men, mean age 65) five of whom has radiographic and/or SF evidence of crystal deposits. SF were also obtained from six patients with psoriatic arthritis (three women, mean age 65; and three men, mean age 53).
nated by the addition of 1 mm sodium azide; ml of PBS, pH 7.3, then added and the change in absorbance at 655 nm was measured. was
Measurement of MPO concentrations in RA SF SF was digested with hyaluronidase (Sigma, Bovine; 100 U/ml SF) for 15 min at 370C and centrifuged for 2 min at 12000 g (Eppendorf centrifuge 3200). After diluting 1:2 with PBS, pH 7 3, SF was assayed for MPO using the spectrophotometric method described above.
-
Isolation of PMNL
Peripheral blood was collected by venipuncture from volunteers or patients in 3-8% sodium citrate and the PMNL were isolated by a modification of the method of Dooley, Simpson & Meryman (1982). Briefly, blood was diluted 1: 1: 3, (v/v) with 6% dextran (Fisons, Lomodex T70, in 0-9% saline) and phosphate-buffered saline (PBS) containing 4% (w/v) sodium citrate, pH 7-3. After 45 min incubation at room temperature, the leucocyte-rich supernatant was centrifuged at 170 g for 10 min. The cells were resuspended in a 50% solution of percoll (Sigma) and layered on to a discontinuous percoll gradient composed of 65% and 82 5% percoll. After centrifugation at 270 g for 20 min the PMNL present at the 65% and 82 5% interface were removed and washed twice prior to use. PMNL were isolated from SF in the same way except that the dextran sedimentation step was omitted. The cells were resuspended in HBSS (136 mm NaCl; 5.4 mm KCI; 1 0 mM MgCI * 6H20; I 0 mM CaCI2H2O; 5 5 mm glucose; 10 mM HEPES) pH 7 3, to give 2 x IO7/ml. The cells prepared by this method were >98% PMNL and 99% viable based on trypan blue exclusion.
LDH assay LDH was assayed using a standard assay kit (Boehringer). The initial rate of oxidation of diphosphopyridine nucleotide in the presence of 0-01 M sodium pyruvate was measured at 340 nm.
Sepharose-protein A chromotography IgG and IgG-containing immune complexes were removed from SF using affinity chromatography (Goudsward et al., 1978). SF was digested with hyaluronidase, diluted 1:2 with PBS, pH 7 3, and applied to a column of sepharose-protein A CL-4B (Sigma). The eluate was assayed for the presence of IgG using radial immunodiffusion kits (Serotec). As a control for nonspecific removal of protein, SF was treated with sepharose CL4B (the matrix to which protein A is attached). PMNL degranulation PMNL were pretreated with 20 ug/ml cytochalasin B (Sigma; 1 mg/ml in DMSO) for 10 min at room temperature. The treated cells (106/50 pl) were incubated with HBSS containing 10 mg/ml human serum albumin (Sigma), and the stimulant HAIgG (0-25-2-0 mg/ml), FMLP (25 x 10-9-25 x 104 M) or SF (120%) for an optimum period, established in preliminary experiments, of 30 min at 370C. The total reaction volume was 500 ,l and each reaction was set up in triplicate. The supernatant was assayed for activities of MPO, LDH and in some cases f,glucuronidase. Enzyme release was expressed as a percentage of the maximum releasable. This was determined by lysing PMNL with 0-05 % triton X- 00 (Sigma) for 2 min. The supernatant was then assayed for MPO, LDH and /3-glucuronidase activities.
Preparation of heat-aggregated IgG (HAIgG)
Aggregated IgG was prepared by dissolving Cohn II globulin fraction (Sigma) in PBS, pH 7-3, at 25 mg/ml and heating at 63°C for 40 min in a glass vessel. The aggregated IgG was cooled at 4°C, aliquoted and stored at 20°C.
Complement-bearing IgG aggregates
Complement-bearing IgG aggregate levels were measured by using an anti-C3d antibody as described by Bedwell et al. (1987).
-
Enzyme assays PMNL degranulation
was measured by assaying for the azurophil granule enzyme MPO (EC 1. 11. 1.7); in some experiments the enzyme beta-glucuronidase (EC 3.2.1.31) was also assayed. As a measure of cell death lactate dehydrogenase (LDH; EC 1.1. 1.27) was assayed.
MPO
MPO was assayed spectrophotometrically using the substrate tetra-methyl benzidine in the presence of hydrogen peroxide by the method of Suzuki et al. (1983). The supernatant to be assayed was added to the assay substrate (20 mm 3,3',5,5' 0 01 M 20 tetra-methyl benzidine (50 p1); 0 03% H202 (w/v, pN); sodium acetate buffer, pH 5-2 (50 p1 and 150 pl PBS) and incubated for exactly 5 min at 37°C. The reaction was termi-
RESULTS Response of RA PMNL to FMLP The degranulation responses of SF and autologous blood PMNL from patients with RA to increasing concentrations of FMLP are summarized in Table 1. There was no difference between the responses of SF and blood-derived PMNL to FMLP at any of the concentrations studied (P>0-05). As differences between PMNL responses are likely to appear at suboptimal doses of the stimulus, the degranulation response of RA blood, SF PMNL and normal blood PMNL to a suboptimal dose of FMLP (2-5 x 10-9 M) was compared. The percentage MPO released was 9-9+8-4% (n=9; range 3-31) for SF PMNL; 15 7+23% (n=9; range 1 8-70%) for RA blood PMNL; and 7 9+6-7% (n= 12; range 1-18%) for normal blood PMNL. The majority of RA SF, blood and normal blood
Depressed degranulation response of polymorphs
197
Table 1. Response to FMLP of synovial fluid (SF) and blood polymorphonuclear leucocytes (PMNL) from patients with rheumatoid arthritis (RA)
Maximum release (%) SF PMNL
Blood PMNL
FMLP
(PM)
Mean ± s.d.
Range
Mean ± s.d.
Range
0-025 0-25 2-50 25-00
9-9+8-4 53-0+33-7 49-6+27-7 55-0+29-9
3-31 18-95
15-7+23-0 42-2+37-2 50-4+34-8 46-3+26-7
2-70 14-95 25-87 32-94
23-93 39-98
P
n
>0-05 9 >0-05 20 > 0-05 11 >0-05 11
0-
'a
"'
Paired RA blood and SF PMNL were stimulated with the specified concentration of FMLP and the release of myeloperoxidase into the medium measured. The results are expressed as a percentage of the maximum releasable enzyme and were analysed using the Wilcoxon rank test.
O
-)
100
o a.
PMNL gave a low response to this suboptimal dose of FMLP (< 5%) but in a small number of patients and healthy donors a higher response was elicited.
Response to HAIgG SF and autologous blood PMNL from six RA patients were stimulated with increasing concentrations of HAIgG (0-25-2-0 mg/ml). It can be seen (Fig. la and lb) that the amount of MPO released increased with the concentration of HAIgG reaching a maximum level at about 0-5 mg/ml. In contrast to FMLP, a greater degranulation response was elicited from blood than SF PMNL in five out of six patients, at all concentrations of HAIgG used. LDH release from PMNL was assayed routinely to measure cell viability. The release was not dose dependent and did not exceed 4 + 2% of the maximum release of LDH for both stimuli, HAIgG and FMLP. In 18 patients, the release of MPO by paired SF and blood PMNL in response to a single dose of HAIgG (0 50 mg/ml) was measured and the results are recorded in Fig. 2. In 10 of the patients, SF PMNL exhibited a decreased response to HAIgG as compared with autologous blood PMNL while in eight patients there was no difference between the responses. The mean response to HAIgG of the RA SF PMNL (27 + 20%, n = 18, range 3-60%) was significantly less (P < 0 01, Wilcoxon rank test) than that of RA blood PMNL (48 + 30%, n = 18, range 14-92%) in these experiments. In other experiments the degranulation response of normal blood PMNL to the same dose of HAIgG was measured and found to be 21 + 5 8%, (n = 8, range 14-1-30%). From this result it appears that both blood and SF PMNL from a few patients are hyper-responsive to HAIgG with respect to normal blood PMNL. It could be argued that the differences between the responses of SF and blood PMNL are due to differences in the MPO content of the cells. To examine this possibility the maximum amounts of MPO that could be released from blood and SF PMNL was compared in 20 patients. The mean MPO content of SF PMNL was 3-4 + 1-4 U (change in absorbance at 655 nm per 5 min/106 PMNL, n=20, range 1-6-5-4), the mean content for blood PMNL was 3 6+ 1-5 U/106 PMNL (n=20, range 1-6 1), and the mean content for normal blood PMNL was 3-2 + 1-3 U/
HAIgG (mg/ml) Fig. 1. Degranulation response of synovial fluid (SF) and autologous blood peripheral blood polymorphonuclear leucocytes (PMNL) to different doses of heat-aggregated IgG (HAIgG). SF and autologous blood PMNL were stimulated with increasing concentrations of HAIgG and the release of myeloperoxidase (MPO) into the medium measured. (a) Patient 1 SF PMNL (v), blood PMNL (v); patient 2 SF PMNL (0), blood PMNL (U); patient 3 SF PMNL (0), blood PMNL (0); (b) patient 4 SF PMNL (O), blood PMNL (*); patient 5 SF PMNL (A), blood (A); patient 6 SF PMNL (*), blood PMNL (*). The results are expressed as a percentage ofthe maximum releasable enzyme and are the mean + s.d. of triplicate determinations.
106 PMNL (n = 20,
range
1 4-6-6). In 14 patients the MPO
content of SF and blood PMNL was comparable, in three it was greater for blood PMNL than for SF PMNL, and in three other patients it was greater for SF PMNL.
Ability of SF to stimulate degranulation by normal blood PMNL The occurrence of large numbers of PMNL and immunoglobulin aggregates in RA SF is well established, as is the ability of immunoglobulin aggregates to stimulate degranulation in vitro. Accordingly, experiments were set up to determine whether SF could stimulate PMNL to degranulate. Normal blood PMNL were incubated with increasing doses of SF (1-25%, v/v) for 30 min at 37°C. The supernatant was assayed for MPO, f,glucuronidase and LDH. Seven out of 20 rheumatoid SF stimulated degranulation as measured by the release of the enzyme f,-glucuronidase and the results for the stimulatory SF are shown in Fig. 3. In these experiments no MPO release (i.e.
198
B. Dularay, P. A. Dieppe & C. J. Elson
100l
40
(a )
0
Ha
30
a~~~~~~~~~~~~~~)
A/
0
0
/i 20
V
a
a
0
m
or
*
a-
;
MI0 '~10V Q1.
Reciprocal dilution of SF SF PMNL
Blood PMNL
Fig. 2. Comparison of myeloperoxidase (MPO) release by synovial fluid (SF) and autologous blood peripheral blood polymorphonuclear leucocytes (PMNL) in response to heat-aggregated IgG (HAIgG). Rheumatoid arthritis (RA) SF and autologous blood PMNL were stimulated with one dose of HAIgG (0-5 mg/ml) and the release of myeloperoxidase (MPO) into the medium was measured. The response of normal blood PMNL from eight donors is also shown (0). The results are expressed as a percentage ofthe maximum releasable enzyme and are the mean of three determinations.
40
(b)
*0 0
30
(A 0
a) to 20
0
I
F
'1
xI 0 05
0
0*10
0-15
0.20
Reciprocal dilution of serum
40 25
n-0
p.
20
0
Depressed degranulation response of synovial fluid polymorphonuclear leucocytes from patients with rheumatoid arthritis to IgG aggregates B. DULARAY, P. A. DIEPPE* & C. J. ELSON Department of Pathology, University of Bristol; and *Department of Rheumatology, Bristol Royal Infirmary, Bristol, England
(Acceptedfor publication 21 September 1989)
SUMMARY No difference was found between the degranulation responses to FMLP of synovial fluid (SF) polymorphonuclear leucocytes (PMNL), from patients with rheumatoid arthritis (RA), and either paired blood PMNL or blood PMNL from a healthy donor. In contrast, the response of SF PMNL to heat-aggregated IgG was often reduced compared with autologous blood PMNL. Similarly, SF from some (35%) RA patients stimulated degranulation of PMNL but the response of SF-derived PMNL to autologous stimulatory SF was reduced compared with the response of blood PMNL. The stimulatory activity of the SF was removed by sepharose-protein A. These results were taken to suggest that the activity is due to immunoglobulin aggregates and that SF PMNL (from some RA patients) are tachyphylactic to stimulation by immunoglobulin aggregates as measured by degranulation because they have been stimulated by immunoglobulin aggregates in vivo. In other studies the concentration of myeloperoxidase (MPO) was measured enzymically in RA SF and was found to be present in varying amounts. However, only a weak relationship was found between MPO levels and either PMNL numbers or levels of complement-bearing IgG aggregates in SF. It is considered that the relationship between MPO and immunoglobulin aggregates levels is obscured by the presence of a peroxidase inhibitor in the fluids and/or because only aggregates bound to tissue stimulate degranulation in vivo. Keywords degranulation synovial fluid rheumatoid arthritis polymorphonuclear leucocytes
INTRODUCTION
demonstrated that interleukin-1 (IL-1), a substance known to be present in RA SF (Fontana et al., 1982; Nouri, Panayi & Goodman, 1984), primed PMNL to give enhanced stimulusinduced degranulation responses. These results raised the possibility that SF PMNL may be capable of enhanced degranulation responses and the primary purpose of the current work was to test this idea. In addition, it is known that stimulants of PMNL degranulation, for example immunoglobulin aggregates (Goldstein et al., 1975; Treadway et al., 1979), leucotriene B4 (Klickstein, Shapleigh & Goetzl, 1980) and platelet-activating factor (Dewald & Baggiolini, 1986) occur in RA SF together with PMNL-derived lysosomal enzymes (Hadler, Spitznagel & Quinet, 1979). Consequently it is often assumed that degranulation of PMNL does take place in rheumatoid joints. To examine this assumption we investigated whether RA SF can actually stimulate RA blood and SF PMNL to degranulate, and sought for evidence that such degranulation had occurred in vivo.
Since the discovery of large numbers of polymorphonuclear leucocytes (PMNL) in the joint fluid of patients with rheumatoid arthritis (RA), considerable efforts have been made to determine how these cells are involved in the disease process. Studies in vitro have revealed that several agents can stimulate PMNL to release lysosomal enzymes (degranulation response) and/or free radicals (oxidative response) into the external environment. Although blood PMNL responses to such agents have been studied in detail there have been few corresponding studies using synovial fluid (SF) PMNL. We therefore (Dularay, Elson & Dieppe, 1988) compared the oxidative responses of SF PMNL from RA patients with that of autologous PMNL and PMNL from healthy donors and showed that the former were primed in that they gave enhanced oxidative responses. Moreover, incubating normal blood PMNL in RA SF primed PMNL to give enhanced oxidative responses suggesting that these SF contained a priming factor. Recently (Dularay et al., 1989) we
MATERIALS AND METHODS
Synovialfluids
Correspondences: Dr C. J. Elson, Department of Pathology, University of Bristol, Bristol BS8 ITD, England.
SF were obtained from patients attending the Rheumatology
195
196
B. Dularay, P. A. Dieppe & C. J. Elson
Out Patients Clinic at the Bristol Royal Infirmary. All patients fulfilled the standard criteria for classical or definite rheumatoid arthritis. The patients were receiving a variety of non-steroidal anti-inflammatory and slow-acting anti-rheumatic drugs. In the degranulation studies, 21 patients were investigated (13 women, mean age 55-7 years, range 32-73; and eight men, mean age 53 8 years, range 37-72). SF were obtained by therapeutic aspiration of inflamed knee joints which were free from bacterial infections. Samples were collected in 0 1-volume acid citrate dextrose and centrifuged at 1 840g for 10 min to remove cells. Cell-free SF 70'C. were aliquoted and stored at Of the 93 SF assayed for myeloperoxidase (MPO) activity, 71 SF were from patients with RA (51 women, mean age 57; 20 men, mean age 55), 16 were from patients with osteoarthritis (seven women, mean age 61; and nine men, mean age 65) five of whom has radiographic and/or SF evidence of crystal deposits. SF were also obtained from six patients with psoriatic arthritis (three women, mean age 65; and three men, mean age 53).
nated by the addition of 1 mm sodium azide; ml of PBS, pH 7.3, then added and the change in absorbance at 655 nm was measured. was
Measurement of MPO concentrations in RA SF SF was digested with hyaluronidase (Sigma, Bovine; 100 U/ml SF) for 15 min at 370C and centrifuged for 2 min at 12000 g (Eppendorf centrifuge 3200). After diluting 1:2 with PBS, pH 7 3, SF was assayed for MPO using the spectrophotometric method described above.
-
Isolation of PMNL
Peripheral blood was collected by venipuncture from volunteers or patients in 3-8% sodium citrate and the PMNL were isolated by a modification of the method of Dooley, Simpson & Meryman (1982). Briefly, blood was diluted 1: 1: 3, (v/v) with 6% dextran (Fisons, Lomodex T70, in 0-9% saline) and phosphate-buffered saline (PBS) containing 4% (w/v) sodium citrate, pH 7-3. After 45 min incubation at room temperature, the leucocyte-rich supernatant was centrifuged at 170 g for 10 min. The cells were resuspended in a 50% solution of percoll (Sigma) and layered on to a discontinuous percoll gradient composed of 65% and 82 5% percoll. After centrifugation at 270 g for 20 min the PMNL present at the 65% and 82 5% interface were removed and washed twice prior to use. PMNL were isolated from SF in the same way except that the dextran sedimentation step was omitted. The cells were resuspended in HBSS (136 mm NaCl; 5.4 mm KCI; 1 0 mM MgCI * 6H20; I 0 mM CaCI2H2O; 5 5 mm glucose; 10 mM HEPES) pH 7 3, to give 2 x IO7/ml. The cells prepared by this method were >98% PMNL and 99% viable based on trypan blue exclusion.
LDH assay LDH was assayed using a standard assay kit (Boehringer). The initial rate of oxidation of diphosphopyridine nucleotide in the presence of 0-01 M sodium pyruvate was measured at 340 nm.
Sepharose-protein A chromotography IgG and IgG-containing immune complexes were removed from SF using affinity chromatography (Goudsward et al., 1978). SF was digested with hyaluronidase, diluted 1:2 with PBS, pH 7 3, and applied to a column of sepharose-protein A CL-4B (Sigma). The eluate was assayed for the presence of IgG using radial immunodiffusion kits (Serotec). As a control for nonspecific removal of protein, SF was treated with sepharose CL4B (the matrix to which protein A is attached). PMNL degranulation PMNL were pretreated with 20 ug/ml cytochalasin B (Sigma; 1 mg/ml in DMSO) for 10 min at room temperature. The treated cells (106/50 pl) were incubated with HBSS containing 10 mg/ml human serum albumin (Sigma), and the stimulant HAIgG (0-25-2-0 mg/ml), FMLP (25 x 10-9-25 x 104 M) or SF (120%) for an optimum period, established in preliminary experiments, of 30 min at 370C. The total reaction volume was 500 ,l and each reaction was set up in triplicate. The supernatant was assayed for activities of MPO, LDH and in some cases f,glucuronidase. Enzyme release was expressed as a percentage of the maximum releasable. This was determined by lysing PMNL with 0-05 % triton X- 00 (Sigma) for 2 min. The supernatant was then assayed for MPO, LDH and /3-glucuronidase activities.
Preparation of heat-aggregated IgG (HAIgG)
Aggregated IgG was prepared by dissolving Cohn II globulin fraction (Sigma) in PBS, pH 7-3, at 25 mg/ml and heating at 63°C for 40 min in a glass vessel. The aggregated IgG was cooled at 4°C, aliquoted and stored at 20°C.
Complement-bearing IgG aggregates
Complement-bearing IgG aggregate levels were measured by using an anti-C3d antibody as described by Bedwell et al. (1987).
-
Enzyme assays PMNL degranulation
was measured by assaying for the azurophil granule enzyme MPO (EC 1. 11. 1.7); in some experiments the enzyme beta-glucuronidase (EC 3.2.1.31) was also assayed. As a measure of cell death lactate dehydrogenase (LDH; EC 1.1. 1.27) was assayed.
MPO
MPO was assayed spectrophotometrically using the substrate tetra-methyl benzidine in the presence of hydrogen peroxide by the method of Suzuki et al. (1983). The supernatant to be assayed was added to the assay substrate (20 mm 3,3',5,5' 0 01 M 20 tetra-methyl benzidine (50 p1); 0 03% H202 (w/v, pN); sodium acetate buffer, pH 5-2 (50 p1 and 150 pl PBS) and incubated for exactly 5 min at 37°C. The reaction was termi-
RESULTS Response of RA PMNL to FMLP The degranulation responses of SF and autologous blood PMNL from patients with RA to increasing concentrations of FMLP are summarized in Table 1. There was no difference between the responses of SF and blood-derived PMNL to FMLP at any of the concentrations studied (P>0-05). As differences between PMNL responses are likely to appear at suboptimal doses of the stimulus, the degranulation response of RA blood, SF PMNL and normal blood PMNL to a suboptimal dose of FMLP (2-5 x 10-9 M) was compared. The percentage MPO released was 9-9+8-4% (n=9; range 3-31) for SF PMNL; 15 7+23% (n=9; range 1 8-70%) for RA blood PMNL; and 7 9+6-7% (n= 12; range 1-18%) for normal blood PMNL. The majority of RA SF, blood and normal blood
Depressed degranulation response of polymorphs
197
Table 1. Response to FMLP of synovial fluid (SF) and blood polymorphonuclear leucocytes (PMNL) from patients with rheumatoid arthritis (RA)
Maximum release (%) SF PMNL
Blood PMNL
FMLP
(PM)
Mean ± s.d.
Range
Mean ± s.d.
Range
0-025 0-25 2-50 25-00
9-9+8-4 53-0+33-7 49-6+27-7 55-0+29-9
3-31 18-95
15-7+23-0 42-2+37-2 50-4+34-8 46-3+26-7
2-70 14-95 25-87 32-94
23-93 39-98
P
n
>0-05 9 >0-05 20 > 0-05 11 >0-05 11
0-
'a
"'
Paired RA blood and SF PMNL were stimulated with the specified concentration of FMLP and the release of myeloperoxidase into the medium measured. The results are expressed as a percentage of the maximum releasable enzyme and were analysed using the Wilcoxon rank test.
O
-)
100
o a.
PMNL gave a low response to this suboptimal dose of FMLP (< 5%) but in a small number of patients and healthy donors a higher response was elicited.
Response to HAIgG SF and autologous blood PMNL from six RA patients were stimulated with increasing concentrations of HAIgG (0-25-2-0 mg/ml). It can be seen (Fig. la and lb) that the amount of MPO released increased with the concentration of HAIgG reaching a maximum level at about 0-5 mg/ml. In contrast to FMLP, a greater degranulation response was elicited from blood than SF PMNL in five out of six patients, at all concentrations of HAIgG used. LDH release from PMNL was assayed routinely to measure cell viability. The release was not dose dependent and did not exceed 4 + 2% of the maximum release of LDH for both stimuli, HAIgG and FMLP. In 18 patients, the release of MPO by paired SF and blood PMNL in response to a single dose of HAIgG (0 50 mg/ml) was measured and the results are recorded in Fig. 2. In 10 of the patients, SF PMNL exhibited a decreased response to HAIgG as compared with autologous blood PMNL while in eight patients there was no difference between the responses. The mean response to HAIgG of the RA SF PMNL (27 + 20%, n = 18, range 3-60%) was significantly less (P < 0 01, Wilcoxon rank test) than that of RA blood PMNL (48 + 30%, n = 18, range 14-92%) in these experiments. In other experiments the degranulation response of normal blood PMNL to the same dose of HAIgG was measured and found to be 21 + 5 8%, (n = 8, range 14-1-30%). From this result it appears that both blood and SF PMNL from a few patients are hyper-responsive to HAIgG with respect to normal blood PMNL. It could be argued that the differences between the responses of SF and blood PMNL are due to differences in the MPO content of the cells. To examine this possibility the maximum amounts of MPO that could be released from blood and SF PMNL was compared in 20 patients. The mean MPO content of SF PMNL was 3-4 + 1-4 U (change in absorbance at 655 nm per 5 min/106 PMNL, n=20, range 1-6-5-4), the mean content for blood PMNL was 3 6+ 1-5 U/106 PMNL (n=20, range 1-6 1), and the mean content for normal blood PMNL was 3-2 + 1-3 U/
HAIgG (mg/ml) Fig. 1. Degranulation response of synovial fluid (SF) and autologous blood peripheral blood polymorphonuclear leucocytes (PMNL) to different doses of heat-aggregated IgG (HAIgG). SF and autologous blood PMNL were stimulated with increasing concentrations of HAIgG and the release of myeloperoxidase (MPO) into the medium measured. (a) Patient 1 SF PMNL (v), blood PMNL (v); patient 2 SF PMNL (0), blood PMNL (U); patient 3 SF PMNL (0), blood PMNL (0); (b) patient 4 SF PMNL (O), blood PMNL (*); patient 5 SF PMNL (A), blood (A); patient 6 SF PMNL (*), blood PMNL (*). The results are expressed as a percentage ofthe maximum releasable enzyme and are the mean + s.d. of triplicate determinations.
106 PMNL (n = 20,
range
1 4-6-6). In 14 patients the MPO
content of SF and blood PMNL was comparable, in three it was greater for blood PMNL than for SF PMNL, and in three other patients it was greater for SF PMNL.
Ability of SF to stimulate degranulation by normal blood PMNL The occurrence of large numbers of PMNL and immunoglobulin aggregates in RA SF is well established, as is the ability of immunoglobulin aggregates to stimulate degranulation in vitro. Accordingly, experiments were set up to determine whether SF could stimulate PMNL to degranulate. Normal blood PMNL were incubated with increasing doses of SF (1-25%, v/v) for 30 min at 37°C. The supernatant was assayed for MPO, f,glucuronidase and LDH. Seven out of 20 rheumatoid SF stimulated degranulation as measured by the release of the enzyme f,-glucuronidase and the results for the stimulatory SF are shown in Fig. 3. In these experiments no MPO release (i.e.
198
B. Dularay, P. A. Dieppe & C. J. Elson
100l
40
(a )
0
Ha
30
a~~~~~~~~~~~~~~)
A/
0
0
/i 20
V
a
a
0
m
or
*
a-
;
MI0 '~10V Q1.
Reciprocal dilution of SF SF PMNL
Blood PMNL
Fig. 2. Comparison of myeloperoxidase (MPO) release by synovial fluid (SF) and autologous blood peripheral blood polymorphonuclear leucocytes (PMNL) in response to heat-aggregated IgG (HAIgG). Rheumatoid arthritis (RA) SF and autologous blood PMNL were stimulated with one dose of HAIgG (0-5 mg/ml) and the release of myeloperoxidase (MPO) into the medium was measured. The response of normal blood PMNL from eight donors is also shown (0). The results are expressed as a percentage ofthe maximum releasable enzyme and are the mean of three determinations.
40
(b)
*0 0
30
(A 0
a) to 20
0
I
F
'1
xI 0 05
0
0*10
0-15
0.20
Reciprocal dilution of serum
40 25
n-0
p.
20
0
