Clin. exp. Immunol. (1992) 87, 435-437
Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids E. FLESCHER*, T. L. BOWLINt & N. TALAL*j *Department of Medicine, The University of Texas Health Science Center, $The Audie L. Murphy Memorial Veterans Hospital, San Antonio, Texas, and tMarion Merrell Dow Research Institute, Cincinnati, Ohio, USA
(Acceptedfor publication 7 October 1991)
SUMMARY Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids. Keywords IL-2 rheumatoid arthritis polyamines
INTRODUCTION T lymphocytes from peripheral blood, synovial fluid (SF) and synovial tissues of rheumatoid arthritis (RA) patients produce low quantities of IL-2 in comparison with T lymphocytes from peripheral blood of healthy individuals. Similar findings were obtained using a variety of stimuli [1-4]. Several products of monocyte activity, prostaglandin E2 (PGE2) [5,6] an IL- I inhibitor [2] and products of polyamine oxidase [7,8], were found to down-regulate IL-2 production and are candidate molecules possibly responsible for the depressed production of IL-2 by RA SF cells.
Polyamine oxidase metabolizes polyamines, like spermidine and spermine, into hydrogen peroxide, ammonia and an aldehyde [8]. RA SF mononuclear cells (MNC) contain elevated levels of polyamines [8] and polyamine oxidase activity is present in RA SF [9]. We looked for a possible relationship between polyamine levels and IL-2 production in a series of individual samples of RA SF MNC. We did this in the presence of monocytes since the poor response of RA SF MNC to mitogens is predominantly monocyte-mediated [2]. In addition, we attempted to correct IL-2 production by RA SF cells using inhibitors of polyamine production and metabolism as well as inhibitors of PGE2 production. PATIENTS AND METHODS Patients Nine patients (seven women, two men) were studied. All patients fulfilled the American College of Rheumatology
criteria for the diagnosis of RA and had a mean age of 55 years. SF was drawn from the patients' knee effusions and incubated with 50 ,ug/ml hyaluronidase (Sigma Chemical Co, St Louis, MO). MNC preparation MNC from SF were prepared by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation. SF samples, diluted 1/1 with RPMI 1640, were layered over Ficoll-Hypaque and centrifuged for 20 min at room temperature, 200 g. The fraction at the interface contained MNC. These cells were washed three times and used for polyamine determination and IL-2 production.
Culture medium Bovine serum contains polyamine oxidase activity [10]. Therefore, a serum-free medium (HBO1, New England Nuclear, Boston, MA) was used throughout this project. Assay for IL-2 secretion MNC were suspended in 24-well plates (Costar, Cambridge, MA) at 2 x 1 06/ml and various reagents were added. Stimulation was always with 2 pg/ml phytohaemagglutinin (PHA) (Burroughs-Wellcome, Durham, NC). After 24 h, supernatants were harvested and stored at 4 C until they were assayed. IL-2 activity was detected in 96-well plates (Costar) using a murine IL-2-dependent cytotoxic T cell line (CTLL-2) in which proliferation (detected by thymidine incorporation) reflected the IL-2 levels in the supernatants assayed [8].
Polyamine analysis
Correspondence: Eliezer Flescher, PhD, Research Assistant Professor, Department of Medicine, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7874, USA.
Samples of 3 x 106 fresh MNC were pelleted and frozen at -20 C until they were assayed in triplicate. The preparation of
435
E. Flescher, T. L. Bowlin & N. Talal
436
20
the cells for reversed-phase HPLC and the subsequent polyamine analysis were done according to Bowlin et al. [11].
(a) m
Reagents
x-methyl ornithine (amo, a polyamine biosynthesis inhibitor) was from Sigma. The N-2,3-butadienyl derivative of putrescine (a polyamine oxidase inhibitor) (MDL 72 527-DA) (MDL) was a gift from Marion Merrell Dow Research Institute, Cincinnati, OH. Piroxicam (Feldene, an inhibitor of prostaglandin biosynthesis) was a gift from Pfizer Chemicals, New York, NY. Sodium diclofenac (Voltaren, an inhibitor of prostaglandin biosynthesis) was a gift from the Ciba Geigy Corporation, Summit, NJ.
R =0 .84 N -J H
a
10
Go
Statistical analysis The statistical significance of the results was calculated using a one-tailed Student's t-test. Linear regression curves were obtained using the cricket graph, version 1.1, software.
0
200
400
600
pmole/million cells
20 (b) (b
RESULTS
The relation between polyamine content and IL-2 production in RA SF MNC Since average polyamine levels in SF MNC are higher than in normal MNC and oxidation products of polyamines downregulate IL-2 production in RA peripheral blood MNC [8], we assessed the relation between polyamine content and IL-2 production in nine RA SF MNC samples. There was a strong inverse correlation between those two parameters (Fig. 1). A similar inverse correlation was found between IL-2 production and putrescine levels (data now shown).
R-0.80 E N -J cD
J~
10
U
0
Inhibitors of polyamine metabolism enhance IL-2 production by RA SF MNC In accordance with the inverse correlation between IL-2 production and polyamine content, inhibitors of polyamine biosynthesis (amo) and polyamine oxidase (MDL) were able to enhance IL-2 production in RA SF MNC (Table 1). We have previously shown [7] that the same concentration of aMO (50 mM) reduces the intracellular levels of putrescine and spermidine by 80%1/o and 70%, respectively. Inhibitors of prostaglandin production were also potent enhancers of IL-2 production in RA SF MNC, as expected (Table 1). The various reagents were used at optimal concentrations [8]. DISCUSSION We have previously reported that IL-2 production by normal and RA peripheral blood MNC is down-regulated by products of polyamine oxidation [7,8]. In the present study we focused on RA synovial fluids, a site of intense inflammation, and found that MNC from this anatomic compartment produce IL-2 in inverse correlation to their polyamine content. The decreased production of IL-2 by RA SF MNC could be up-regulated by inhibitors of polyamine biosynthesis and metabolism as well as by inhibitors of prostaglandin production. The interaction of polyamine oxidase with polyamines generates products that suppress IL-2 production [7,8]. Average polyamine levels are elevated in RA SF MNC [8] and significant
100
200
300
pmole/million cells Fig. 1. Inverse correlation between IL-2 production and polyamine concentrations in rheumatoid arthritis (RA) synovial fluid (SF) mononuclear cells (MNC). RA SF MNC were assayed in two ways: (i) Three million cells were assayed for polyamine content; (ii) cells were stimulated with phytohaemagglutinin (PHA) for 24 h, supernatants were harvested and assayed for IL-2 activity. Nine SF MNC samples were studied and the linear regression curve is presented. (a) Spermine. (b) Spermidine.
levels of polyamine oxidase are present in RA SF [9]. Thus, both the enzyme and its products are present in the RA inflammatory site and may contribute to the decreased IL-2 production characteristic of RA SF MNC. We have shown that monocytes are the source of polyamine oxidase activity in the MNC population [8]. The hypothesis that IL-2 production in the inflamed joint is regulated by a polyamine-dependent mechanism is supported by our finding that IL-2 production by RA SF MNC is lower when polyamine levels are higher. Moreover, blocking polyamine biosynthesis (by ocMO [7,8]), or polyamine oxidation (by MDL [7,8]) were both effective in enhancing IL-2 production by RA SF MNC. In a previous publication [8], we reported that 3 days rest enhanced significantly the production of IL-2 by peripheral blood MNC while no such effect was observed with SF MNC
437
IL-2 production in RA synovialfluids Table 1. Modulation of IL-2 production in rheumatoid arthritis synovial fluid mononuclear cells
Condition None amo (50 mM) MDL (10-6 M)
Piroxicam (10-5 M) Sodium diclofenac (10-5 M)
IL-2 (U/ml)
68+1-1
I1-9+0.9* 10.0+ l-Ot 12 1 + 1l3t 11-7+ 1.0*
Rheumatoid arthritis synovial fluid mononuclear cells were stimulated for 24 h by phytohaemagglutinin (PHA) in the presence of various reagents. Supernatants were harvested and assayed for IL-2 activity. The results shown are means ofsix synovial fluid (SF) samples + s.e.m. Significance of difference in relation to 'none'. *P
Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids E. FLESCHER*, T. L. BOWLINt & N. TALAL*j *Department of Medicine, The University of Texas Health Science Center, $The Audie L. Murphy Memorial Veterans Hospital, San Antonio, Texas, and tMarion Merrell Dow Research Institute, Cincinnati, Ohio, USA
(Acceptedfor publication 7 October 1991)
SUMMARY Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids. Keywords IL-2 rheumatoid arthritis polyamines
INTRODUCTION T lymphocytes from peripheral blood, synovial fluid (SF) and synovial tissues of rheumatoid arthritis (RA) patients produce low quantities of IL-2 in comparison with T lymphocytes from peripheral blood of healthy individuals. Similar findings were obtained using a variety of stimuli [1-4]. Several products of monocyte activity, prostaglandin E2 (PGE2) [5,6] an IL- I inhibitor [2] and products of polyamine oxidase [7,8], were found to down-regulate IL-2 production and are candidate molecules possibly responsible for the depressed production of IL-2 by RA SF cells.
Polyamine oxidase metabolizes polyamines, like spermidine and spermine, into hydrogen peroxide, ammonia and an aldehyde [8]. RA SF mononuclear cells (MNC) contain elevated levels of polyamines [8] and polyamine oxidase activity is present in RA SF [9]. We looked for a possible relationship between polyamine levels and IL-2 production in a series of individual samples of RA SF MNC. We did this in the presence of monocytes since the poor response of RA SF MNC to mitogens is predominantly monocyte-mediated [2]. In addition, we attempted to correct IL-2 production by RA SF cells using inhibitors of polyamine production and metabolism as well as inhibitors of PGE2 production. PATIENTS AND METHODS Patients Nine patients (seven women, two men) were studied. All patients fulfilled the American College of Rheumatology
criteria for the diagnosis of RA and had a mean age of 55 years. SF was drawn from the patients' knee effusions and incubated with 50 ,ug/ml hyaluronidase (Sigma Chemical Co, St Louis, MO). MNC preparation MNC from SF were prepared by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation. SF samples, diluted 1/1 with RPMI 1640, were layered over Ficoll-Hypaque and centrifuged for 20 min at room temperature, 200 g. The fraction at the interface contained MNC. These cells were washed three times and used for polyamine determination and IL-2 production.
Culture medium Bovine serum contains polyamine oxidase activity [10]. Therefore, a serum-free medium (HBO1, New England Nuclear, Boston, MA) was used throughout this project. Assay for IL-2 secretion MNC were suspended in 24-well plates (Costar, Cambridge, MA) at 2 x 1 06/ml and various reagents were added. Stimulation was always with 2 pg/ml phytohaemagglutinin (PHA) (Burroughs-Wellcome, Durham, NC). After 24 h, supernatants were harvested and stored at 4 C until they were assayed. IL-2 activity was detected in 96-well plates (Costar) using a murine IL-2-dependent cytotoxic T cell line (CTLL-2) in which proliferation (detected by thymidine incorporation) reflected the IL-2 levels in the supernatants assayed [8].
Polyamine analysis
Correspondence: Eliezer Flescher, PhD, Research Assistant Professor, Department of Medicine, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7874, USA.
Samples of 3 x 106 fresh MNC were pelleted and frozen at -20 C until they were assayed in triplicate. The preparation of
435
E. Flescher, T. L. Bowlin & N. Talal
436
20
the cells for reversed-phase HPLC and the subsequent polyamine analysis were done according to Bowlin et al. [11].
(a) m
Reagents
x-methyl ornithine (amo, a polyamine biosynthesis inhibitor) was from Sigma. The N-2,3-butadienyl derivative of putrescine (a polyamine oxidase inhibitor) (MDL 72 527-DA) (MDL) was a gift from Marion Merrell Dow Research Institute, Cincinnati, OH. Piroxicam (Feldene, an inhibitor of prostaglandin biosynthesis) was a gift from Pfizer Chemicals, New York, NY. Sodium diclofenac (Voltaren, an inhibitor of prostaglandin biosynthesis) was a gift from the Ciba Geigy Corporation, Summit, NJ.
R =0 .84 N -J H
a
10
Go
Statistical analysis The statistical significance of the results was calculated using a one-tailed Student's t-test. Linear regression curves were obtained using the cricket graph, version 1.1, software.
0
200
400
600
pmole/million cells
20 (b) (b
RESULTS
The relation between polyamine content and IL-2 production in RA SF MNC Since average polyamine levels in SF MNC are higher than in normal MNC and oxidation products of polyamines downregulate IL-2 production in RA peripheral blood MNC [8], we assessed the relation between polyamine content and IL-2 production in nine RA SF MNC samples. There was a strong inverse correlation between those two parameters (Fig. 1). A similar inverse correlation was found between IL-2 production and putrescine levels (data now shown).
R-0.80 E N -J cD
J~
10
U
0
Inhibitors of polyamine metabolism enhance IL-2 production by RA SF MNC In accordance with the inverse correlation between IL-2 production and polyamine content, inhibitors of polyamine biosynthesis (amo) and polyamine oxidase (MDL) were able to enhance IL-2 production in RA SF MNC (Table 1). We have previously shown [7] that the same concentration of aMO (50 mM) reduces the intracellular levels of putrescine and spermidine by 80%1/o and 70%, respectively. Inhibitors of prostaglandin production were also potent enhancers of IL-2 production in RA SF MNC, as expected (Table 1). The various reagents were used at optimal concentrations [8]. DISCUSSION We have previously reported that IL-2 production by normal and RA peripheral blood MNC is down-regulated by products of polyamine oxidation [7,8]. In the present study we focused on RA synovial fluids, a site of intense inflammation, and found that MNC from this anatomic compartment produce IL-2 in inverse correlation to their polyamine content. The decreased production of IL-2 by RA SF MNC could be up-regulated by inhibitors of polyamine biosynthesis and metabolism as well as by inhibitors of prostaglandin production. The interaction of polyamine oxidase with polyamines generates products that suppress IL-2 production [7,8]. Average polyamine levels are elevated in RA SF MNC [8] and significant
100
200
300
pmole/million cells Fig. 1. Inverse correlation between IL-2 production and polyamine concentrations in rheumatoid arthritis (RA) synovial fluid (SF) mononuclear cells (MNC). RA SF MNC were assayed in two ways: (i) Three million cells were assayed for polyamine content; (ii) cells were stimulated with phytohaemagglutinin (PHA) for 24 h, supernatants were harvested and assayed for IL-2 activity. Nine SF MNC samples were studied and the linear regression curve is presented. (a) Spermine. (b) Spermidine.
levels of polyamine oxidase are present in RA SF [9]. Thus, both the enzyme and its products are present in the RA inflammatory site and may contribute to the decreased IL-2 production characteristic of RA SF MNC. We have shown that monocytes are the source of polyamine oxidase activity in the MNC population [8]. The hypothesis that IL-2 production in the inflamed joint is regulated by a polyamine-dependent mechanism is supported by our finding that IL-2 production by RA SF MNC is lower when polyamine levels are higher. Moreover, blocking polyamine biosynthesis (by ocMO [7,8]), or polyamine oxidation (by MDL [7,8]) were both effective in enhancing IL-2 production by RA SF MNC. In a previous publication [8], we reported that 3 days rest enhanced significantly the production of IL-2 by peripheral blood MNC while no such effect was observed with SF MNC
437
IL-2 production in RA synovialfluids Table 1. Modulation of IL-2 production in rheumatoid arthritis synovial fluid mononuclear cells
Condition None amo (50 mM) MDL (10-6 M)
Piroxicam (10-5 M) Sodium diclofenac (10-5 M)
IL-2 (U/ml)
68+1-1
I1-9+0.9* 10.0+ l-Ot 12 1 + 1l3t 11-7+ 1.0*
Rheumatoid arthritis synovial fluid mononuclear cells were stimulated for 24 h by phytohaemagglutinin (PHA) in the presence of various reagents. Supernatants were harvested and assayed for IL-2 activity. The results shown are means ofsix synovial fluid (SF) samples + s.e.m. Significance of difference in relation to 'none'. *P
