0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ©1992 International Society for Immanopharmacology.
Int. J. lmmunopharmac., Vol. 14, No. 7, pp. 1279-1284, 1992. Printed in Great Britain.
SHORT COMMUNICATION I N V I T R O A N D E X VIVO EFFECT OF T I A P R O F E N I C A C I D ON H U M A N P E R I P H E R A L BLOOD M O N O N U C L E A R CELLS WILMA BARCELLINI,*+ MARIA ORIETTA BORGHI,* CRISTINA FAIN,* NICOLETTA DEL PAPA,* PATRIZIA FAVINI* and PIER LUIGI MERONI* *Insitute of Internal Medicine, Infectious Diseases and Immunopathology, University of Milan, Milan; and *Medical Department, Roussel-Pharma, Milan, Italy (Received 26 March 1992 and in final form 10 May 1992)
Abstract -- The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different
human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (1L-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.
Non-steroidal anti-inflammatory drugs (NSAIDs) are powerful and widely used drugs for the treatment of different rheumatic diseases. Although the antiinflammatory as well as the analgesic properties of these compounds are thought to depend mainly on the inhibition of prostaglandin (PG) synthesis, their exact mechanism of action is not fully understood (Abramson & Weissmann, 1989; Cortet & Duquesnoy, 1991). It is widely accepted that PGs can function not only as mediators involved in inflammation, but also as regulators of the immune response (Bray, 1980). In this context, interest has focused on the possible activity of NSAIDs on the immune system. NSAIDs were shown to inhibit polymorphonuclear leukocytes (PMN) functions (Best, Lewis & Patel, 1985; Divincenzo & Venzio, 1987) as well as the production of the rheumatoid factor (Goo0win, Ceuppens & Rodriguez, 1983; Forre, Thoen, Helgetveit & Haile, 1984). At variance, no clear effects were demonstrated on natural killer (NK) activity and interleukin 1 (IL-1) and IL-2 production (Cortet & Duquesnoy, 1991).
These effects were partially related to prostaglandinmediated regulation of the immune response, even if a direct action of NSAIDs on immune cells could not be excluded (Abramson & Weissmann, 1989). Tiaprofenic acid, a propionic acid derivative, is a NSAID which was shown to possess both antiinflammatory and analgesic activity similar to that of several other NSAIDs (Sorkin & Brogden, 1985). The drug was shown to be a powerful inhibitor of PG synthesis, at least twice as potent as diclofenac or indomethacin (Sorkin & Brogden, 1985). However, no data are available on the effect of tiaprofenic acid on the immune system. The aim of this study was to investigate the effect of tiaprofenic acid on different human immune parameters either in vitro or following in vivo administration. Results from the in vitro study show that the drug increases mitogen-induced proliferation and IL-2 production, whereas it decreases polyclonal lg synthesis by normal human mononuclear cells. After an oral treatment in healthy volunteers we found no significant changes of the
*Author to whom correspondence should be addressed at: Istituto di Medicina Interna, Malattie Infettive e Immunopatologia, Padiglione Granelli, Ospedale Policlinico, Via F. Sforza 35, 20122 Milano, Italy. 1279
1280
Short Communication
following parameters: unstimulated and mitogeninduced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels. The significance of these results is discussed.
levels of IgG, IgM and IgA, complement fractions C3 and C4, detected by radial immunodiffusion, and rheumatoid factor, detected by the baemagglutination test
L y m p h o c y t e isolation a n d culture conditions EXPERIMENTAL PROCEDURES
E x p e r i m e n t a l design
The effect of tiaprofenic acid was investigated either in vitro or ex vivo. For the in vitro studies, tiaprofenic acid (Surgamyl R), kindly provided by Roussel-Pharma, Italy, was tested on different immune responses by normal peripheral blood mononuclear cells (PBM). The drug was dissolved in sterile saline at a concentration of 100 mg/ml and then diluted to the final concentrations tested (25, 50 and 100 gg/ml) in RPMI 1640 (Gibco-Life Technologies, Paisley, U.K.) supplemented with 10% foetal calf serum (FCS, Flow-Labs, Irvine, U.K.), penicillin 100 U/ml, streptomycin 100 txg/ml and t.-glutamine 1%, hereafter referred as culture medium (CM). The drug was added at the beginning of the culture period. The following immune parameters were evaluated in the presence or absence of the different concentrations of tiaprofenic acid: (i) spontaneous and phytohaemagglutinin (PHA, Gibco-Life Technologies)-induced PBM-proliferative response; (ii) spontaneous and PHA-stimulated IL-2 production; and (iii) unstimulated and pokeweed mitogen (PWM, Gibco-Life Technologies)induced Ig production. For the ex vivo study, blood samples were collected before (TO) and 2 h after the last administration (T1) of a 7-day oral treatment with tiaprofenic acid (300 mg b.i.d, at 12 h intervals) from eight healthy volunteers (mean age -+ S.D., 35 _+ 7 yr), whose informed consent had been obtained previously. None of the subjects was receiving any other drug at the time of the study. The following parameters were investigated at TO and T 1: (i) PBM spontaneous blastogenesis; (ii) PBMproliferative response induced by different concentrations of P H A and PWM; (iii) spontaneous and PHA-induced IL-2 production; (iv) spontaneous and PWM-induced IgG and IgM synthesis; (v) peripheral blood lymphocyte subpopulations, detected by immunofluorescence with OKT3, OKT4 and OKT8 monoclonal antibodies (Ortho Diagnostic, Raritan, N J), as described previously (Meroni, Barcellini, De Bartolo, Invernizzi & Zanussi, 1984); and (vi) serum
Normal human PBM were obtained from fresh heparinized venous blood by F i c o l - H y p a q u e (Lymphoprep, Nycomed AS, Oslo, Norway) density gradient centrifugation, according to Boyum (Boyum, 1968). For proliferation assays mitogenic stimuli were provided by: (i) P H A at the final concentrations of 4, 1 and 0.25 ~g/ml; and (ii) PWM at the final dilution of 1°70. The PBM-proliferative response was assessed by 3H-thymidine (3H-TdR) uptake after 72 h of culture in a 5% CO2 humidified incubator, as described previously (Meroni, Barcellini, Messina, De Bartolo, Capsoni & Invernizzi, 1982). Cell viability was estimated by the trypan blue exclusion test. For the evaluation of IL-2 production, 2 x 10" PBM were cultured in 12 x 75 mm round-bottomed glass tubes fitted with Morton closures (Sterilin, Feltham, Middlesex, U.K.) in a final volume of 1 ml with or without P H A 4/xg/ml at 37°C in a humidified atmosphere of 5% CO2 and air for 48 h. The supernatants were collected, centrifuged at 900 g for 10 rain and filtered through 0.22 txm filters (Millipore S.A., Molsheim, France). The IL-2 content of the supernatants was estimated either by a CTLL proliferation assay or by competitive radioimmunoassay (Medgenix, Benelux N.V./S.A). A positive linear correlation was demonstrated between biological and immunochemical determinations, as reported elsewhere (Barcellini, Borghi & Meroni, 1988). The CTLL proliferation assay was based on the method of Gillis et al. (Gillis, Ferm, Ou & Smith, 1978) and involved statistical validation of titration data according to the principles of the biological assay by parallel lines as applied to probit analysis of quantitative response, as described previously (Frasca, Adorini, Landolfo & Doria, 1985; Meroni et al., 1987). The radioimmunoassay was performed according to the manufacturer's instructions. Briefly, 100 txl of each sample were incubated overnight with 100 txl of polyclonal rabbit anti-human IL-2 antiserum. Then, 100 tA of ~25I-labelled-recombinant human IL-2 were dispensed in each tube and incubated for 4 h. After addition of anti-rabbit gamma globulin antiserum
1281
Short Communication mixed with polyethylene glycol (PEG), the radioactivity of the precipitate was determined. The IL-2 content of supernatants was calculated referring to a curve, constructed using known amounts of human recombinant IL-2 (from 0 to 50 U/ml). For in vitro immunoglobulin synthesis, 1 × l 0 6 PBM were cultured in 12 × 75 mm round-bottomed glass tubes fitted with Morton closures (Sterilin) in a final volume of 1 ml with or without PWM mitogen (1°70 final concentration) at 37°C in a 5°7o CO2 humidified incubator for 8 days. The amounts of IgG and IgM in the culture supernatants were determined by ELISA, as described previously (Meroni, Barcellini, Sguotti, Capsoni, Palmieri, Guidi & Zanussi, 1987).
A)
tcJ
100000 (b)
(b)
80000
z~ IZ d 6oo0o40000
i
i
25 50 Tiaprofenic Acid ~g/ml)
i
100
(a)
Statistical analysis
Statistical analysis was carried out by using Student's t-test for paired data.
RESULTS
In vitro s t u d y Tiaprofenic acid had no effect on spontaneous 3H-TdR incorporation, IL-2 production and polyclonal Ig synthesis by normal PBM (data not shown). In PHA-stimulated cultures, the drug increased proliferation induced by mitogenic and sub-mitogenic concentrations of this lectin (1 and 0.25 tag/ml, respectively); the variations were statistically significant, even if their magnitude was not marked. On the contrary PHA 4/~g/ml-induced blastogenesis was unchanged by the addition of tiaprofenic acid at the concentrations tested [Fig. I(A)]. PHA-induced IL-2 production was significantly increased by tiaprofenic acid, with a dose-dependent profile [Fig. I(B)]. As regards PWM-stimulated Ig production, we found that tiaprofenic acid induced a dosedependent decrease of both IgG and IgM synthesis, with a statistical significance for drug concentrations of 50 and 100/ag/ml (Fig. 2). Viability studies, performed with the trypan blue exclusion test, showed that, at all the concentrations tested, tiaprofenic acid did not affect cell viability.
Ex vivo s t u d y Data from the e x vivo study showed that treatment with tiaprofenic acid had no significant effects on the in vitro immune parameters investigated (Table 1). In fact, spontaneous and mitogen-stimulated PBM proliferation was comparable at TO and T1
B)
250 200 ~"
150
cJ _~
100'
(a)
50
25 50 Tiaprofenic Acid (,ug/rnl)
100
Fig. 1. (A) In vitro effect of tiaprofenic acid on lymphocyte proliferation induced by different concentrations of PHA: 4 ~g/ml (U]); 1 /~g/ml(11);0.25 /ag/ml (A). Values are given as counts/min, mean _+ S.E. of 19 subjects. (B) In vitro effect of tiaprofenic acid on PHAqnduced IL-2 production. Values are given as U/ml, mean ± S.E. of 10 subjects. Statistical significance by Student's t-test for paired data: (a) P
Int. J. lmmunopharmac., Vol. 14, No. 7, pp. 1279-1284, 1992. Printed in Great Britain.
SHORT COMMUNICATION I N V I T R O A N D E X VIVO EFFECT OF T I A P R O F E N I C A C I D ON H U M A N P E R I P H E R A L BLOOD M O N O N U C L E A R CELLS WILMA BARCELLINI,*+ MARIA ORIETTA BORGHI,* CRISTINA FAIN,* NICOLETTA DEL PAPA,* PATRIZIA FAVINI* and PIER LUIGI MERONI* *Insitute of Internal Medicine, Infectious Diseases and Immunopathology, University of Milan, Milan; and *Medical Department, Roussel-Pharma, Milan, Italy (Received 26 March 1992 and in final form 10 May 1992)
Abstract -- The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different
human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (1L-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.
Non-steroidal anti-inflammatory drugs (NSAIDs) are powerful and widely used drugs for the treatment of different rheumatic diseases. Although the antiinflammatory as well as the analgesic properties of these compounds are thought to depend mainly on the inhibition of prostaglandin (PG) synthesis, their exact mechanism of action is not fully understood (Abramson & Weissmann, 1989; Cortet & Duquesnoy, 1991). It is widely accepted that PGs can function not only as mediators involved in inflammation, but also as regulators of the immune response (Bray, 1980). In this context, interest has focused on the possible activity of NSAIDs on the immune system. NSAIDs were shown to inhibit polymorphonuclear leukocytes (PMN) functions (Best, Lewis & Patel, 1985; Divincenzo & Venzio, 1987) as well as the production of the rheumatoid factor (Goo0win, Ceuppens & Rodriguez, 1983; Forre, Thoen, Helgetveit & Haile, 1984). At variance, no clear effects were demonstrated on natural killer (NK) activity and interleukin 1 (IL-1) and IL-2 production (Cortet & Duquesnoy, 1991).
These effects were partially related to prostaglandinmediated regulation of the immune response, even if a direct action of NSAIDs on immune cells could not be excluded (Abramson & Weissmann, 1989). Tiaprofenic acid, a propionic acid derivative, is a NSAID which was shown to possess both antiinflammatory and analgesic activity similar to that of several other NSAIDs (Sorkin & Brogden, 1985). The drug was shown to be a powerful inhibitor of PG synthesis, at least twice as potent as diclofenac or indomethacin (Sorkin & Brogden, 1985). However, no data are available on the effect of tiaprofenic acid on the immune system. The aim of this study was to investigate the effect of tiaprofenic acid on different human immune parameters either in vitro or following in vivo administration. Results from the in vitro study show that the drug increases mitogen-induced proliferation and IL-2 production, whereas it decreases polyclonal lg synthesis by normal human mononuclear cells. After an oral treatment in healthy volunteers we found no significant changes of the
*Author to whom correspondence should be addressed at: Istituto di Medicina Interna, Malattie Infettive e Immunopatologia, Padiglione Granelli, Ospedale Policlinico, Via F. Sforza 35, 20122 Milano, Italy. 1279
1280
Short Communication
following parameters: unstimulated and mitogeninduced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels. The significance of these results is discussed.
levels of IgG, IgM and IgA, complement fractions C3 and C4, detected by radial immunodiffusion, and rheumatoid factor, detected by the baemagglutination test
L y m p h o c y t e isolation a n d culture conditions EXPERIMENTAL PROCEDURES
E x p e r i m e n t a l design
The effect of tiaprofenic acid was investigated either in vitro or ex vivo. For the in vitro studies, tiaprofenic acid (Surgamyl R), kindly provided by Roussel-Pharma, Italy, was tested on different immune responses by normal peripheral blood mononuclear cells (PBM). The drug was dissolved in sterile saline at a concentration of 100 mg/ml and then diluted to the final concentrations tested (25, 50 and 100 gg/ml) in RPMI 1640 (Gibco-Life Technologies, Paisley, U.K.) supplemented with 10% foetal calf serum (FCS, Flow-Labs, Irvine, U.K.), penicillin 100 U/ml, streptomycin 100 txg/ml and t.-glutamine 1%, hereafter referred as culture medium (CM). The drug was added at the beginning of the culture period. The following immune parameters were evaluated in the presence or absence of the different concentrations of tiaprofenic acid: (i) spontaneous and phytohaemagglutinin (PHA, Gibco-Life Technologies)-induced PBM-proliferative response; (ii) spontaneous and PHA-stimulated IL-2 production; and (iii) unstimulated and pokeweed mitogen (PWM, Gibco-Life Technologies)induced Ig production. For the ex vivo study, blood samples were collected before (TO) and 2 h after the last administration (T1) of a 7-day oral treatment with tiaprofenic acid (300 mg b.i.d, at 12 h intervals) from eight healthy volunteers (mean age -+ S.D., 35 _+ 7 yr), whose informed consent had been obtained previously. None of the subjects was receiving any other drug at the time of the study. The following parameters were investigated at TO and T 1: (i) PBM spontaneous blastogenesis; (ii) PBMproliferative response induced by different concentrations of P H A and PWM; (iii) spontaneous and PHA-induced IL-2 production; (iv) spontaneous and PWM-induced IgG and IgM synthesis; (v) peripheral blood lymphocyte subpopulations, detected by immunofluorescence with OKT3, OKT4 and OKT8 monoclonal antibodies (Ortho Diagnostic, Raritan, N J), as described previously (Meroni, Barcellini, De Bartolo, Invernizzi & Zanussi, 1984); and (vi) serum
Normal human PBM were obtained from fresh heparinized venous blood by F i c o l - H y p a q u e (Lymphoprep, Nycomed AS, Oslo, Norway) density gradient centrifugation, according to Boyum (Boyum, 1968). For proliferation assays mitogenic stimuli were provided by: (i) P H A at the final concentrations of 4, 1 and 0.25 ~g/ml; and (ii) PWM at the final dilution of 1°70. The PBM-proliferative response was assessed by 3H-thymidine (3H-TdR) uptake after 72 h of culture in a 5% CO2 humidified incubator, as described previously (Meroni, Barcellini, Messina, De Bartolo, Capsoni & Invernizzi, 1982). Cell viability was estimated by the trypan blue exclusion test. For the evaluation of IL-2 production, 2 x 10" PBM were cultured in 12 x 75 mm round-bottomed glass tubes fitted with Morton closures (Sterilin, Feltham, Middlesex, U.K.) in a final volume of 1 ml with or without P H A 4/xg/ml at 37°C in a humidified atmosphere of 5% CO2 and air for 48 h. The supernatants were collected, centrifuged at 900 g for 10 rain and filtered through 0.22 txm filters (Millipore S.A., Molsheim, France). The IL-2 content of the supernatants was estimated either by a CTLL proliferation assay or by competitive radioimmunoassay (Medgenix, Benelux N.V./S.A). A positive linear correlation was demonstrated between biological and immunochemical determinations, as reported elsewhere (Barcellini, Borghi & Meroni, 1988). The CTLL proliferation assay was based on the method of Gillis et al. (Gillis, Ferm, Ou & Smith, 1978) and involved statistical validation of titration data according to the principles of the biological assay by parallel lines as applied to probit analysis of quantitative response, as described previously (Frasca, Adorini, Landolfo & Doria, 1985; Meroni et al., 1987). The radioimmunoassay was performed according to the manufacturer's instructions. Briefly, 100 txl of each sample were incubated overnight with 100 txl of polyclonal rabbit anti-human IL-2 antiserum. Then, 100 tA of ~25I-labelled-recombinant human IL-2 were dispensed in each tube and incubated for 4 h. After addition of anti-rabbit gamma globulin antiserum
1281
Short Communication mixed with polyethylene glycol (PEG), the radioactivity of the precipitate was determined. The IL-2 content of supernatants was calculated referring to a curve, constructed using known amounts of human recombinant IL-2 (from 0 to 50 U/ml). For in vitro immunoglobulin synthesis, 1 × l 0 6 PBM were cultured in 12 × 75 mm round-bottomed glass tubes fitted with Morton closures (Sterilin) in a final volume of 1 ml with or without PWM mitogen (1°70 final concentration) at 37°C in a 5°7o CO2 humidified incubator for 8 days. The amounts of IgG and IgM in the culture supernatants were determined by ELISA, as described previously (Meroni, Barcellini, Sguotti, Capsoni, Palmieri, Guidi & Zanussi, 1987).
A)
tcJ
100000 (b)
(b)
80000
z~ IZ d 6oo0o40000
i
i
25 50 Tiaprofenic Acid ~g/ml)
i
100
(a)
Statistical analysis
Statistical analysis was carried out by using Student's t-test for paired data.
RESULTS
In vitro s t u d y Tiaprofenic acid had no effect on spontaneous 3H-TdR incorporation, IL-2 production and polyclonal Ig synthesis by normal PBM (data not shown). In PHA-stimulated cultures, the drug increased proliferation induced by mitogenic and sub-mitogenic concentrations of this lectin (1 and 0.25 tag/ml, respectively); the variations were statistically significant, even if their magnitude was not marked. On the contrary PHA 4/~g/ml-induced blastogenesis was unchanged by the addition of tiaprofenic acid at the concentrations tested [Fig. I(A)]. PHA-induced IL-2 production was significantly increased by tiaprofenic acid, with a dose-dependent profile [Fig. I(B)]. As regards PWM-stimulated Ig production, we found that tiaprofenic acid induced a dosedependent decrease of both IgG and IgM synthesis, with a statistical significance for drug concentrations of 50 and 100/ag/ml (Fig. 2). Viability studies, performed with the trypan blue exclusion test, showed that, at all the concentrations tested, tiaprofenic acid did not affect cell viability.
Ex vivo s t u d y Data from the e x vivo study showed that treatment with tiaprofenic acid had no significant effects on the in vitro immune parameters investigated (Table 1). In fact, spontaneous and mitogen-stimulated PBM proliferation was comparable at TO and T1
B)
250 200 ~"
150
cJ _~
100'
(a)
50
25 50 Tiaprofenic Acid (,ug/rnl)
100
Fig. 1. (A) In vitro effect of tiaprofenic acid on lymphocyte proliferation induced by different concentrations of PHA: 4 ~g/ml (U]); 1 /~g/ml(11);0.25 /ag/ml (A). Values are given as counts/min, mean _+ S.E. of 19 subjects. (B) In vitro effect of tiaprofenic acid on PHAqnduced IL-2 production. Values are given as U/ml, mean ± S.E. of 10 subjects. Statistical significance by Student's t-test for paired data: (a) P
