Stimulatory effects of anti-rheumatic drugs on human neutrophil functions

lmmunopharmacoloKv, 16 (1988) 157 165 Elsevier

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Stimulatory effects of anti-rheumatic drugs on human neutrophil functions J.A. Oben a n d J.C. F o r e m a n Department qf Pharmacology, University Colh,ge London, Gower Street, London WCIE 6BT, U.K. (Received [ February 1988; accepted 16 August 1988)

Auranofin (AF), D-penicillamine (D-pen) and thiola are prescribed as disease-modifyingdrugs in the treatment of rheumatoid arthritis (RA). We have shown here that auranofin, 10 .8 to [ 0 - 6 M, D-penicillamine, [0 6 to 10 -3 M, thiola, 10 7 to 10 3 M, and the tripeptide thiol, glutathione, 10 ~' to l0 3 M, enhanced f-met-leu-phe-induced lysosomal enzyme release and the phagocytic uptake of bacteria by up to 40%. The previously reported inhibitory effects of AF were only observed at concentrations in excess of those likely to be available to effector cells in vivo. The stimulatory effects of thiola and D-pen occurred at concentrations likely to be available to effector cells in vivo and, therefore, may be of greater clinical relevance. There is evidence that the drugs used in this study exert their effects via a thiol moiety and their therapeutic effect is preceded by an elevation of intracellular thiol levels. Abstract:

Key words:

Human neutrophil: Enzyme release; Phagocytosis; Auranofin; D-Penicillamine; Thiol compound; Rheumatoid arthritis

Introduction In the treatment of rheumatoid arthritis (RA) auranofin (AF), D-penicillamine (D-pen) and thiola or tiopronin are some of the drugs prescribed as second-line or disease-modifying drugs following the failure of the non-steroidal anti-inflammatory drugs (NSAIDs) to control the progression of the disease (Goddard, 1984). The introduction of these and other disease-modifying anti-rheumatic drugs (DMARDs) has largely been empirical and, to date, there has not been a Correspondence: Dr J.C. Foreman, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, U.K. Abbreviations." AF, auranofin; D-pen, D-penicillamine; RA, rheumatoid arthritis; NSA1D, non-steroidal anti-inflammatory drug; DMARD, disease-modifying anti-rheumatic drug; OMPI, DL-2-oxo-3-(2-mercaptoethyl-5-phenylimidazolidine); ATM, aurothiomalate; TATG, tetraacetylthioglucose; DMSO, dimethyl sulphoxide; FITC, fluorescein isothiocyanate: LDH, lactate dehydrogenase.

systematic synthesis of compounds targeted against specific factors known to be of aetiological and pathological relevance in RA. What is required at present, therefore, is an appraisal of these structurally diverse compounds to determine any similarities which might indicate their mode of action and thus allow a more rational approach to the design of anti-rheumatic drugs. Besides the slow onset of clinical effects, the D M A R D s also have similar toxicity profiles: rashes and taste disturbances occur in nearly all cases (Goddard, 1984). The most striking structural similarity between many of these compounds is the presence of a thiol group either in the native compound or in its metabolites. Thus the active metabolite of levamisole, OMPI (DL-2-oxo-3-(2-mercaptoethyl-5-phenylimidazolidine)), is a thiol, as are the metabolites of azathioprine, 6-mercaptopurine and methylnitrothiomidazole. Thiol participation in the activity of aurothiomalate (ATM) is also indicated by the detection of the free thiomalate moeity both in the plasma and urine

0162-3109/88/$03.50 (.~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)

158 of patients receiving ATM (Rudge et al., 1984). There are also reports that sodium thiomalate alone is effective in RA (Munthe and Jellum, 1980). With auranofin one of the proposed metabolites is tetraacetylthioglucose (TATG) (Crooke et al., 1986; Snyder et al,, 1987). The angiotensin-converting enzyme inhibitor, captopril, which is also a thiol, has recently been shown to possess an anti-rheumatic effect (Martin et al., 1984). Neutrophils are the predominant cells in synovial fluid in the acute process involving rheumatoid joints, and various studies have been undertaken to determine the role of neutrophils in the inflammation of rheumatoid arthritis (Weissmann and Korchak, 1984). These cells, since they release degradative lysosomal enzymes, highly reacti'Je oxygen metabolites and pro-inflammatory eicosanoids, have also been implicated in the chronicity of RA (Lunec et al., 1985). We have examined in this study the actions of some thiols on neutrophil function and we report that AF, ATM, D-pen and thiola at concentrations attainable in vivo paradoxically stimulate neutrophil functions. Inhibitory effects, when seen, occurred only with AF at higher concentrations in excess of those likely to be free to effector cells in vivo.

(ii) Release of fl-glueuroni&lse, lysozyme and lactaw dehydrogenase

Methods

Drug solutions were added to pre-incubated cells (37°C, 10 min, 3 4 x 10(' cells) suspended in Tyrode solution (composition: 137 mM NaCI, 2.7 mM KCI, I mM MgCI, 1 mg/ml glucose and 20 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) pH 7.2) and followed 30 min later by cytochalasin B, 10 mg/ml, and after a further 10 min by f-met-leu-phe (f-met), 10 ~' M. Reactions were terminated after 20 rain by immersing the tubes in ice and centrifuging at 300 x g for 10 min. Supernatants were assayed For enzymes as below. Appropriate controls, detailed in the results, were always undertaken. The dose of f-met was shown previously to elicit approximately 70% of maximal response. Although inflammation in rheumatoid arthritis is sterile, t-met was used as a stimulus because previous studies have been done with it in this context (Wolach et al., 1982: Hafstrom et al., 1984a; Marcolongo et al., 1985) and some reported effects of auranofin have been stimulus-independent (Hafstrom et al.. 1984a). The peptide l-met is also chemically a well-defined stimulus, where the transduction of ligand-receptor interaction at the cell surface into intracellular biochemical information is well understood (Snyderman and Pike, 1984; Sklar et al., 1984).

(i) Separation of neutrophils

(iii) Enzyme assa.vs

Neutrophils were prepared from human whole blood essentially by the method of Boyum (1968). Briefly, following dextran sedimentation of erythrocytes, neutrophils were separated from remaining plasma by density-gradient centrifugation over hypaque-ficoll, and contaminating erythrocytes were removed by hypotonic lysis at 4°C. The procedure yielded neutrophils with purity above 95% and a viability, assessed by trypan blue exclusion, of at least 95%.

fl-Glucuronidase was assayed by measuring fluorimetrically the release of methylumbelliferone from methylumbelliferyl-fi-D-glucuronide (Bennett et al., 1980). Lysozyme was assayed by spectrophotometrically following the rate of lysis of Mieroeoccus lysodeikticus (Becker et al., 1985), and lactate dehydrogenase (LDH) was assayed by spectrophotometrically measuring the conversion of NADH to NAD during the reduction of pyruvate to lactate by LDH (Gomperts et al., 1983; Barrett, 1972).

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(iv) Drugs 150

With the exception of auranofin and thiola, which were gifts from Hoechst (UK) Ltd., all other drugs and reagents were obtained from Sigma. Stock solutions of cytochalasin B and f-met-leuphe, stored at 20°C, were prepared in dimethyl sulphoxide (DMSO) and appropriate dilutions were obtained with Tyrode solution. Stock solutions of auranofin were prepared for each experiment with DMSO and similarly diluted with Tyrode solution. The concentration of DMSO in the reaction mixture was always less than 0.4% (v/v). At this concentration the effects of DMSO were not different from those observed in Tyrode solution alone. All other drugs were water-soluble and prepared fresh for each experiment in Tyrode solution.

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(v) Phagocytosis of fluorescein-isothiocTanate (FITC) labelled Micrococcus lysodeikticus ( M L ) The protocol for this assay has been thoroughly described previously (Vray et al., 1980; Oben and Foreman, 1988) but, briefly, drugs were pre-incubated at 37°C with neutrophils for 45 rain and then labelled bacterial suspension was added for a further 1 h. The reaction was then quenched by addition of ice-cold Tyrode solution. Following two washes and centrifugations, the non-ingested bacteria were removed by incubation with a solution of lysozyme. After another two centrifugations and washes, the ensuing pellet was resuspended, the cells were disrupted with Triton X-100 and the fluorescence was read at 490 nm excitation and 520 nm emission.

Results

(i) Auranofin on f met induced fl-glucuronidase release Fig, I shows that auranofin (AF) at concentrations of 10 ~ to 10 6 M caused a dose-dependent increase in the amount of fl-glucuronidase released by f-met (10 -6 M). This enhancement ranged from about 10% at 10 -8 M to about 40% at 10 6 M.

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Fig. 1. AF on f-met-induced fl-glucuronidase release. AF at the concentrations shown was preincubated at 37°C with neutrophils for 40 min before induction of enzyme release by 10 ~' M f-met. The graph represents data from at least three donors, each acting as their own control; each point is the mean ± S.E.M. of at least three such determinations. Values are expressed as a percentage of the control response to 10 6 M f-met, which was 20 4- 3% of the amount of enzyme released by 0.1% TX-100. Statistical significance from control was determined with the paired Student t-test;*p < 0.05.

A b o v e 10 -6 M, AF reduced the response to f-met. AF also had qualitatively similar effects on the fmet-induced release of lysozyme.

( ii) Thiola ( tiopronin ) on f-met-induced fl-glucuronidase release Fig. 2 shows that thiola caused a dose-dependent enhancement of f-met-induced fl-glucuronidase release. This effect ranged from about 7% at 10 _7 M to about 30% at 10 .3 M. Thiola also had qualitatively similar effects on the f-met-induced release of lysozyme.

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(iv) Qvtotoxici O, o[ AF, lhiola, D~pen and reduced glutathione

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Concentration of thiola (MI Fig. 2. Thiola on f-met-induced fi-glucuronidase release. Thiola at the concentrations shown was preincubated at 37°C with neutrophils for 40 min be%re induction of enzyme release by 10 " M f-met. The graph represents data from at least three donors, each acting as their own control: each point is the mean ± S.E.M. of at least three such determinations. Values are expressed as a percentage of the control response to l0 " M f-met, which was 33 :t: 8% of the amount of enzyme release by 0.1% TX-100. Statistical signillcance from control was determined with the paired Student /-test: *p < 0.05.

Over the time-course of these experiments the unstimulated release of fi-glucuronidase was 6.0 ± 0.4% of total cell enzyme (n = 51). The enhanced levels of enzymes released by f-met in the presence of AF, thiola, D-pen and reduced glutathione (GSH) were not due to cytotoxic effects of these drugs because, under the reaction conditions used, the drugs did not release quantities of L D H at concentrations up to 10 3 M (D-pen, thiola) and 10 4 M (AF) which were significantly greater than the release in the presence of Tyrode solution. The L D H release in Tyrode solution was 7.0 :k 0.3% of total (n = 4).

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Fig. 3 shows that D-penicillamine (D-pen) dose-dependently enhanced f-met-induced release of fi-glucuronidase. This effect ranged from about 10% at 10 ~'M to about 20% at 10 a M. D-pen also had qualitatively similar effects on the f-met-induced release of lysozyme. Fig. 3 also shows that reduced glutathione (GSH) dose-dependently enhanced f-met-induced release of fi-glucuronidase. This effect ranged from about 8% at 10 6 M to about 20% a t 1 0 3M. Thedisul_ phide form of glutathione (GSSG) at concentrations of 10 a M and 10 s M had effects which were not statistically significantly different from those of GSH. GSH also had qualitatively similar effects on the f-met-induced release of lysozyme.

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Concentration (MI Fig. 3. D-pen and GSH on f-met-induced fl-glucuronidasc release, D-pen ( O ) and G S | | (11) at tile concentrations shown were pre-incubated at 37°C with neutrophils l\)r 40 rain bclk)re induction of enzyme release by 10 ~' M f-met, The graphs represent data from at least three donors, each acting as their own control: each point is the mean i S.E.M. of at least three such determinations. Values arc expressed as a percentage of tile control response to 10 (' M f-met, which was 27 :k 3(!/o(O) and 42 :k 8% (11) of the amount of enzyme released by 0.1% TX-100. Statistical significance from control was determined with the paired Student /-test; *p > 0,05.

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(v) Secretory ability olAF, thiola and D-pen 130

To determine whether these drugs were secretagogues, the release experiments were repeated but f-met was omitted. These studies showed that, in the absence off-met, AF at 10 - 9 t o 10 - 4 M , thiola at 10 -9 to 10 - 3 M and D-pen at 10 9 t o 10 - 3 M did not significantly increase the levels of fl-glucuronidase, lysozyme and L D H above those released by Tyrode solution alone.

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(vi) AF, thiola and D-pen ~ff'ects on released em

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To ascertain that the apparent enhancing and inhibitory effects of AF, thiola and D-pen on enzyme release were not due to actions of these drugs directly on the released enzymes, TX-100 cell lysates were incubated with appropriate concentrations of the drug. The studies showed that AF at concentrations between 10 9 M and 10 4 M and thiola and D-pen at 10 3 M did not affect the activities offl-glucuronidase, lysozyme or L D H .

(vii) AF on uptake of M L - F I T C Fig. 4 shows that A F at concentrations less than or equal to 3 × 10 7 M enhanced uptake of MLF I T C whilst inhibiting uptake only at concentrations in excess of this. At 3 x 10 7 M AF elicited an enhancement of about 30% above control. AF at concentrations between 10 9 M and 10 -4 M has no intrinsic fluorescence at the wavelengths used. Moreover, the concentration of D M S O in the reaction mixtures was always less than 0.005% (v/v) and its effects at this concentration were not different from those elicited by Tyrode solution. At the concentrations used, AF did not quench the fluorescence of FITC.

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Fig. 4. Modulation of uptake of ML-FITC by auranofin. AF at the concentrations shown was pre-incubated at 37°C with neutrophils for 40 min. ML-FITC was then added at a bacterial/ neutrophil ratio of 300:1 for a further ! h. The graph represents data from at least three donors, each acting as their own control; each point is the mean • S.E.M. of at least three such determinations. Values are expressed as a percentage of the control response. Statistical significance from control was determined with the paired Student t-test; * p < 0.05 ** p < 0,01.

these concentrations A T M has no intrinsic fluorescence at the wavelengths used. Fig. 5 also shows that thiola dose-dependently enhanced the uptake of M L - F I T C . This effect ranged from about 6% at 10 - 4 M to about 23% at 10 _3 M. At these concentrations thiola has no intrinsic fluorescence at the wavelengths used.

(viii) A TM and thiola on uptake of M L - F I T C

(ix) D-pen and GSH on uptake of M L - F I T C

Fig. 5 shows that A T M dose-dependently enhanced the uptake of M L - F | T C . This effect ranged from about 6% at 10 - s M to about 22% at 10 a M . At

Fig. 6 shows that D-pen dose-dependently enhanced the uptake of M L - F I T C . This effect ranged from about 4% at 10 - 9 M to about 20% at 10 - 4

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Concentration M Fig. 5. ATM ( • ) and thiola (11) on uptake of ML-FITC. ATM and thiola at the concentrations shown were pre-incubated at 37°C with neutrophils for 40 min. ML-FITC was then added at a bacterial/neutrophil ratio of 300:1 for a further I h. The graphs represent data from at least three donors, each acting as their own control: each point is the mean ± S.E.M. of at least three such determinations. Values are expressed as a percentage ofthc control response. Statistical significance from control was determined with the paired Student l-test: *p < 0.05.

M. At these concentrations D-pen has no intrinsic fluorescence at the wavelength used. Fig. 6 also shows that G S H dose-dependently enhanced the uptake of M L - F I T C . This effect ranged from about 19% at 10 5 M to about 40% at 10 3 M. At these concentrations G S H has no intrinsic fluorescence at the wavelengths used.

Discussion

The present results demonstrate that AF at concentrations up to 10 -6 M enhances f-met-induced release of azurophilic granules from human neutrophils. Above 10 6 M, AF inhibits release. The thiols GSH, D-pen and thiola between 10 8 and 10 `3 M also enhanced f-met-induced enzyme re-

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Concentration (M) Fig. 6. D-pcn ( • ) and GSH ( • ) on uptake of M L- FITC. D-pen and GSH at the concentrations shown were pro-incubated at 37°C with neutrophils for 40 rain. ML-F1TC was then added at a bacterial,,neutrophil ratio of 300:1 for a further I h. The graphs represent data from at least three donors, each acting as their own control: each point is the mean ± S.E.M. of at least three such determinations. Values are expressed as a percentage of the control response. Statistical significance from control was determined with the paired Student t-test: *p < (I.05, **p < 0.01.

lease. At the concentrations used. none of the compounds was cytotoxic as judged by measurements of L D H release. Also they did not affect the activities of the released enzymes nor cause release by themselves. Furthermore, phagocytosis was potentiated by these drugs, whereas if they had a significant cytotoxic effect an inhibition of phagocytosis would have been expected. Besides lysosomal enzyme release, superoxide radical generation and adherence of human neutrophils were also stimulated by low concentrations of AF in the studies of Hafstrom et al. (1985). MarcoIongo et al. (1985) further extended these studies to

163 include a stimulatory effect of AF on the chemotaxis, in vitro, of neutrophils from rheumatoid arthritic patients. Using a highly quantitative fluorescent assay of phagocytosis, in vitro, improved in our laboratory (Vray et al,, 1980; Oben and Foreman, 1988) we have also shown here a stimulatory effect of AF on the uptake of labelled bacteria by human neutrophils. In these studies AF was stimulatory at concentrations as low as 10 + M to 3 x 10 7 M and D-pen and GSH were similarly stimulatory at concentrations as low as 10 s M. ATM was also stimulatory at concentrations in excess of 10 8 M and thiola at concentrations above 10 -s M. Now, although human serum levels of gold following oral administration of AF at 6 mg/day range between 0.3 and 1 /xg/ml (equivalent to 2 5 /~M AF), a very high proportion ( ~ 99%) is bound to serum components or associated with erythrocytes (Davis, 1984; Blodgett et al., 1984). In fact, the quantity of AF gold which is present unbound and thus likely to be available to eflEctor cells is of the order of 3 40 ng/ml (equivalent to 15 200 nM AF) (Lorber et al., 1982, 1983). Similarly with ATM at a dose of 50 mg intramuscularly each week serum gold levels are 2-7/,g/ml, but again the levels unbound are in the range 3 40 ng/ml (equivalent to 15-200 nM ATM) (Lorber et al., 1982, 1983). To date, no evidence has been found to show a relationship between the clinical efficacies of these drugs and their total blood gold levels (Gerber et al., 1972; Bandilla et al., 1982). Lorber et al. (1982) have therefore suggested that the unbound serum gold levels may be more relevant to an understanding of the actions of these drugs in vivo. We have shown here that with AF at concentrations likely to be free and therefore available to effector cells in vivo, there is an apparent enhancement of neutrophil function. ATM at similar concentrations was also stimulatory. The consistently reported inhibitory effects of AF (e.g. Stephanus et al., 1985; Dimartino et al., 1977) occur at concentrations in excess of the lower ranges shown here to be stimulatory and correspond more to the total blood levels rather than the unbound (free) levels. In extrapolating the free concentrations of the native

compound from the unbound gold levels, it has been assumed that the complex and its gold moiety remain in the same molar proportions as they are metabolised. The proposal that the D M A R D s may function by a unitary mechanism possibly involving their thiol groups is supported further by the fact that, like GSH, D-pen and thiola also enhanced neutrophil functions. These stimulatory effects of D-pen and thiola may also be therapeutically relevant, since the concentration of these drugs present unbound in vivo, about 30/~M (Capasso, 1981; Butler et al., 1982), is within the ranges shown here to enhance neutrophil functions. This study does not indicate the role of intracellular thiol/disulphide ratios in the effects described, but no difference in the activity of GSSG compared with GSH was observed. A previous study by Broome and Jeng (1973) on lymphoid cell replication found no difference between the activity of thiols and disulphides. GSH itself does not permeate the cell membrane (Meister, 1983). Hence, it may act at the cell surface or alternatively it may be broken down by membrane GSH-transpeptidase to yield amino acids which are taken up into the cell. The amino acids taken up could be utilized as substrates in the 7-glutamyl cycle and hence alter the GSH and mixed disulphide levels within the cell (Meister, 1983). A separate study of the actions of the drugs on intracellular thiol and disulphide levels is being undertaken. The data show that auranofin is more potent than the other thiols used in this study and a different mechanism of action for this drug cannot be ruled out. Auranofin is rapidly metabolized to tetraacetylthioglucose by macrophages (Snyder et al., 1986) but it is not known at present whether it is this thiol compound which is responsible for the potentiation of enzyme release and phagocytosis. That the serum from patients with RA contains a significantly lowered thiol concentration compared with controls and that this deficiency correlates with the disease severity (Banford et al., 1982) has been interpreted as corroborating the role of thiols in the aetiology and pathogenesis of RA (Banford et al., 1982). Moreover, D-pen and other D M A R D s elevate intracellular thiol (GSH) concentrations

164 prior to a clinical response to these drugs (Munthie et al., 1981). The immunomodulatory effect of n o n - D M A R D thiols is very well documented (Fanger et al., 1970; Ohmori et al., 1982" G o o d m a n et al., 1981: Meisten 1983). Recently Eidelus et al. (1986) showed that depletion of murine lymphocyte glutathione reduced their transformation and proliferation responses to mitogens. They concluded that intracellular thiols are involved in the regulation eL at least these, immune cells. A stirnulatory effect of increased intracellular glutathione levels was also reported by these workers. At first sight there appears to be a paradox created by the results in this paper, since D M A R D s are used clinically to suppress a chronic inflamrnatory disease and yet we have shown that they stimulate IL-2 production at clinically relevant concentrations (Oben et al., 1988). However. it is too simple to consider rheumatoid arthritis as a straightforward inflammatory process. There is evidence of defective T cell function in rheumatoid arthritis compared to normal subjects (Felder et al., 1985). Furthermore, IL-2 production and the response to IL-2 appear depressed in rheumatoid arthritis (see Henderson and Edwards, 1987, for review). Hence, D M A R D s may act clinically to modify the disease by correcting defects in I L-2 production and other inflammatory processes. It may be that to call these drugs 'anti-inflammatory" is a misnomer.

Acknowledgement J.A.O. is in receipt of an S.E.R.C. Casc Award with Hoechst (UK) Ltd. as industrial sponsors.

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