Vesnarinone is a selective inhibitor of macrophage TNFα release

Int. J. Immunopharmac., Vol. 18,No. 617,pp. 371-378,1996 Copyright 0 1996International Societyfor Immunopharmacology Published by ElsevierScienceLtd. Printed in Great Britain 0192-0561/%$15.00+ .OO

Pergamon

PII: s0192+561(%)ooo37-9

VESNARINONE

IS A SELECTIVE INHIBITOR RELEASE

OF MACROPHAGE

TNFct

TAKU KAMBAYASHI,* NACHMAN MAZUREK,* CHAIM 0. JACOB,t NATHAN WEI,$ MIRANDA FONG* and GIDEON STRASSMANN*P *Department of Immunology, Otsuka-America Pharmaceutical Inc., 9900 Medical Center Drive, Rockville, MD 20850, U.S.A.; Division of Rheumatology and Immunology, University of Southern California, School of Medicine, Los Angeles, CA 90033, U.S.A.; IDepartment of Medicine, University of Maryland, Baltimore, MD 21201, U.S.A. (Received 27 February 1996; revised 29 May 1996)

Abstract-Vesnarinone is an experimental drug that has been used successfully in the treatment of congestive heart failure patients. In this report we investigate the effect of vesnarinone on the cytokine secretory products of mononuclear phagocytes. In a concentration-dependent manner, the drug inhibits the endotoxin(LPS)stimulated release of tumor necrosis factor (TNF)u and suppresses interleukin(IL)-6 release, but does not affect the release of IL-la, IL-10 and leukemia inhibitory factor (LIF) by mouse peritoneal macrophages. Using competitive polymerase chain reaction (PCR) analyses, we find that vesnarinone significantly reduces TNFa. but not IL-10 mRNA. In addition to LPS. the drug inhibits TNFa release induced by several other stimuli. The inhibitory effect of the drug on the TNFa biosynthesis can be observed in differentiated human monocytes, in macrophage cell lines, and in synovial adherent cells from rheumatoid arthritis patients. Although the precise mode of action of vesnarinone in the signal transduction pathway leading to the selective inhibition of TNFa is not known, the drug might be useful in the treatment of diseases involving that cytokine. Copyright 0 1996 International Society for Immunophannacology Keywords: macrophage, monocyte, TNFa, IL-6, vesnarinone.

Mononuclear phagocytes (macrophages) are widely recognized as cells that play a key role in the regulation of immune and inflammatory activities, as well as in tissue remodelling. The execution of these activities is mediated by complex, multifactorial processes involving macrophage secretory products (reviewed by Nathan & Cohn, 1985). Cytokines such as tumor necrosis factor (TNF)u, interleukin(IL)-1, IL-6, IL-10 and leukemia inhibitory factor (LIF) are important macrophage secretory products. A large body of evidence has implicated the excessive or uncontrolled production of cytokines in general, and of TNFu in particular, as contributing to the pathophysiological changes associated with chronic and acute inflammatory conditions, including septic shock, autoimmune diseases, wasting and AIDS (reviewed by Beutler & Cerami, 1989; Vassa!i, 1992). A selective pharmacological intervention aimed at inhibiting the release or acti_,n of TNFcY, may therefore prove beneficial to the treatment of a variety of pathological conditions such as septic shock, rheumatoid arthritis (RA), multiple sclerosis, etc. This in turn led to a

recent research focused on several compounds that inhibit the release of “pro-inflammatory” cytokines, including TNFu. These compounds can be divided into two categories; selective and non-selective inhibitors. Dexamethasone, for example, inhibits the release of several cytokines, including TNFa, by reducing translation post-transcriptionally (Han et al., 1990). Thalidomide, on the other hand, selectively inhibits the release of TNFu from bacterial endotoxin (LPS) stimulated human monocytes (Sampaio et al., 1991), but does not significantly affect the release of other cytokines, including IL-I, IL-6 and GM-CSF. It has recently been shown that thalidomide accelerates the degradation of monocytic TNFa mRNA (Moreira et al., 1993). In yet another example, the selective phosphodiesterase (PDE) type IV inhibitor rolipram is known to be a potent inhibitor of TNFu production (Semmler et al., 1993; Fischer et al., 1993). It has been recently shown that the inhibitory effect of rolipram on TNFu is in fact indirect, and involves the augmentation of IL-10 mRNA and protein (Kambayashi et al., 1995a; Kambayashi et al., 1995b).

?Author to whom correspondence should be addressed. Tel.: 301-424-9055, Fax: 301-424-9054. 371

T. KAMBAYASHI et al.

372

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4dimethoxybenzoyl)-I-piperazinyll-2-quinolinone), is a positive inotropic compound that augments myocardial contractility. It has been shown to improve the quality of life and reduce mortality of congestive heart failure patients (Feldman et al., 1993). In addition to its cardiac effects, vesnarinone possesses several other biological activities. It inhibits viral replication of HIV-l in culture (Maruyama et al., 1993) and at relatively high concentrations reduces both nucleoside and nucleobase transport in several cell types (Kumakura et al., 1995). A preliminary report has shown that the drug inhibits cytokine release from LPS stimulated blood from normal volunteers (Matsumori et ul., 1994). In the present report, we examine the effect of vesnarinone on cytokine release by stimulated macrophages. We show that vesnarinone is a selective inhibitor of macrophage TNFcr mRNA and protein release, irrespective of the inducer used for cell stimulation.

EXPERIMENTAL PROCEDURES Mice

Specific pathogen free, male C57BL/6, 69weeks of age were purchased from Charles River Breeding Laboratories (Wilmington, MA, U.S.A.), and were housed under conventional conditions. Reagents

Recombinant murine interferon (IFN)y was purchased from Biosource International (Camarillo, CA, U.S.A.). LPS (Salmonella enteritides) and hydrogen peroxide were purchased from Sigma Chemical Co (St Louis, MO, U.S.A.). Peptidoglycan polysaccharide (PGPS) was purchased from Leeland Labs (Chapel Hill, NC, U.S.A.). Okadaic acid was purchased from Calbiochem (La Jolla, CA, U.S.A.). Vesnarinone (OPC-8212) was obtained from Otsuka Pharmaceutical Co. (Tokushima, Japan). The drug was dissolved in dimethyl sulfoxide (DMSO) and was immediately diluted in culture medium. In vitro culture and stimulation macrophages

of’murine peritoneal

Thioglycollate broth-elicited macrophages were prepared as reported previously (Strassmann et al., 1994; Kambayashi et al., 1995a,b). After collection, peritoneal lavage cells were centrifuged, resuspended in culture medium (RPM1 1640 supplemented with 10% low-endotoxin fetal bovine serum, Hyclone,

Logan, UT) and 1 x lo6 cells/well were plated in tissue culture 12-well dishes (Costar, Cambridge, MA, U.S.A.). After a 2 h incubation period at 37°C the monolayers were washed twice with complete medium and then treated as indicated. Cell free supernatants were harvested kept frozen (-20°C) until further analysis. Macrophage monolayers were then washed with phosphate-buffered saline (PBS) and cellular protein was quantified. Results are expressed as mean + S.E.M of duplicate determinations of duplicate cultures produced per 100 ug cell protein. Human macrophages and synovial cells

Blood from normal volunteers was diluted in PBS and centrifuged over a Ficoll-Hypaque as described by the manufacturer (Sigma). The layer containing mononuclear cells was collected, washed three times in PBS and resuspended in culture medium. PBMC (3 x 106) were incubated in 24-well culture dishes for 1 h at 37°C washed three times in culture medium and stimulated as indicated. Monocytes were dissociated by vigorous pipetting with ice-cold medium and tested for purity by FACS analysis using F(AB’)2 anti-CD14 antibody and estimated at approximately 90%. To obtain human macrophages, we differentiated these adherence-purified cells in the presence of long/ml recombinant human macrophage colony stimulating factor (M-CSF, Strassmann et al., 1991) given on days 0 and 3. After an incubation period of 5days, the monolayers were washed twice and stimulated as indicated. The M-CSF dependent differentiation of macrophages from monocytes was characterized by a dramatic change in morphology which is characteristic of macrophages. HL-60 and THP-1 were differentiated by incubating 1 x 106cells for 3 days with PMA (1 nM) +vitamin D3( IOnM), or with vitamin D3 alone, respectively. Synovial fluids were obtained from the knees of rheumatoid arthritic patients by needle aspiration. The fluid was diluted with two volumes of PBS and mononuclear cells were obtained and purified by adherence as described above. RNA isolution, cDNA synthesis and competitive polymerase chain reaction (PCR) analyses

These procedures were performed as previously described by us (Kambayashi et al., 1995a; Kambayashi et al., 1995b). Briefly, macrophage monolayers were treated as indicated, lysed and RNA was isolated according to standard protocols, and complementary DNA was synthesized from RNA. The primer sequences of mouse IL-IO, mouse TNFcx and corresponding MIMIC sequence primers were syn-

Vesnarinone Inhibits TNFc( Release thesized on an Applied Biosystem-391 thesizer (Applied Biosystems, Foster U.S.A.) and further purified before use. Quantitation analyses

of cytokine mRNA

DNA synCity, CA,

by competitive

PCR

This procedure was performed as previously described by us (Kambayashi et al., 1995a; Kambayashi et al., 1995b). To accurately compare cytokine mRNA expression in different samples we used competitive PCR (Perkin-Elmer, Foster City, CA, U.S.A.) analyses, during which one set of primers is used to amplify both a target gene and another DNA fragment (MIMIC)-in essence the second DNA fragment competes with the target DNA for the same primers and thus acts as an internal standard. We used a non-homologous DNA fragments with primer templates that are recognized by a pair of gene-specific primers that generates a PCR product of a size different from that of the target DNA. Serial dilutions of known amounts of MIMIC cDNA were added to a PCR amplification reaction containing a constant amount of the experimental DNA sample. By knowing the amount of MIMIC added to the reaction, mRNA levels were determined. For each sample, aliquots containing approximately 0.2 ug of starting DNA were used as a substrate and amplified by PCR by primers for the specific cytokine DNA. Enzyme-linked

immunosorbent

assays (ELISAs)

mTNFa, mIL-10 and mIL-6 ELISA were purchased from Endogen (Boston, MA, U.S.A.). mIL-la ELISA was purchased from Genzyme (Cambridge, MA, U.S.A.). Human TNFcl ELISA kits were purchased from Biosource. The levels of LIF were quantitated by ELISA, courtesy of Dr H.R. Alexander (NCI, NIH).

313

the drug suppresses IL-6 release by about 50% whereas the levels of IL-la and IL-10 remain largely unchanged. The levels of LIF were 168f 11 and 142 f 19 pg/lOO ug cell protein, in LPS and in LPS plus vesnarinone (30 PM)-treated macrophages, respectively. TNFa, IL-10 and LIF could not be detected in the absence of LPS stimulation. This experiment was reproduced 15 times with similar results. In about 30% of the experiments, the levels of IL-10 produced by vesnarinone-treated cells were modestly higher (between 20 and 50%) than in vehicle-treated cells. Because IL-10 is well known as a potent autocrine inhibitor of macrophage TNFcr release (reviewed by Ho & Moore, 1994) we investigated this point further. Since IFNy up-regulates TNFcl production via the inhibition of IL-10 mRNA and protein (Chomarat et al., 1993; Donnelly et al., 199.5) we examined whether vesnarinone would also inhibit TNFcl release in LPS plus IFNy stimulated cells. Figure 2 shows that vesnarinone also inhibited the augmented TNFcl release in LPS plus IFNy treated cells, under conditions where the release of IL-10 was blocked. Of note, although the amount of TNFcl produced in LPS plus IFNy treated cells is about 25-fold higher than in LPS-treated macrophages, the IQ,, of vesnarinone is comparable in both conditions. Maximal release of TNFcl in LPS-stimulated peritoneal macrophages occurred at about 6 h post-stimulation (Kambayashi et al., 1995a). Figure 3 describes the time-dependent inhibition of TNFcl release by vesnarinone. The drug reduces TNFcl release in all of the time points tested, including the 6 h post-LPS stimulation where TNFcl level was maximal. We next determined whether the effect of vesnarinone involves transcription of the murine TNFcl gene. Table 1 demonstrates that vesnarinone significantly reduced TNFa mRNA at 3 and 6 h, post-LPS stimulation. By contrast, the drug does not significantly affect the LPS-dependent up-regulation of IL-10

RESULTS

Effect of vesnarinone macrophages

on cytokine

release by mouse

In our experiments, we treated cells with a DMSOdissolved vesnarinone or with a vehicle (control) solution containing DMSO; the final concentration of the solvent did not exceed 0.02%. To assess the effect of vesnarinone on macrophage secretory products, cells were stimulated with LPS in the presence of increasing concentrations of the drug and the culture medium was harvested 16 h post-LPS. Figure 1 demonstrates that the drug inhibited TNFcl release with an IC,, of approximately 12 PM. The highest concentration of

Table 1. Vesnarinone in LPS-stimulated

Treatment

(h)

Medium LPS LPS + Vesnarinone

inhibits TNFa, but not IL-10 mRNA murine peritoneal macrophages

I

TNFa 3

6

1

IL-10 3

6

82 66

1 76 14

70 36

5 5

0 10 13

54 70

Macrophages were treated in the presence or absence of LPS (1 ug/ml) for the indicated time. Vesnarinone was dissolved in DMSO. Final concentration of the drug was 30 FM. An identical concentration of DMSO was added to LPS stimulated cells. The results of competitive PCR analysis are expressed in arbitrary units.

T. KAMBAYASHI et al.

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Fig. 1. In a dose-dependent manner vesnarinone inhibits LPS induced TNFcl release. Mouse peritoneal macrophages were treated with LPS (0.5pg/ml) in the presence of increasing concentrations of vesnarinone. Cell-free supernatants were harvested 16 h post-stimulation and cytokine levels were quantified as described in Experimental Procedures. *P
mRNA. As an additional control in this experiment we used rolipram. As previously published (Kambayashi et al., 1995a), this specific-PDEIV inhibitor dramatically up-regulates IL-IO mRNA, but does not change TNF~L mRNA levels (data not shown). Together these results suggest that vesnarinone is a selective inhibitor of LPS stimulated TNFcr biosynthesis by mouse peritoneal macrophage. The ability of the drug to inhibit macrophage TNFcr release by various stimuli was also addressed. Both PGPS and okadaic acid (a potent protein phosphatase inhibitor, ppl and pp2a), induced significant production of TNFcr (Higuchi & Aggarwal, 1993; Gupta et al., 1995). Table 2 shows that the stimulation of macrophages with the exotoxin PGPS and okadaic

acid stimulated TNFcl release and that vesnarinone inhibits the stimulation of macrophage TNFc( release by all the stimuli used. Thus, the inhibitory effect of vesnarinone on TNFcl release appears to be stimulusindependent.

Vesnarinone inhibits TNFa release by human macrophages We determined the effect of vesnarinone on cytokine release by human macrophages. The differentiated human monocytic cell lines HL-60 and THP1 produce a significant level of TNFcl (Fig. 4). Undifferentiated HL-60 and THP-1 cells do not produce significant levels of TNFu upon LPS stimulation

I

Vesnarinone Inhibits TNFcl Release Table 2. Vesnarinone

inhibits various stimuli of TNFa release

from several RA patients. Table 3 demonstrates that in a dose-dependent manner, vesnarinone inhibits the release of TNFcl produced by adherence-purified synovial cells from the majority of the patients tested (6/7). In contrast, FACS analyses shows that vesnarinone (at 30uM) does not modulate the IFNydependent up-regulation of CD25 and DR expression on CD14+ synovial cells (not shown). Although the number of patients tested is rather small, these results support the hypothesis that vesnarinone is a selective inhibitor of macrophage derived TNFa.

TNFa

Treatment

ND 378 ND 183 1280 ND 455 1247 191 921

Medium PGPS (1 ug/ml) PGPS + vesnarinone (30 FM) PGPS + vesnarinone (3 FM) Okadaic (30 nM) Okadaic + vesnarinone (30 uM) Okadaic+ vesnarinone (3 uM) LPS (0.5 ug/ml) LPS + vesnarinone (30 PM) LPS + vesnarinone (3 uM)

315

Mouse peritoneal macrophages were treated as indicated. Culture medium was harvested 16 h post-stimulation. Positive control cultures received an identical concentration of DMSO. Results are expressed as mean pg/lOO ug cell protein. S.E.M. of triplicate cultures did not exceed 10%. ND indicates a level below the detection limit.

(not shown). The treatment of these cells with LPS plus vesnarinone (30 PM) reduces the level of TNFo: release by about 90% (Fig. 4). In the next set of experiments we obtain PBMC and adherence-purified monocytes from healthy donors. These monocytes were further differentiated to macrophages by M-CSF (see Experimental Procedures). A representative experiment is shown in Fig. 5. Vesnarinone inhibits the release of TNFu release from human macrophages with an ICso of about 10 PM. Next we examined the effect of the drug on tissue macrophages obtained A.

DISCUSSION

Several lines of evidence suggest that vesnarinone is a non-toxic and selective inhibitor of macrophage TNFa. First, the drug inhibits the release of TNFcY in a dose-related manner. Second, the release of other cytokines, including IL-la, IL-10 and LIF remains largely unchanged, and the drug only partially inhibits IL-6 levels. Third, the drug inhibits TNFu mRNA but does not significantly change IL-10 mRNA levels. Whether vesnarinone affects TNFu mRNA stability by accelerating degradation, or directly regulates a component in the TNFcl promoter, remains to be established. Fourth, FACS analyses of mIFNy stimulated mouse peritoneal macrophages (10 U/ml for 16 h, in suspension) show that vesnarinone (at 30 PM) does not reduce the IFNy-dependent up-regulation of

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316

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(30 PM). Ia antigen. A preliminary report by Matsumori et ~11. (1994) showed that vesnarinone inhibits the release of several cytokines including IL-6, IL-lu and B, IFNy and TNFu produced by human blood. In our study, vesnarinone appears to selectively inhibit TNFcr release by both mouse and human macrophages. It is possible that the inhibitory effect of the drug on monocytes is more universal than on macrophages.

The inhibitory

effect of vesnarinone

on the release of

TNFcl is not restricted to mononuclear phagocytes. Previous reports show that the drug inhibits the release of TNFcl from splenocytes (Matsui et al., 1994) and from a human T cell line (Shioi et al., 1993). An interesting feature of vesnarinone is its ability to inhibit TNFcl release by several stimuli, including LPS, PGPS, LPS plus IFNy and okadaic acid. These

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311

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Fig. 5. Vesnarinone inhibits TNFa release from human macrophages. Monocytes from the same donor (0.5 x 106/we11/1ml) were obtained and differentiated as described in Experimental Procedures and stimulated by LPS. TNFa levels were quantified 16 h post-LPS stimulation. *Pi 0.01.

results suggest that vesnarinone inhibits an intracellular element common to all of these stimuli. We attempted to identify the direct or indirect site(s) of action of vesnarinone in LPS signal transduction. It has been shown that LPS signaling involves the phosphorylation of several kinases like p561y”,Vav, Ras, Map and the nuclearization of NFkB (reviewed by Levitzki & Gazit, 1995). However, vesnarinone, at 30pM, does not affect LPS-stimulation of macrophage PS6’Y”and Map kinase activity or the translocation of NFkB (not shown). Vesnarinone has been previously shown to inhibit cardiac PDE type III activity (Rapundalo et al., 1988). We confirmed these results by using recombinant bovine PDE III. Halfmaximal inhibition of vesnarinone on this enzyme occurs at about ~FM. It is unlikely, however, that

vesnarinone inhibits TNFu by blocking macrophage PDE type III, because fractionation of LPS stimulated macrophage lysate reveals the activation of two forms of PDE, type IV and type II, but not of type III. Furthermore, the drug has no effect on LPS-stimulation of intracellular CAMP (data not shown). In summary, these data suggest that vesnarinone may facilitate TNFu mRNA degradation or intervenes with a signaling pathway that remains to be discovered. Irrespective of the precise mechanism by which vesnarinone inhibits TNFcl release, the drug might be useful in treating diseases involving this cytokine. For example, the ability of the drug to inhibit TNFcl release by LPS-stimulated synovial cells from nearly all RA patients in vitro might prove beneficial in the management of that pathological condition.

Table 3. Vesnarinone inhibits TNFa release from LPS-stimulated synovial cells Condition/Patient Medium LPS LPS + Ves. 30 PM LPS+Ves. 10 I.IM LPS + Ves. 3 pM

no.

1

2

3

4

5

6

I

46 1644 SO* 792* 1060

ND 414 ND* 226* 334

ND 3806 900* 1756* 4001

ND 662 5* 94* 466

296 2400 1060* NT NT

19 846 42* NT NT

ND 2360 2008 NT NT

Mononuclear cells (0.5 x 106)from synovia of RA patients were treated with LPS (0.5 pg/ml) in the presence or absence of the indicated concentration of vesnarinone. After 16 h, cell-free supernatants were harvested and TNFa levels were quantified by ELBA. Results are expressed as mean pg/ml. S.E.M. of triplicate determinations did not exceed 10%. *PiO.Olwas obatined between LPS and LPS + vesnarinone treated cultures.

378

T. KAMBAYASHI

et al.

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