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Immunoregulatory properties of novel specific inhibitors of 5-lipoxygenase
Immunopharmacology, 17 (1989) 1-9 Elsevier
1
IMO 00431
Immunoregulatory properties of novel specific inhibitors of 5-1ipoxygenase D.S. Liu*, F.Y. Liew and J. Rhodes Department q["Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. (Received 13 June 1988; accepted 7 October 1988)
Abstract: The immunoregulatory properties of novel specific inhibitors of 5-1ipoxygenase (5-LO) were investigated in vitro. These novel acetohydroxamic acids were found to inhibit the proliferation of lymphocytes in response to Interleukin-l (IL-l) and Interleukin-2 (IL-2). Mitogen- and antigen-induced lymphoproliferation were likewise inhibited and so too was the spontaneous proliferation of transformed cells of myeloid and lymphoid origin. In this respect the compounds were one-tenth as potent as azathioprine on a molar basis. The production of IL-1 and IL-2 remained unaffected. Inhibition of lymphocyte proliferation was not related to inhibition of 5-LO. The latter occurred at 40-fold lower concentrations of 5-LO inhibitor. Moreover, inhibition of lymphoproliferation was not reversible in the presence of excess arachidonic acid. IL-l-induced collagenase production by chondrocytes was unaffected by 5-LO inhibitors. Contrary to a number of published reports, products of 5-LO do not appear to be obligatory mediators in IL-1 and IL-2 signal transduction. The immunosuppressive action of the 5-LO inhibitors described here seems to be a novel secondary property of the compounds. Key words:
5-Lipoxygenase inhibitor; Immunoregulation; lnterleukin-l; Interleukin-2; Collagenase
Introduction 20-carbon atom fatty acids esterified in membrane phospholipids provide the source for two important groups of inflammatory mediators. The most common of these fatty acids is arachidonic acid. Its metabolism begins with cleavage from phospholipids catalysed by calcium-dependent phospholipases A2 and C (Kuehl and Egan, 1980; Dennis et al., 1985). The free acid is then metabolized via two major pathways. The cyclooxygenase pathway gives rise to prostaglandins and this pathway is readily susceptible to inhibition by aspirin and related nonCorrespondence: J. Rhodes, Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. * Present address: Changchun Institute of Biological Products, Ministry of Public Health, Changchun, The Peoples Republic of China.
steroidal anti-inflammatory drugs (Flower, 1985). The lipoxygenase pathway produces leukotrienes and other hydroxyeicosatetraenoic acids (HETES) (Samuelsson et al., 1980). Of particular interest is the 5-1ipoxygenase pathway which converts arachidonic acid into 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) which is in turn converted to the 5,6-epoxide leukotriene A4 (LTA4), a highly unstable compound which serves as a common intermediate for synthesis of either (5S-12R)-dihydroxy-6,14-cis-8-10-trans-eicosatetraenoic acid ( l e u k o t r i e n e B4, LTB4) or the thiol-ether leukotrienes C4 D4 and E4. The addition of glutathione to LTA4 by glutathione-S-transferase produces LTC4 which can be converted to LTD4 by removal of the terminal glutamine by 7-glutamyl transpeptidase. In turn, LTD4 can be converted to LTE4 by loss of the terminal glycine. A substantial body of published work now shows that it is a mixture of
0162-3109/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
2 LTC4, LTD4 and LTE4 that constitute the biological activity defined as slow reacting substance of anaphylaxis (Samuelsson, 1983). Several cell-types are known to contain 5-1ipoxygenase and to produce leukotrienes and HETES. They include polymorphonuclear leukocytes (PMN), macrophages, basophilic leukaemia cells and bone marrow-derived mast cells. Whether or not 5-1ipoxygenase fulfills a significant role in lymphocyte function is less certain. Because of the evidence implicating products of the 5-1ipoxygenase pathway in some of the pathological events associated with acute inflammation and the reversible airways obstruction of asthma, considerable effort has been directed towards the discovery of specific inhibitors of this pathway (Bach, 1984). Although compounds such as BW755C which inhibit both cyclooxygenase (CO) and 5-1ipoxygenase pathways with equal efficacy and nafazatrom and nordihydroguairetic acid (NDGA) which inhibit the 5-LO pathway preferentially, have been exploited experimentally, no potent selective inhibitors of 5-LO with favourable therapeutic potential have been available. Recently, Garland and colleagues have described a series of novel acetohydroxamic acid derivatives that selectively block 5-LO and which possess favourable therapeutic potential in terms of bioavailability and inhibitory effects on acute inflammation and anaphylactic bronchoconstriction (Tateson et al., 1988; Payne et al., 1988; Higgs et al., 1988). While the evidence for the involvement of leukotrienes in these processes is clear-cut, products of the 5-1ipoxygenase pathway have also been reported to exert numerous and varied effects in vitro on lymphocyte proliferation and lymphokine production (Rola-Pleszczynski et al., 1982; Payan and Goetzl, 1983; Atluru and Goodwin, 1984; Gualde et al., 1985; Payan et al., 1984; Webb et al., 1982; Rola-Pleszczynski and Lemaire, 1985). In particular, it has been reported that products of the 5-LO pathway may participate as an essential component of the signal transduction process stimulated by Interleukin-1 and Interleukin-2 (Dinarello et al., 1983; Farrar and Humes, 1985). These conclusions are based on the inhibition of
interleukin-induced proliferation and lymphokine production by partially selective inhibitors of 5-LO. Here, we investigate the immunoregulatory properties of novel acetohydroxamic acid inhibitors of 5lipoxygenase shown to be selective for 5-LO in vitro and ex vivo, and to be inhibitory towards leukotriene-mediated events in vivo.
Experimental procedures Preparation of lymphocytes Blood from healthy donors was obtained by venipuncture and defibrinated with a U-shaped rod in a siliconized tube. Autologous serum was obtained at this stage by centrifugation. The mononuclear leukocyte fraction was obtained by Metrizoate/Ficoll gradient separation and the monocyte and lymphocyte fractions were separated by differential adhesion to glass or plastic in the presence of 10% autologous serum. Two cycles of adherence were used to deplete lymphocytes of monocytes in some experiments. All procedures were carried out in RPMI 1640 medium (Flow Laboratories, Ayeshire, Scotland) containing antibiotics and 10% autologous serum. In some experiments, B lymphocytes were removed by differential adherence to nylon fibre columns (Fenwal Laboratories, Deerfield, IL, U.S.A.). Cells were cultured in 96-well microtitre plates (U-shaped wells) (Titertek, Flow Laboratories) in the presence of mitogen or antigen as specified with or without acetohydroxamic acids. After 3 5 days, cultures were pulsed with [3H]thymidine (Amersham International) for 24 h after which cells were harvested onto glass microfibre paper by means of a semi-automatic cell-harvester (Skatron). The degree of thymidine incorporation was determined by liquid scintillation spectrometry.
Assays for IL~I IL~I production Adherent resident peritoneal macrophages in Linbro 24-well plates were cultured in RPMI 1640 containing 10% FCS in the presence or absence of bacterial lipopolysaccharide (Serotype 055-B5, Sigma). 5-Lipoxygenase inhibitors or vehicle alone (DMSO) were added at time 0. After
24 h, cell-free supernatants were harvested and dialysed against PBS for 24 h. Dialysed supernatants were stored at 20°C and subsequently tested in the thymocyte co-mitogenesis assay.
IL-1 thymocyte comitogenesis assay C3H/He thymocytes from 3-6-week-old mice were extracted from the thymus by gentle homogenization followed by sieving. The cells were adjusted to 5 x 106 per ml and cultured in 96-well microtitre plates in the presence of phytohaemagglutinin at a suboptimal concentration (1 /~g per ml). IL-l-containing supernatants were added at a 1/10 dilution to triplicate cultures. After 24 h the cultures were pulsed with [3H]thymidine (1 pCi per well) and incorporation was measured after a further 24 h by liquid scintillation spectrometry.
Assays for IL~2 IL-2production Spleen cells from normal BALB/c mice 6 12 weeks old were prepared and adjusted to 10 7 cells per ml in RPMI 1640 medium supplemented with 2 mercaptoethanol at 10 5 M. One ml aliquots of the suspension in Linbro 24-well plates were cultured with or without Con-A (Sigma) at a final concentration of 2 #g/ml at 37°C in an atmosphere of 5% CO2 in air. Twenty-four hours later, the supernatants were collected and supplemented with 20 mg ~-methyl-o-mannoside (Sigma) per ml in order to inactivate residual Con-A. The cell-free supernatants were stored at -20°C until testing.
IL-2 activity
The IL-2 activity in supernatants was measured by their capacity to maintain in vitro proliferation of Con-A blasts. T-cell blasts were prepared by incubation of spleen cells from normal mice at a concentration of 10 v cells per ml with 3 #g/ml of Con-A in medium supplemented with 5% FCS for 48 h at 37°C in 5% CO2 in air. Cells were then harvested, washed and resuspended at a concentration of 2 x 105 cells per ml. 100 #1 aliquots of this cell suspension were then combined with 100/A of IL-2-containing supernatants and the cells were cultured for a further 24 h with [3H]thymidine present for the last 4 h. Incorporation was determined by liquid scintillation spectrometry. IL-2 activity
was also measured by the capacity to maintain the proliferation of a murine IL-2-dependent cytotoxic T-cell line (CTLL). 100 /~1 containing 1 × 104 CTLL cells were cultured in RPMI 1640 containing 10% FCS in triplicate microtitre wells with 100 pl of the sample to be tested for 24 h at 37°C in an atmosphere of 5% CO2 in air. Six hours before harvesting, the cultures were pulsed with [3H]thymidine (1 #Ci per well). Incorporation was measured by liquid scintillation spectrometry.
Assay for mononuclear cell 5-1ipoxygenase Human blood mononuclear cells prepared by Metrizoate/Ficoll gradient separation were homogenized by sonication and pre-incubated with 5-LO inhibitor at 37°C for 5 min. Reactions were initiated by the addition of 5/~M arachidonic acid and 1 mM CaCI2 and terminated after 5 rain by boiling. LTB4 was measured by specific radioimmunoassay.
Assay for chondrocyte collagenase production Articular cartilage was dissected from the knee and shoulder joints of young (<1 kg) New Zealand white rabbits using a method based on that described by Plaas et al. Briefly, cartilage slices were digested for 4 h at 37°C in Dulbecco's modified Eagles medium (DMEM) (Flow Laboratories) containing 0.5% BSA (Sigma) and antibiotics, to which collagenase (type II, Sigma) had been added. Cells were then washed three times in calcium-free PBS, filtered through a Nitex filter (Cadish, London) to remove undigested cartilage and were aliquoted into 24-well Linbro plates. After 6-8 days, incubation at 37°C in 5% CO2 in air in DMEM containing 10% FCS, the cultures had become confluent and were then used in assays for IL-I activity. The washed confluent monolayers were treated in triplicate with various concentrations of IL-1 with or without acetohydroxamic acids. After 24 h cell-free supernatants were harvested and stored at -20°C until assayed for collagenase content. Tritiated type 1 collagen from rat tail tendon (New England Nuclear) was induced to form fibrils by incubation for 2 h at 4°C in 20 nM Tris-HC1 buffer, pH 7.6, containing 5 mM CaC13. Collagenase-containing samples were activated with 4-aminophenylmercuric
acetate. The acetate was first dissolved in 0.5 M N a O H and then diluted with the calcium buffer to give a 0.5 mM solution. Supernatants were tested at a 1/10 dilution. Collagen fibril plus test supernatant were incubated at 37°C for 5-24 h. Tubes were then microfuged and the amount of solubilized collagen was measured by liquid scintillation spectrometry. Procedures not detailed here were as described in the N E N technical manual NEK-016. Results were expressed as a percentage of total digestion, the latter being determined with an excess of bacterial collagenase (NEN).
Results
Effect of 5-LO &hibitors on the production of IL-1 and IL-2 When freshly isolated resident peritoneal macrophages were stimulated with LPS to induce IL-1 production over a 24 h period in vitro, the addition of 5-LO inhibitors in the concentration range of 10 40 pM had no significant inhibitory effect on the production of Interleukin-l. In obtaining this result
tO
it was, of course, important to remove the drug by extensive dialysis of IL-l-containing supernatants before testing in the biological assay. The data are shown in Fig. 1. Similarly, when murine spleen cells were stimulated with Con-A to induce IL-2 production over a 24 h period in vitro, the addition of 5-LO inhibitors in the concentration range 10-40 /~mol had no significant effects on the production of IL-2 detectable in dialysed supernatants. The data are shown in Fig. 2. Effect o)C5-LO inhibitors on the thymocyte co-mitogenic response to IL-1 and the T-cell mitogenic response to IL-2 When IL-1 produced by LPS-stimulated murine macrophages or human blood monocytes was tested in the co-mitogenesis assay using murine thymocytes stimulated with a suboptimal dose of PHA (1 #g per ml), the addition of 5-LO inhibitors in the 10-40/~M range produced a dose-dependent inhibition of thymocyte proliferation (Fig. 3). The same picture of dose-dependent inhibition was observed when recombinant human IL-1 ~ or/~ were used to induce thymocyte proliferation. 5-LO inhibitors in
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Fig. 1. Resident peritoneal macrophages from BALB/c mice were cultivated for 24 h in the presence of LPS (1/~g/ml) with or without 5-LO inhibitor at the concentrations specified. Dialysed supernatants were tested in the thymocyte co-mitogenesis assay. O, BW A137C; II, BW A1C; &, BW A4C. Similar results were obtained in four independent experiments. Typically, proliferative responses measured by [3H]thymidine incorporation produced I ~ 1 5 × 103 cpm.
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(lag per ml) Fig. 2. Spleen cells from BALB/c mice were cultured for 24 h in the presence of Con-A with or without 5-LO inhibitors at the concentration range specified. Dialysed, c~-methyl-o-mannosidetreated supernatants were then tested for their ability to maintain the proliferation of Con-A spleen cell blasts. O, BW A 137C; II, BW A1C; A, BW A4C. Similar results were obtained in three independent experiments. Typically, responses to 1L-2 were in the range of 5-10 × 104 cpm.
the same dose range also inhibited the response of murine T-cells to Interleukin 2 (Fig. 4).
100
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~_ 60
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10 5 2.5 1.25 Concentration of 5 - - L O inhibitor (pg per ml)
Fig. 3. Thymocytes from C3H/He mice were cultured for 72 h in the presence of a suboptimal dose of P H A (1 gg/ml) and a standard dose of IL-1 with or without 5-LO inhibitors at the concentration specified. O, BW A137C; I , BW AIC; A, BW A4C. These effects were highly consistent in a series of five experiments. One hundred per cent inhibition corresponds to the response obtained with P H A alone ( ~ 500 cpm).
A 100. o~
Effect of 5~LO inhibitors on lymphocyte proliferation in response to antigen and mitogen When normal human lymphocytes were stimulated with the recall antigen tetanus toxoid at a dose which produced near optimal responses (10/tg/ml) the addition of 5-LO inhibitors produced a dosedependent inhibition of the proliferative response. In order to investigate whether the observed effects were directly related to inhibition of 5-1ipoxygenase, excess substrate in the form of the arachidonic acid sodium salt was added. Although the addition of arachidonic acid generally enhanced lymphocyte proliferation in response to antigen, 40 160 /~M concentrations did not reverse the inhibition of proliferation produced by 5-LO inhibitors. Representative data are shown in Fig. 5. In order to compare the inhibitory effects of acetohydroxamic acids with standard immunosuppressive drugs, experiments were performed using azathioprine and mercaptopurine. There drugs were approximately ten-times more potent than 5-LO inhibitors on a molar basis, in suppressing mitogen-induced lymphocyte proliferation (Fig. 6)
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Antigen Antigen +AA24pg per ml BW A137C 10lag per ml
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BW A137C 10lJg per m~ + AA
10 5 2.5 1.25 Concentration of 5 - - L O inhibitor (~Jg per ml)
Fig. 4. Splenic Con-A blasts from BALB/c mice were cultured for 24 h in the presence o f a standard IL-2 containing supernatant with or without 5-LO inhibitors at the concentration specified. Similar results (not shown) were obtained by using recombinant 1L-2 and the CTLL-IL-2 dependent cell line. Q, BW A137C; II, BW A1C; A, BW A4C. Similar results were obtained in three independent experiments. One hundred per cent inhibition corresponds to a background response of 2 × 103 cpm.
BW A137C 20~g per ml BW A137C 20~Jg per ml + AA
0 B I
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I 200 Antigen-induced lymphocyte proliferation (percentage of control)
Fig. 5. Normal h u m a n lymphocytes were cultivated for 5 days in the presence of tetanus toxoid (Wellcome Research Laboratories, Beckenham, Kent, U.K.) at 10 #g per ml. Arachidonic acid (sodium salt, Sigma) and 5-LO inhibitors were present throughout the culture at the concentrations and in the combinations specified. The same picture was obtained in three independent experiments. One hundred per cent corresponds to 3 x 104 cpm.
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Fig. 6. Normal human lymphocytes were cultured for 72 h in the presence of PHA at 6/tg per ml. 5-kO inhibitors, azathioprine and mercaptopurine were present at the concentrations specified. T, BW A4C; &, BW A137C; O, azathioprine; B, mercaptopurine. Similar results were obtained in two experiments.
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Effect of 5~LO inhibitors on the spontaneous prol([eration of lymphoid and non-lymphoid cells
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In order to ask whether the inhibitory effect o f 5L O inhibitors on cell proliferation was confined to lymphoid cells responding to immunologic signals, the effect o f these c o m p o u n d s on spontaneously dividing lymphoid and n o n - l y m p h o i d cells was investigated. A dose-dependent inhibition o f spontaneous proliferation similar to that observed with IL-1-induced proliferation was obtained with a murine lymphoid t u m o u r of B-cell origin (A20/2J) with h u m a n lymphoid t u m o u r lines (HSB2 M O L T 4), and with the murine m a c r o p h a g e derived cell-line R A W 264.7. The data are shown in Fig. 7. Inhibition o f proliferation was not due to non-specific toxicity since the c o m p o u n d s caused no release of 5~chromium from pre-labelled cells in cytotoxicity assays.
genase production by rabbit chondrocytes was investigated. Collagenase production in response to IL-1 was shown to be unaffected by 5-LO inhibitors in the dose-range which produced complete inhibition of lymphoproliferative responses (Fig. 8).
Effect of 5-LO inhibitors on IL-l-induced chondrocyte collagenase production
Effect of 5-LO inhibitors on blood mononuclear cell 5-lipoxygenase
In order to determine whether the inhibitory effects o f acetohydroxamic acids on I L - l - i n d u c e d cellular responses were confined to proliferative responses the effect o f 5-LO inhibitors on I L - l - i n d u c e d colla-
5-LO inhibitors BW A 4 C and BW A137C were found to be potent inhibitors of 5-1ipoxygenase activity in sonicates o f h u m a n blood m o n o n u c l e a r cells. The data are shown in Table I. Greater than
2e~ 0
I 1.25
I 2.5
I 5
I 10
Concentration of BW A 4 C (ug per ml)
Fig. 7. Cell-lines were cultured for 48 h with or without 5-LO inhibitors at the concentration specified. A, BW A137C; B, BW A4C; 0, HSB2; i , A20/2J; A, RAW 264.7; S, MOLT 4. Similar results were obtained in two experiments. Average maximum incorporation of[3H]thymidine corresponding to 100% was 8 x 104cpminAand5 x 104cpminB.
ition of lymphocyte proliferation by 5-LO inhibitors required concentrations in the 10-80/~M range (2.5-20 yg/ml).
Untreated control rlL-1/3
5 units per ml
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+
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Discussion
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+ 2.51Jg 1.251Jg per Perm' ml I
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20
40
60
80
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Digestion of 3H-collagen (%1
Fig. 8. Primary chondrocyte cultures derived from articular cartilage of New Zealand white rabbits were stimulated with recombinant IL-fl for 24 h with or without 5-LO inhibitors at the concentration specified. Collagenase activity in cell-free supernatants was then assayed in a tritiated type 1 collagen fibril assay system. Similar results were obtained in three independent experiments. One hundred per cent digestion corresponds to the amount of soluble tritiated material produced by bacterial collagenase at 10 #g/ml which yielded 11 x 103 cpm.
80% inhibition was observed in the concentration range 0.1-1 #M (0.03-0.3 #g/ml). No inhibitory effects on lymphocyte proliferation in response to interleukins were observed in this dose range. InhibTABLE I Inhibition of mononuclear cell 5-LO by acetohydroxamic acids Treatment
LTB,, produced (ng)
Mean percentage inhibition
Boiled blank
0.0 0.3 1.85
--
Untreated sonicate
1.78
BW A4C (0.1 /zM) BW A4C (1.0 #M) BW A137C (1 #M) BW A137C (10 ,uM)
2.56 1.59 0.26 0.23 0.36 0.07 0.29 0.41 0.06 0.07
88 89 82 96
The present studies focussed on the immunomodulatory properties of a new class of specific 5-1ipoxygenase inhibitors. These novel acetohydroxamic acids were shown to consistently inhibit the proliferation of lymphoid cells in response to a wide range of signals without affecting the production of interleukins by myeloid and lymphoid cells. The compounds were found to inhibit the proliferation of lymphoid ceils in response to IL-1 and IL-2, at concentrations of 10 40/~M. The production of IL-1 in response to LPS by murine peritoneal macrophages was unaffected by the compounds. Similarly, the production of IL-2 in response to Con-A by murine splenocytes was unaffected by acetohydroxamic acids at the concentrations which completely inhibited the response of splenocytes and the CTLL line to Interleukin-2. The inhibitory effects of acetohydroxamic acids on lymphocyte proliferation were not confined to responses induced by IL-1 and IL-2. Thus the polyclonal proliferation of human peripheral blood lymphocytes induced by PHA, and the specific oligoclonal expansion of lymphocyes in response to tetanus toxoid, were inhibited to a comparable degree by 5-LO inhibitors in the 10-40 yM range. The spontaneous proliferation of transformed cell lines of T lymphocyte origin and the non-lymphoid line RAW 264.7 were likewise inhibited by acetohydroxamic acids in the same dose range. In order to investigate the possibility that these suppressive effects were due to inhibition of 5-1ipoxygenase in the responding cells, we measured the conversion of arachidonic acid to LTB4 by human blood mononuclear cell 5-1ipoxygenase in the presence and absence of 5-LO inhibitors. These novel 5-LO inhibitors were developed and characterized using polymorphonuclear 5-1ipoxygenase; nevertheless, these compounds produced almost complete inhibition of mononuclear cell 5-LO at concentrations of
0.1-1 #M. In this dose range lymphocyte proliferative responses were entirely unaffected. In order to achieve substantial inhibition of proliferation concentrations forty-fold greater than this were necessary. The inhibitory effects of 5-LO inhibitors on polymorphonuclear leukocyte 5-LO is reversible in the presence of excess substrate in the form of arachidonic acid. We therefore wished to know whether a molar excess of substrate could reverse the inhibition of lymphoproliferative responses by acetohydroxamic acids. Interestingly, the addition of arachidonic acid in the 40-160 /~M range modestly enhanced the proliferative response of lymphocytes to antigen. However, the addition of a four-fold molar excess of arachidonic acid did not reverse the inhibitory effects of 5-LO inhibitors on lymphocyte proliferation. Taken together these data indicate that the marked suppressive effect of these novel specific 5LO inhibitors on lymphoproliferative responses is unlikely to be due to their effect on 5-1ipoxygenase. Indeed, the data show that it is possible to completely inhibit 5-LO in mononuclear cells using low doses of inhibitor without affecting the proliferation of these cells in response to IL-1 and IL-2. This finding cannot be consistent with the suggestion made by others that products of the 5-1ipoxygenase pathway are obligatory mediators of the biological effects of interleukins (Dinarello et al., 1983; Farrar and Humes, 1985). Others have shown that Interleukin-I can stimulate lipoxygenation of arachidonic acid in certain T cell lines which proliferate and/or secrete lymphokines in response to interleukins (Farrar and Humes, 1985). However, neither exogenous 5H E T E nor leukotrienes B4, C4, D , and E4 could reconstitute the proliferative response to IL-2. The lack of correlation between inhibition of 5-LO and inhibition of lymphoproliferation by novel selective inhibitors of 5-LO demonstrated in the present study argues strongly against 5-LO products being essential components of the signal transduction process for IL-1 and IL-2. Consistent with this is the finding that the suppressive effects of 5-LO inhibitors were not confined to IL-1- and IL-2-in-
duced responses. While it could be argued that lymphocyte response to mitogen and specific antigen are dependent upon the production of interleukins 1 and 2, this is unlikely to be true for the spontaneous proliferation of transformed T cell lines and it is not true for the spontaneous proliferation of the non-lymphoid cell-line RAW 264.7. Moreover, signal transduction in rabbit chondrocytes where IL-1 induces the synthesis of collagenase was unaffected by high concentrations of 5-LO inhibitors. We conclude that the immunosuppressive properties of the selective 5-LO inhibitors described here are a novel secondary characteristic of acetohydroxamic acids. The suppression these compounds achieve in vitro is quite substantial. They are approximately one-tenth as potent as the conventional immunosuppressive drugs azathioprine and mercaptopurine on a molar basis. Their mode of action in this context is presently unknown. One possibility is that acetohydroxamic acids in the higher dose range inhibitory towards cell-proliferation, are affecting pathways of arachidonic acid metabolism other than 5-1ipoxygenase and that the products of these pathways are a contributory element in the regulation of cell proliferation.
Acknowledgements We are grateful to Mr R.W, Randall, Department of Biochemistry, Wellcome Research Laboratories, for performing assays for mononuclear cell 5-1ipoxygenase activity. We thank Drs L.G. Garland and G.A. Higgs for valuable discussion.
References Atluru D, Goodwin JS. Control of polyclonal immunoglobulin production from human lymphocytesby leukotrienes: leukotriene B4 induces an OKT9(+ ), radiosensitivesuppressor cell from resting human OKT8(-) T cells. J Clin Invest !984;74:1444-1450. Bach MK, Prospectsfor the inhibition of leukotrienesynthesis. Biochem Pharmacol 1984;33:515-521. Dennis EA, Hazlett TL, Deems RA, Ross MI, Ulevitch, RJ. Phospholipases in the macrophage. In: BaileyJM. ed. Pros-
taglandins, leukotrienes and lipoxins. New York: Plenum, 1985;213-220. Dinarello CA, Marnoy SO, Rosenwasser LJ. Role of arachidonate metabolism in the immunoregulatory function of human leukocyte pyrogen/lymphocyte-activating factor/interleukin 1. J lmmunol 1983;130:890 895. Dinarello CA, Bishai I, Rosenwasser L J, Coceani, F. The influence of lipoxygenase inhibitors on the in vitro production of human leukocytic pyrogen and lymphocyte activating factor (interleukin 1). Int J Immunopharmacol 1984;6:43-50. Farrar, WL, H umes JL. The role of arachidonic acid metabolism in the activities of interleukin I and 2. J lmmunol 1985;135:1153-1159. Flower RJ. Cyclo-oxygenase inhibitors: an overview. In: Bailey JM, ed. Prostaglandins, leukotrienes and lipoxins. New York: Plenum, 1985;583-591. Gualde N, Atluru D, Goodwin JS. Effect oflipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets. J Immunol 1985;134:1125 1129. Higgs GA, Follenfant RL, Garland LG. Selective inhibition of arachidonate 5-1ipoxygenase by novel acetohydroxamic acids. III. Effects on acute inflammatory responses. Br J Pharmacol 1988;in press. Kuehl FAJ, Egan RW. Prostaglandins, arachidonic acid and inflammation. Science 1980;210:978-984. Payan DG, Goetzl EJ. Specific suppression of human T lymphocyte function by leukotriene B4. J Immunol 1983;131:551-553. Payan DG, Missirian-Bastian A, Goetzl EJ. Human lymphocyte
subset specificity of the regulatory effects of leukotriene B4. Proc Natl Acad Sci USA 1984;81:3501-3505. Payne AN, Garland LG, Lees IW, Salmon JA. Selective inhibition of arachidonate 5-1ipoxygenase by novel acetohydroxamic acids. II. Effects on bronchial anaphylaxis in anaethetised guineas-pigs. Br J Phamacol 1988;in press. Plaas AHK, Sandy JD, Muir H. Proteoglycan-aggregate formation by articular chondrocytes. Biochem J 1983;214:855 858. Rola-Pleszczynski M, Borgeat P, Sirois P. Leukotriene B4 induces human suppressor lymphocytes. Biochem Biophys Res Commun 1982;I08:1531-1537. Rola-Pleszczynski M, Lemaire I. Leukotrienes augment interleukin 1 production by human monocytes. J Immunol 1985;135:3958 3961. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 1983;220:568 575. Samuelsson B, Borgeat P, Hammarstrom S, Murphy RC. Leukotrienes: a new group of biologically active compounds. Adv Prostaglandin Thromboxane Res 1980;6:1-18. Tateson JE, Randall RW, Reynolds CH, Jackson WP, Bhattacherjee P, Salmon JA, Garland LG. Selective inhibition of arachidonate 5-1ipoxygenase by novel acetohydroxamic acids. I. Biochemical assessment in vitro and ex vivo. Br J Pharmacol 1988; in press. Webb DR, Nowowiejski I, Healy C, Rogers TJ. lmmunosuppressive properties of leukotriene D 4 and E 4 in vitro. Biochem Biophys Res Commun 1982;194:1617-1622.
1
IMO 00431
Immunoregulatory properties of novel specific inhibitors of 5-1ipoxygenase D.S. Liu*, F.Y. Liew and J. Rhodes Department q["Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. (Received 13 June 1988; accepted 7 October 1988)
Abstract: The immunoregulatory properties of novel specific inhibitors of 5-1ipoxygenase (5-LO) were investigated in vitro. These novel acetohydroxamic acids were found to inhibit the proliferation of lymphocytes in response to Interleukin-l (IL-l) and Interleukin-2 (IL-2). Mitogen- and antigen-induced lymphoproliferation were likewise inhibited and so too was the spontaneous proliferation of transformed cells of myeloid and lymphoid origin. In this respect the compounds were one-tenth as potent as azathioprine on a molar basis. The production of IL-1 and IL-2 remained unaffected. Inhibition of lymphocyte proliferation was not related to inhibition of 5-LO. The latter occurred at 40-fold lower concentrations of 5-LO inhibitor. Moreover, inhibition of lymphoproliferation was not reversible in the presence of excess arachidonic acid. IL-l-induced collagenase production by chondrocytes was unaffected by 5-LO inhibitors. Contrary to a number of published reports, products of 5-LO do not appear to be obligatory mediators in IL-1 and IL-2 signal transduction. The immunosuppressive action of the 5-LO inhibitors described here seems to be a novel secondary property of the compounds. Key words:
5-Lipoxygenase inhibitor; Immunoregulation; lnterleukin-l; Interleukin-2; Collagenase
Introduction 20-carbon atom fatty acids esterified in membrane phospholipids provide the source for two important groups of inflammatory mediators. The most common of these fatty acids is arachidonic acid. Its metabolism begins with cleavage from phospholipids catalysed by calcium-dependent phospholipases A2 and C (Kuehl and Egan, 1980; Dennis et al., 1985). The free acid is then metabolized via two major pathways. The cyclooxygenase pathway gives rise to prostaglandins and this pathway is readily susceptible to inhibition by aspirin and related nonCorrespondence: J. Rhodes, Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. * Present address: Changchun Institute of Biological Products, Ministry of Public Health, Changchun, The Peoples Republic of China.
steroidal anti-inflammatory drugs (Flower, 1985). The lipoxygenase pathway produces leukotrienes and other hydroxyeicosatetraenoic acids (HETES) (Samuelsson et al., 1980). Of particular interest is the 5-1ipoxygenase pathway which converts arachidonic acid into 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) which is in turn converted to the 5,6-epoxide leukotriene A4 (LTA4), a highly unstable compound which serves as a common intermediate for synthesis of either (5S-12R)-dihydroxy-6,14-cis-8-10-trans-eicosatetraenoic acid ( l e u k o t r i e n e B4, LTB4) or the thiol-ether leukotrienes C4 D4 and E4. The addition of glutathione to LTA4 by glutathione-S-transferase produces LTC4 which can be converted to LTD4 by removal of the terminal glutamine by 7-glutamyl transpeptidase. In turn, LTD4 can be converted to LTE4 by loss of the terminal glycine. A substantial body of published work now shows that it is a mixture of
0162-3109/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
2 LTC4, LTD4 and LTE4 that constitute the biological activity defined as slow reacting substance of anaphylaxis (Samuelsson, 1983). Several cell-types are known to contain 5-1ipoxygenase and to produce leukotrienes and HETES. They include polymorphonuclear leukocytes (PMN), macrophages, basophilic leukaemia cells and bone marrow-derived mast cells. Whether or not 5-1ipoxygenase fulfills a significant role in lymphocyte function is less certain. Because of the evidence implicating products of the 5-1ipoxygenase pathway in some of the pathological events associated with acute inflammation and the reversible airways obstruction of asthma, considerable effort has been directed towards the discovery of specific inhibitors of this pathway (Bach, 1984). Although compounds such as BW755C which inhibit both cyclooxygenase (CO) and 5-1ipoxygenase pathways with equal efficacy and nafazatrom and nordihydroguairetic acid (NDGA) which inhibit the 5-LO pathway preferentially, have been exploited experimentally, no potent selective inhibitors of 5-LO with favourable therapeutic potential have been available. Recently, Garland and colleagues have described a series of novel acetohydroxamic acid derivatives that selectively block 5-LO and which possess favourable therapeutic potential in terms of bioavailability and inhibitory effects on acute inflammation and anaphylactic bronchoconstriction (Tateson et al., 1988; Payne et al., 1988; Higgs et al., 1988). While the evidence for the involvement of leukotrienes in these processes is clear-cut, products of the 5-1ipoxygenase pathway have also been reported to exert numerous and varied effects in vitro on lymphocyte proliferation and lymphokine production (Rola-Pleszczynski et al., 1982; Payan and Goetzl, 1983; Atluru and Goodwin, 1984; Gualde et al., 1985; Payan et al., 1984; Webb et al., 1982; Rola-Pleszczynski and Lemaire, 1985). In particular, it has been reported that products of the 5-LO pathway may participate as an essential component of the signal transduction process stimulated by Interleukin-1 and Interleukin-2 (Dinarello et al., 1983; Farrar and Humes, 1985). These conclusions are based on the inhibition of
interleukin-induced proliferation and lymphokine production by partially selective inhibitors of 5-LO. Here, we investigate the immunoregulatory properties of novel acetohydroxamic acid inhibitors of 5lipoxygenase shown to be selective for 5-LO in vitro and ex vivo, and to be inhibitory towards leukotriene-mediated events in vivo.
Experimental procedures Preparation of lymphocytes Blood from healthy donors was obtained by venipuncture and defibrinated with a U-shaped rod in a siliconized tube. Autologous serum was obtained at this stage by centrifugation. The mononuclear leukocyte fraction was obtained by Metrizoate/Ficoll gradient separation and the monocyte and lymphocyte fractions were separated by differential adhesion to glass or plastic in the presence of 10% autologous serum. Two cycles of adherence were used to deplete lymphocytes of monocytes in some experiments. All procedures were carried out in RPMI 1640 medium (Flow Laboratories, Ayeshire, Scotland) containing antibiotics and 10% autologous serum. In some experiments, B lymphocytes were removed by differential adherence to nylon fibre columns (Fenwal Laboratories, Deerfield, IL, U.S.A.). Cells were cultured in 96-well microtitre plates (U-shaped wells) (Titertek, Flow Laboratories) in the presence of mitogen or antigen as specified with or without acetohydroxamic acids. After 3 5 days, cultures were pulsed with [3H]thymidine (Amersham International) for 24 h after which cells were harvested onto glass microfibre paper by means of a semi-automatic cell-harvester (Skatron). The degree of thymidine incorporation was determined by liquid scintillation spectrometry.
Assays for IL~I IL~I production Adherent resident peritoneal macrophages in Linbro 24-well plates were cultured in RPMI 1640 containing 10% FCS in the presence or absence of bacterial lipopolysaccharide (Serotype 055-B5, Sigma). 5-Lipoxygenase inhibitors or vehicle alone (DMSO) were added at time 0. After
24 h, cell-free supernatants were harvested and dialysed against PBS for 24 h. Dialysed supernatants were stored at 20°C and subsequently tested in the thymocyte co-mitogenesis assay.
IL-1 thymocyte comitogenesis assay C3H/He thymocytes from 3-6-week-old mice were extracted from the thymus by gentle homogenization followed by sieving. The cells were adjusted to 5 x 106 per ml and cultured in 96-well microtitre plates in the presence of phytohaemagglutinin at a suboptimal concentration (1 /~g per ml). IL-l-containing supernatants were added at a 1/10 dilution to triplicate cultures. After 24 h the cultures were pulsed with [3H]thymidine (1 pCi per well) and incorporation was measured after a further 24 h by liquid scintillation spectrometry.
Assays for IL~2 IL-2production Spleen cells from normal BALB/c mice 6 12 weeks old were prepared and adjusted to 10 7 cells per ml in RPMI 1640 medium supplemented with 2 mercaptoethanol at 10 5 M. One ml aliquots of the suspension in Linbro 24-well plates were cultured with or without Con-A (Sigma) at a final concentration of 2 #g/ml at 37°C in an atmosphere of 5% CO2 in air. Twenty-four hours later, the supernatants were collected and supplemented with 20 mg ~-methyl-o-mannoside (Sigma) per ml in order to inactivate residual Con-A. The cell-free supernatants were stored at -20°C until testing.
IL-2 activity
The IL-2 activity in supernatants was measured by their capacity to maintain in vitro proliferation of Con-A blasts. T-cell blasts were prepared by incubation of spleen cells from normal mice at a concentration of 10 v cells per ml with 3 #g/ml of Con-A in medium supplemented with 5% FCS for 48 h at 37°C in 5% CO2 in air. Cells were then harvested, washed and resuspended at a concentration of 2 x 105 cells per ml. 100 #1 aliquots of this cell suspension were then combined with 100/A of IL-2-containing supernatants and the cells were cultured for a further 24 h with [3H]thymidine present for the last 4 h. Incorporation was determined by liquid scintillation spectrometry. IL-2 activity
was also measured by the capacity to maintain the proliferation of a murine IL-2-dependent cytotoxic T-cell line (CTLL). 100 /~1 containing 1 × 104 CTLL cells were cultured in RPMI 1640 containing 10% FCS in triplicate microtitre wells with 100 pl of the sample to be tested for 24 h at 37°C in an atmosphere of 5% CO2 in air. Six hours before harvesting, the cultures were pulsed with [3H]thymidine (1 #Ci per well). Incorporation was measured by liquid scintillation spectrometry.
Assay for mononuclear cell 5-1ipoxygenase Human blood mononuclear cells prepared by Metrizoate/Ficoll gradient separation were homogenized by sonication and pre-incubated with 5-LO inhibitor at 37°C for 5 min. Reactions were initiated by the addition of 5/~M arachidonic acid and 1 mM CaCI2 and terminated after 5 rain by boiling. LTB4 was measured by specific radioimmunoassay.
Assay for chondrocyte collagenase production Articular cartilage was dissected from the knee and shoulder joints of young (<1 kg) New Zealand white rabbits using a method based on that described by Plaas et al. Briefly, cartilage slices were digested for 4 h at 37°C in Dulbecco's modified Eagles medium (DMEM) (Flow Laboratories) containing 0.5% BSA (Sigma) and antibiotics, to which collagenase (type II, Sigma) had been added. Cells were then washed three times in calcium-free PBS, filtered through a Nitex filter (Cadish, London) to remove undigested cartilage and were aliquoted into 24-well Linbro plates. After 6-8 days, incubation at 37°C in 5% CO2 in air in DMEM containing 10% FCS, the cultures had become confluent and were then used in assays for IL-I activity. The washed confluent monolayers were treated in triplicate with various concentrations of IL-1 with or without acetohydroxamic acids. After 24 h cell-free supernatants were harvested and stored at -20°C until assayed for collagenase content. Tritiated type 1 collagen from rat tail tendon (New England Nuclear) was induced to form fibrils by incubation for 2 h at 4°C in 20 nM Tris-HC1 buffer, pH 7.6, containing 5 mM CaC13. Collagenase-containing samples were activated with 4-aminophenylmercuric
acetate. The acetate was first dissolved in 0.5 M N a O H and then diluted with the calcium buffer to give a 0.5 mM solution. Supernatants were tested at a 1/10 dilution. Collagen fibril plus test supernatant were incubated at 37°C for 5-24 h. Tubes were then microfuged and the amount of solubilized collagen was measured by liquid scintillation spectrometry. Procedures not detailed here were as described in the N E N technical manual NEK-016. Results were expressed as a percentage of total digestion, the latter being determined with an excess of bacterial collagenase (NEN).
Results
Effect of 5-LO &hibitors on the production of IL-1 and IL-2 When freshly isolated resident peritoneal macrophages were stimulated with LPS to induce IL-1 production over a 24 h period in vitro, the addition of 5-LO inhibitors in the concentration range of 10 40 pM had no significant inhibitory effect on the production of Interleukin-l. In obtaining this result
tO
it was, of course, important to remove the drug by extensive dialysis of IL-l-containing supernatants before testing in the biological assay. The data are shown in Fig. 1. Similarly, when murine spleen cells were stimulated with Con-A to induce IL-2 production over a 24 h period in vitro, the addition of 5-LO inhibitors in the concentration range 10-40 /~mol had no significant effects on the production of IL-2 detectable in dialysed supernatants. The data are shown in Fig. 2. Effect o)C5-LO inhibitors on the thymocyte co-mitogenic response to IL-1 and the T-cell mitogenic response to IL-2 When IL-1 produced by LPS-stimulated murine macrophages or human blood monocytes was tested in the co-mitogenesis assay using murine thymocytes stimulated with a suboptimal dose of PHA (1 #g per ml), the addition of 5-LO inhibitors in the 10-40/~M range produced a dose-dependent inhibition of thymocyte proliferation (Fig. 3). The same picture of dose-dependent inhibition was observed when recombinant human IL-1 ~ or/~ were used to induce thymocyte proliferation. 5-LO inhibitors in
50
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Fig. 1. Resident peritoneal macrophages from BALB/c mice were cultivated for 24 h in the presence of LPS (1/~g/ml) with or without 5-LO inhibitor at the concentrations specified. Dialysed supernatants were tested in the thymocyte co-mitogenesis assay. O, BW A137C; II, BW A1C; &, BW A4C. Similar results were obtained in four independent experiments. Typically, proliferative responses measured by [3H]thymidine incorporation produced I ~ 1 5 × 103 cpm.
~. - 3 0 ¢n
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(lag per ml) Fig. 2. Spleen cells from BALB/c mice were cultured for 24 h in the presence of Con-A with or without 5-LO inhibitors at the concentration range specified. Dialysed, c~-methyl-o-mannosidetreated supernatants were then tested for their ability to maintain the proliferation of Con-A spleen cell blasts. O, BW A 137C; II, BW A1C; A, BW A4C. Similar results were obtained in three independent experiments. Typically, responses to 1L-2 were in the range of 5-10 × 104 cpm.
the same dose range also inhibited the response of murine T-cells to Interleukin 2 (Fig. 4).
100
•~, 80
~_ 60
40
•=
.o_ 20 e~ 03
0
10 5 2.5 1.25 Concentration of 5 - - L O inhibitor (pg per ml)
Fig. 3. Thymocytes from C3H/He mice were cultured for 72 h in the presence of a suboptimal dose of P H A (1 gg/ml) and a standard dose of IL-1 with or without 5-LO inhibitors at the concentration specified. O, BW A137C; I , BW AIC; A, BW A4C. These effects were highly consistent in a series of five experiments. One hundred per cent inhibition corresponds to the response obtained with P H A alone ( ~ 500 cpm).
A 100. o~
Effect of 5~LO inhibitors on lymphocyte proliferation in response to antigen and mitogen When normal human lymphocytes were stimulated with the recall antigen tetanus toxoid at a dose which produced near optimal responses (10/tg/ml) the addition of 5-LO inhibitors produced a dosedependent inhibition of the proliferative response. In order to investigate whether the observed effects were directly related to inhibition of 5-1ipoxygenase, excess substrate in the form of the arachidonic acid sodium salt was added. Although the addition of arachidonic acid generally enhanced lymphocyte proliferation in response to antigen, 40 160 /~M concentrations did not reverse the inhibition of proliferation produced by 5-LO inhibitors. Representative data are shown in Fig. 5. In order to compare the inhibitory effects of acetohydroxamic acids with standard immunosuppressive drugs, experiments were performed using azathioprine and mercaptopurine. There drugs were approximately ten-times more potent than 5-LO inhibitors on a molar basis, in suppressing mitogen-induced lymphocyte proliferation (Fig. 6)
;S_ 80 t ~
60
~
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Antigen Antigen +AA24pg per ml BW A137C 10lag per ml
u
20
0
BW A137C 10lJg per m~ + AA
10 5 2.5 1.25 Concentration of 5 - - L O inhibitor (~Jg per ml)
Fig. 4. Splenic Con-A blasts from BALB/c mice were cultured for 24 h in the presence o f a standard IL-2 containing supernatant with or without 5-LO inhibitors at the concentration specified. Similar results (not shown) were obtained by using recombinant 1L-2 and the CTLL-IL-2 dependent cell line. Q, BW A137C; II, BW A1C; A, BW A4C. Similar results were obtained in three independent experiments. One hundred per cent inhibition corresponds to a background response of 2 × 103 cpm.
BW A137C 20~g per ml BW A137C 20~Jg per ml + AA
0 B I
I
I
I
0
50
100
150
I 200 Antigen-induced lymphocyte proliferation (percentage of control)
Fig. 5. Normal h u m a n lymphocytes were cultivated for 5 days in the presence of tetanus toxoid (Wellcome Research Laboratories, Beckenham, Kent, U.K.) at 10 #g per ml. Arachidonic acid (sodium salt, Sigma) and 5-LO inhibitors were present throughout the culture at the concentrations and in the combinations specified. The same picture was obtained in three independent experiments. One hundred per cent corresponds to 3 x 104 cpm.
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Compound concentration (ug m1-1) B
Fig. 6. Normal human lymphocytes were cultured for 72 h in the presence of PHA at 6/tg per ml. 5-kO inhibitors, azathioprine and mercaptopurine were present at the concentrations specified. T, BW A4C; &, BW A137C; O, azathioprine; B, mercaptopurine. Similar results were obtained in two experiments.
100
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80 s0
Effect of 5~LO inhibitors on the spontaneous prol([eration of lymphoid and non-lymphoid cells
.~
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In order to ask whether the inhibitory effect o f 5L O inhibitors on cell proliferation was confined to lymphoid cells responding to immunologic signals, the effect o f these c o m p o u n d s on spontaneously dividing lymphoid and n o n - l y m p h o i d cells was investigated. A dose-dependent inhibition o f spontaneous proliferation similar to that observed with IL-1-induced proliferation was obtained with a murine lymphoid t u m o u r of B-cell origin (A20/2J) with h u m a n lymphoid t u m o u r lines (HSB2 M O L T 4), and with the murine m a c r o p h a g e derived cell-line R A W 264.7. The data are shown in Fig. 7. Inhibition o f proliferation was not due to non-specific toxicity since the c o m p o u n d s caused no release of 5~chromium from pre-labelled cells in cytotoxicity assays.
genase production by rabbit chondrocytes was investigated. Collagenase production in response to IL-1 was shown to be unaffected by 5-LO inhibitors in the dose-range which produced complete inhibition of lymphoproliferative responses (Fig. 8).
Effect of 5-LO inhibitors on IL-l-induced chondrocyte collagenase production
Effect of 5-LO inhibitors on blood mononuclear cell 5-lipoxygenase
In order to determine whether the inhibitory effects o f acetohydroxamic acids on I L - l - i n d u c e d cellular responses were confined to proliferative responses the effect o f 5-LO inhibitors on I L - l - i n d u c e d colla-
5-LO inhibitors BW A 4 C and BW A137C were found to be potent inhibitors of 5-1ipoxygenase activity in sonicates o f h u m a n blood m o n o n u c l e a r cells. The data are shown in Table I. Greater than
2e~ 0
I 1.25
I 2.5
I 5
I 10
Concentration of BW A 4 C (ug per ml)
Fig. 7. Cell-lines were cultured for 48 h with or without 5-LO inhibitors at the concentration specified. A, BW A137C; B, BW A4C; 0, HSB2; i , A20/2J; A, RAW 264.7; S, MOLT 4. Similar results were obtained in two experiments. Average maximum incorporation of[3H]thymidine corresponding to 100% was 8 x 104cpminAand5 x 104cpminB.
ition of lymphocyte proliferation by 5-LO inhibitors required concentrations in the 10-80/~M range (2.5-20 yg/ml).
Untreated control rlL-1/3
5 units per ml
"
+
BW A137C lOpg per ml
+
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+
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+
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t;~ili!,!i:i::~ii.ili:ii~!:i !; ii.~:.iii::.(~!i':i~:!~.~4 [ :iiii~:,ii!~ !i.i!.",!~:i!(i:! :i!::i'::i:!:i:.; .:!if:~4
Discussion
ii:!i!i;~;}:!:,..!!i'i!.;:ii!.i'.iiii!i:i:!i!i;!~ i;il, [:!i!,.i!'!.i.;iiliii!.!~i:i~:i!:::i..!:ii.:i~~il..i.: i i~ !'::'::!::!i:! ::':::"- :" "."i;~':::; "'~
+ 2.51Jg 1.251Jg per Perm' ml I
I
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I
I
I
0
20
40
60
80
100
Digestion of 3H-collagen (%1
Fig. 8. Primary chondrocyte cultures derived from articular cartilage of New Zealand white rabbits were stimulated with recombinant IL-fl for 24 h with or without 5-LO inhibitors at the concentration specified. Collagenase activity in cell-free supernatants was then assayed in a tritiated type 1 collagen fibril assay system. Similar results were obtained in three independent experiments. One hundred per cent digestion corresponds to the amount of soluble tritiated material produced by bacterial collagenase at 10 #g/ml which yielded 11 x 103 cpm.
80% inhibition was observed in the concentration range 0.1-1 #M (0.03-0.3 #g/ml). No inhibitory effects on lymphocyte proliferation in response to interleukins were observed in this dose range. InhibTABLE I Inhibition of mononuclear cell 5-LO by acetohydroxamic acids Treatment
LTB,, produced (ng)
Mean percentage inhibition
Boiled blank
0.0 0.3 1.85
--
Untreated sonicate
1.78
BW A4C (0.1 /zM) BW A4C (1.0 #M) BW A137C (1 #M) BW A137C (10 ,uM)
2.56 1.59 0.26 0.23 0.36 0.07 0.29 0.41 0.06 0.07
88 89 82 96
The present studies focussed on the immunomodulatory properties of a new class of specific 5-1ipoxygenase inhibitors. These novel acetohydroxamic acids were shown to consistently inhibit the proliferation of lymphoid cells in response to a wide range of signals without affecting the production of interleukins by myeloid and lymphoid cells. The compounds were found to inhibit the proliferation of lymphoid ceils in response to IL-1 and IL-2, at concentrations of 10 40/~M. The production of IL-1 in response to LPS by murine peritoneal macrophages was unaffected by the compounds. Similarly, the production of IL-2 in response to Con-A by murine splenocytes was unaffected by acetohydroxamic acids at the concentrations which completely inhibited the response of splenocytes and the CTLL line to Interleukin-2. The inhibitory effects of acetohydroxamic acids on lymphocyte proliferation were not confined to responses induced by IL-1 and IL-2. Thus the polyclonal proliferation of human peripheral blood lymphocytes induced by PHA, and the specific oligoclonal expansion of lymphocyes in response to tetanus toxoid, were inhibited to a comparable degree by 5-LO inhibitors in the 10-40 yM range. The spontaneous proliferation of transformed cell lines of T lymphocyte origin and the non-lymphoid line RAW 264.7 were likewise inhibited by acetohydroxamic acids in the same dose range. In order to investigate the possibility that these suppressive effects were due to inhibition of 5-1ipoxygenase in the responding cells, we measured the conversion of arachidonic acid to LTB4 by human blood mononuclear cell 5-1ipoxygenase in the presence and absence of 5-LO inhibitors. These novel 5-LO inhibitors were developed and characterized using polymorphonuclear 5-1ipoxygenase; nevertheless, these compounds produced almost complete inhibition of mononuclear cell 5-LO at concentrations of
0.1-1 #M. In this dose range lymphocyte proliferative responses were entirely unaffected. In order to achieve substantial inhibition of proliferation concentrations forty-fold greater than this were necessary. The inhibitory effects of 5-LO inhibitors on polymorphonuclear leukocyte 5-LO is reversible in the presence of excess substrate in the form of arachidonic acid. We therefore wished to know whether a molar excess of substrate could reverse the inhibition of lymphoproliferative responses by acetohydroxamic acids. Interestingly, the addition of arachidonic acid in the 40-160 /~M range modestly enhanced the proliferative response of lymphocytes to antigen. However, the addition of a four-fold molar excess of arachidonic acid did not reverse the inhibitory effects of 5-LO inhibitors on lymphocyte proliferation. Taken together these data indicate that the marked suppressive effect of these novel specific 5LO inhibitors on lymphoproliferative responses is unlikely to be due to their effect on 5-1ipoxygenase. Indeed, the data show that it is possible to completely inhibit 5-LO in mononuclear cells using low doses of inhibitor without affecting the proliferation of these cells in response to IL-1 and IL-2. This finding cannot be consistent with the suggestion made by others that products of the 5-1ipoxygenase pathway are obligatory mediators of the biological effects of interleukins (Dinarello et al., 1983; Farrar and Humes, 1985). Others have shown that Interleukin-I can stimulate lipoxygenation of arachidonic acid in certain T cell lines which proliferate and/or secrete lymphokines in response to interleukins (Farrar and Humes, 1985). However, neither exogenous 5H E T E nor leukotrienes B4, C4, D , and E4 could reconstitute the proliferative response to IL-2. The lack of correlation between inhibition of 5-LO and inhibition of lymphoproliferation by novel selective inhibitors of 5-LO demonstrated in the present study argues strongly against 5-LO products being essential components of the signal transduction process for IL-1 and IL-2. Consistent with this is the finding that the suppressive effects of 5-LO inhibitors were not confined to IL-1- and IL-2-in-
duced responses. While it could be argued that lymphocyte response to mitogen and specific antigen are dependent upon the production of interleukins 1 and 2, this is unlikely to be true for the spontaneous proliferation of transformed T cell lines and it is not true for the spontaneous proliferation of the non-lymphoid cell-line RAW 264.7. Moreover, signal transduction in rabbit chondrocytes where IL-1 induces the synthesis of collagenase was unaffected by high concentrations of 5-LO inhibitors. We conclude that the immunosuppressive properties of the selective 5-LO inhibitors described here are a novel secondary characteristic of acetohydroxamic acids. The suppression these compounds achieve in vitro is quite substantial. They are approximately one-tenth as potent as the conventional immunosuppressive drugs azathioprine and mercaptopurine on a molar basis. Their mode of action in this context is presently unknown. One possibility is that acetohydroxamic acids in the higher dose range inhibitory towards cell-proliferation, are affecting pathways of arachidonic acid metabolism other than 5-1ipoxygenase and that the products of these pathways are a contributory element in the regulation of cell proliferation.
Acknowledgements We are grateful to Mr R.W, Randall, Department of Biochemistry, Wellcome Research Laboratories, for performing assays for mononuclear cell 5-1ipoxygenase activity. We thank Drs L.G. Garland and G.A. Higgs for valuable discussion.
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