Regulation of 5-lipoxygenase activity in mononuclear phagocytes: characterization of an endogenous cytosolic inhibitor

Regulation of 5-Lipoxygenase Activity in Mononuclear Phagocytes: Characterization of an Endogenous Cytosolic Inhibitor Michael J. Coffey,1 Steven E. Wilcoxen, Peter H. Sporn,2 and Marc Peters-Golden Divisions of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109 The proinflammatory leukotrienes (LT) play important roles in host defense and disease states. However, no endogenous mechanisms to downregulate 5-lipoxygenase (5-LO), the enzyme catalyzing LT synthesis, have been described. We observed that the cytosolic fraction of rat alveolar macrophages (AMs) and peritoneal macrophages (PMs), and of peripheral blood monocytes (PBMs) contain substantial amounts of 5-LO protein, but little detectable 5-LO activity. We therefore examined these mononuclear phagocyte (MNP) cytosolic fractions for inhibitory activity against 5-LO. MNP cytosol dose-dependently reduced the 5-LO activity in neutrophil (PMN) cytosol and AM membrane. Furthermore, MNP cytosol dose-dependently prolonged the lag phase of soybean lipoxygenase (LO) without affecting the rate of product formation. This effect was overcome by subsequent addition of 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD), suggesting that the active factor scavenges hydroperoxides. Inactivation by boiling and proteinase K suggest that is a protein. We speculate that this cytosolic factor(s) may serve as an endogenous means for the down-regulation of 5-LO in macrophages. Keywords: Macrophage; monocyte; lung; leukotriene; eicosanoid 1 Address correspondence to: Michael J. Coffey, Assistant Professor of Internal Medicine, 6301 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 481090642; Phone: (313) 764-4554; Fax: (313) 764-4556; E-mail: [email protected] 2 Current address: Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL.

Prostaglandins & other Lipid Mediators 56:103–117, 1998 © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0090-6980/98/$19.00 PII S0090-6980(98)00046-X

Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. Introduction Leukotrienes (LT) play important roles in both normal host defense and in the pathogenesis of a multitude of inflammatory disease states, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and a variety of pulmonary disorders, including asthma (1). 5-lipoxygenase (5LO) catalyzes the first two steps in the synthesis of LT: oxygenation of arachidonic acid (AA) to 5-hydroperoxyeicosatetraenoic acid (5-HpETE) and subsequent dehydration of 5-HpETE to LTA4. In view of the importance of this key enzyme in LT synthesis, the molecular regulation of its activation has been studied extensively. Most investigators have utilized granulocytic cells, including human neutrophil (PMN), rat basophilic leukemia (RBL-1) cells, and dimethyl sulfoxide-differentiated HL-60 cells. In these cells, greater than 90% of the 5-LO activity and immunoreactive protein are localized to the cytosol. In response to agonists which increase intracellular Ca21, the enzyme translocates to the nuclear membrane, with resultant LT synthesis from AA. Another cell type which synthesizes LT in substantial amounts is the mononuclear phagocytic (MNP) cell, the macrophage. Despite being the main resident inflammatory cell type in most organs, little is known about the regulation of 5-LO in macrophages. Recent studies from our laboratory have delineated a novel activation scheme for 5-LO in alveolar macrophages (AMs) which is fundamentally different from that described for granulocytic cells (2– 4). Interestingly, we also observed in the course of these studies that the cytosolic fraction of both AMs and peritoneal macrophages (PMs) contained substantial amounts of 5-LO protein, but little or no measurable 5-LO activity as determined by cell-free assay. The present study was undertaken to examine the apparent discrepancy between the amount of immunoreactive 5-LO protein and its enzymatic activity in the cytosol fraction of these two MNP populations. We hypothesized that there was an endogenous “inhibitor” in the cytosol of these MNPs which regulated LT synthesis by reducing cytosolic 5-LO activity. Therefore, we examined the effect of a variety of MNP cytosol on 5-LO cell free activity.

Materials and Methods Cell Isolation and Culture Fresh rat AMs and PMs were obtained by lavage of the lung and peritoneum, respectively, as previously described (2). 96% of the cells lavaged from the lung and 81% of the resident cells obtained from the peritoneum (after hypotonic lysis) were macrophages as determined by modified Wright-Giemsa stain (Diff-Quik, American Scientific Products, McGaw Park, IL). Type II alveolar epithelial cells were isolated by elastase digestion of rat lung and harvested following culture in Dulbecco’s Minimum

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. Essential Medium (GIBCO) containing 10% fetal calf serum (FCS) for 48 h (5). Ficoll-Hypaque centrifugation of venous blood from normal volunteers yielded PMNs (98% pure as assessed by Diff-Quik staining) and peripheral blood monocytes (PBMs) (80% pure as assessed by non-specific esterase staining). The following cell lines were obtained from American Type Culture Collection (Rockville, MD). RBL-1 cells were cultured in Minimum Essential Medium (GIBCO) containing 10% FCS. Cells of the A549 human alveolar epithelial carcinoma line and human monoblastlike U937 cells were cultured in RPMI. HL-60 human promyelocytic leukemia cells were cultured for 7 days in RPMI alone or RPMI containing 1.2% DMSO for 4 days (Sigma, St. Louis, MO) (to induce myeloid differentiation) (6). Adherent cells were scraped into buffer (50 mM potassium phosphate, 0.1 M NaCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, and 60 mg/mL soybean trypsin inhibitor, pH 7.1) and freshly isolated cells were resuspended in the same buffer. Preparation of Cytosol and Membrane Fractions The above cells were suspended in 1 mL of buffer and sonicated using a Model 250 Sonifier (Branson Ultrasonics, Danbury, CT). Cell sonicates were centrifuged at 10,000 3 g for 10 min at 4°C to remove nuclei and unbroken cells. The supernatant was centrifuged again at 100,000 3 g for 60 min at 4°C. After removal of the 100,000 3 g supernatant (cytosol fraction), the pellet was rinsed to remove residual cytosol. The washed pellet (membrane fraction) was then resuspended in 1 mL of buffer by homogenization on iced ethanol, using a Potter-Elvehjem homogenizer. Both cellular fractions were stored at 270°C in buffer containing 10% glycerol as a cryoprotective agent. Immunoblot Analysis of 5-LO Immunoblot analysis of 5-LO in cytosol fractions was performed as described (2). Aliquots of protein samples (20 – 40 mg) were denatured, boiled and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels overlaid with a 5% stacking gel by the method of Laemmli (7). Proteins were transferred overnight to nitrocellulose membranes, and probed with rabbit polyclonal antibody against either human leukocyte 5-LO (1:3,000 dilution). Antisera against 5-LO were kindly provided by Dr. J. Evans, Merck Frosst (Pointe Claire-Dorval, Quebec). After washing, blots were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham) at a dilution of 1:5000. Membranes were then washed and incubated for 1 min with chemiluminescence detection reagents (ECL, Amersham), and exposed to film for varying time periods to ensure that densitometric quantitation was performed under conditions in which band density and exposure time were linearly related. Video densitometry was performed using Image (NIH) software.

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. Assay of Cell-Free 5-LO Activity 5-LO activity was determined in 1-mL reaction mixtures containing 0.2 M Tris-HCl, 3 mM CaCl2, 1.6 mM EDTA, 1.8 mM ATP, and aliquots (;100 mg) of PMN cytosol, RBL-1 cytosol, or AM membrane, as described (2,8). Assay mixtures contained final concentrations of 20 mM AA (NuChek Prep. Elysian, MN), including ;100,000 dpm of [3H]AA (sp act 76 Ci/mmol)(Du Pont-New England Nuclear) per assay tube, and 2.6 mM 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD) (Cayman Chemicals, Ann Arbor, MI). The reaction was stopped after 10 min by adding 1 mL absolute ethanol and 10 mL formic acid per tube. The acidified mixture was then extracted twice with 1 mL of chloroform, evaporated under nitrogen and stored at 270°C. Lipid residues were dissolved in 400 mL methanol and analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a 5-mm Bondapak C18 column (30 3 0.4 cm; Waters Associates, Milford, MA) using a mobile phase of acetonitrile/water/trifluoroacetic acid, at a flow rate of 2 mL/min. 5-LO metabolites and free AA were eluted during a series of linear gradient increases of acetonitrile from initial conditions of 50:50:0.1 (v/v/v) to 73:27:0.1 (v/v/v) at 7 min, then to 85:15:0.1 (v/v/v) at 9 min, and finally to 100:0:0.1 (v/v/v) at 15 min. The eluate was continuously monitored for UV absorbance (280 nm for LTB4 and its isomers, 235 nm for 5-HpETE and 5-HETE, and 210 nm for AA). Retention times were as follows: LTB4 and its isomers, co-eluting at 5.5 min; 5-HpETE and 5-hydroxyeicosatetraenoic acid (5-HETE), co-eluting at 9.0 min; and AA, 12.5 min. Radioactivity in 1 mL eluate fractions was quantitated by liquid scintillation counting. 5-LO specific activity was calculated based on conversion of AA to 5-HpETE/5-HETE plus LTB4/LTB4 isomers, and was expressed as nmol/mg protein/10 min. Soybean Lipoxygenase (LO) Assay Activity of soybean LO (Sigma) was assayed at 23°C using a continuous spectrophotometric method as described (9). Soybean LO was suspended in 0.2 M sodium borate buffer, pH 9.0 at a final concentration of 30 units per one mL final volume. The reaction was initiated by the addition of sodium arachidonate (final concentration 20 mM). Formation of products, predominantly hydroperoxides, was monitored continuously at wavelength 238 nm on a Beckman Model 35 Spectrophotometer. Preliminary experiments revealed that activity was linearly related to enzyme concentration over the range 10 to 100 units, and maximal at AA concentrations . 20 mM. The rate of enzyme activity was determined by the amount of hydroperoxide product formed in nmoles per unit of enzyme per min, using an extinction coefficient of 2.5 3 104 M21 cm21. Cytosol fractions to be tested for “inhibitory” activity were added to the reaction mixture, stirred and the reaction initiated.

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. Protein Assay Protein was quantitated by the method of Bradford (10) using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin (BSA) as standard. Data Analysis All data for which n $ 3 are expressed as mean values 6 SEM. The significance of differences between means was assessed by analysis of variance and the Scheffe F-test. A p value , 0.05 was considered significant.

Results Discrepancy Between 5-LO Protein and Activity in MNP Cytosol In the prior investigations of the molecular regulation of 5-LO in macrophages, we noticed that little 5-LO activity was detectable in rat AM and PM cytosol fractions, although there was considerable 5-LO protein present (2). Therefore, we determined 5-LO activity and immunoreactive protein levels in the cytosolic fractions of these cells as well as a number of other cell types. Representative data for cytosolic 5-LO protein and cell-free activity from human PMNs, human PBMs, rat AMs, and rat PMs are shown in Figure 1. As reported previously (8), human PMN cytosol contained relatively large amounts of both 5-LO protein and activity. Only rat AMs and PMs, and to a lesser extent, human PBMs, contained relatively more 5-LO protein than specific enzymatic activity. Inhibition of 5-LO Activity by MNP Cytosol One possible explanation for the relatively large amounts of inactive 5-LO protein in the three MNP cytosols was the presence of an inhibitor of 5-LO. We evaluated this by performing mixing studies utilizing MNP cytosol added to various sources of active 5-LO (PMN cytosol, RBL-1 cytosol, and AM membrane). Figure 2 displays representative HPLC analyses of radioactive products formed by aliquots (90 mg protein) of PMN cytosol in the presence and absence of added rat AM cytosol. Rat AM cytosol alone had little 5-LO activity, as demonstrated previously (Fig. 1). The major product of the PMN cytosol cell-free assay was 5-HETE/5-HpETE (eluted at 9 min) along with a minor LTB4 peak (5.5 min). An earlier peak eluting at ;2 min remains to be identified, but is unlikely to represent prostaglandins since the cyclooxygenase enzyme is generally not considered to be localized to the cytosol fraction, and because the addition of indomethacin to the reaction mixture did not decrease this peak. Rat AM cytosol added to PMN cytosol decreased the 5-HpETE/5-HETE peak substantially. The apparent increase in the early peak was not a reproducible finding. The inhibitory effects of AM cytosol against PMN cytosol 5-LO were dose-dependent (Fig. 3), and similar

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FIGURE 1. Relationship between levels of immunoreactive 5-LO protein and activity in the cytosolic fractions of various cell types. Aliquots of cytosol were subjected to immunoblot analysis (top panel) and assay for cell-free 5-LO activity (bottom panel), as described in Material and Methods. Results are from experiments representative of several performed for each cell type.

results were obtained against the 5-LO activity present in resting AM membrane (Fig. 3) and RBL-1 cytosol (not shown). As with AM cytosol, similar results were obtained in mixing studies using PM and PBM cytosol as the source of “inhibitory” activity against 5-LO (data not shown). Incubation of radiolabeled LTB4 and 5-HpETE with MNP cytosol did not decrease the quantities of these eicosanoids identified by HPLC, indicating that the above results are not the consequence of degradation of the products of the 5-LO reaction. It is also unlikely that this cytosolic inhibitory factor(s) is a protease, since the buffer contains a mixture of anti-proteases. Furthermore, the incubation of MNP cytosol with sources of 5-LO protein at 37°C failed to reveal any evidence of increased 5-LO protein degradation, as determined by immunoblot analysis of 5-LO in the cytosol of rat AM (Fig. 1). MNP Cytosol Prolongation of Soybean LO Lag Phase Soybean LO forms hydroperoxides from AA via an oxygenation reaction which is similar to that catalyzed by 5-LO. In addition, it is readily

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FIGURE 2. Representative 3H-labeled HPLC elution profiles from 5-LO reaction mixtures containing PMN cytosol alone (left) and together with rat AM cytosol (right). Cell-free assays were performed as described in Materials and Methods. Peaks of radioactivity were identified by co-elution of authentic standards. 50 mg of PMN cytosol protein was utilized yielding a specific activity of 10 nmoles/mg/min. With the addition of 50 mg of rat AM cytosol the calculated specific activity for 50 mg of PMN cytosol is 1.2 nmoles/mg/10 min.

available and has been extensively studied. However, since soybean LO does not require Ca21, the contribution of 5-LO activity contained in cellular cytosols can be eliminated. Therefore we performed mixing studies to determine the effects of cellular cytosols on the kinetics of soybean LO activity assessed using a continuous spectrophotometric assay. The

FIGURE 3. Dose-dependent inhibitory effect of rat AM cytosol on cellular 5-LO activity. AM cytosol fractions were added to the cell-free 5-LO assay of two test cellular fractions known to contain 5-LO activity, PMN cytosol (90 mg, left) and AM membrane (30 mg, right), as described in Material and Methods. Ratios of AM cytosolic protein to test fraction protein are depicted on the abscises.

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FIGURE 4. Dose-dependent effect of rat AM cytosol on soybean LO reaction curves. Tracings from a representative experiment show dose-dependent prolongation of the control soybean LO lag (a) and with the addition of (b) 25 mg, 30 mg, and 35 mg of rat AM cytosol.

rate of soybean LO product formation under standard conditions (30 units, 20 mM AA) was 1.8 6 0.2 nmoles per mg (136,000 units) per min (n 5 10). The addition of MNP cytosol dose-dependently prolonged the control lag phase (usually 2–3 min) of soybean LO, as shown for AM cytosol in Figure 4. Once the reaction had commenced, after the prolonged lag phase, the rate of product formation was unchanged from control. Figure 5 demonstrates the dose-dependent prolongation of the soybean LO lag phase by different batches of AM and PM cytosol. Notably, there was a steep increase in potency of “inhibition” at 20 –30 mg of MNP. Preparations of MNP cytosol containing 20 – 40 mg protein prolonged the lag phase up to 1 h, but reactions were generally not monitored beyond 45 min. Similar patterns were observed with the addition of PBM cytosol. The potency of “inhibitory” activity was expressed by defining 1 unit as the reciprocal of the amount of MNP cytosol protein in mg required to prolong the control lag phase fivefold. It was expressed in this fashion because this degree of prolongation of the lag phase approximates the mid-point of the linear portion of dose-response curves, as seen in Figure 5. While different batches of MNP cytosol varied in potency, we observed rather consistently that cytosol from PM was slightly more potent per mg of protein than AM cytosol, which in turn was substantially more potent than PBM cytosol (Table 1). The potency of the “inhibitory” activity of each preparation declined after storage for 4 weeks. Importantly, cells which had no discrepancy between 5-LO protein and activity, PMN, demonstrated no “inhibitory” activity. Furthermore, cells which have no 5-LO protein, rat type II alveolar epithelial cells (AECs), had no inhibition of 5-LO activity.

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FIGURE 5. Dose-dependent prolongation of soybean LO lag phase by rat AM and PM cytosol. Soybean LO reaction was performed as described in Material and Methods. This graph summaries the effect of different batches of AM cytosol (top) (n 5 4) and PM cytosol (bottom) (n 5 3) on the soybean LO lag phase.

Hydroperoxides Reverse the Effect of MNP Cytosol on Soybean LO The lag phase constitutes the period necessary for the generation of hydroperoxides, which are required for activation of soybean LO and 5-LO. The prolonged lag phase was immediately overcome by the addition of a lipid hydroperoxide, 13-HpOD, resulting in product accumulation at a rate which parallels that of the control reaction (Fig. 6). This lipid hydroperoxide did not absorb at 238 nm at the concentration used and so its addition to the reaction does not account for the increased absorbance at this wavelength. A concentration of 0.5 mM was sufficient, and was as

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. TABLE 1. Effect of cellular cytosols on the lag phase and rate of product formation of soybean LO

Cell cytosols

Effect on lag phase (inhibitory units)

Effect on reaction rate (% of control slope)

Rat AM (n 5 4) Rat PM (n 5 3) Human PBM (n 5 3) Human PMN Rat type II AEC

75.1 6 12.4* 92.2 6 18.1* 17 6 11.8* ,5 ,5

97.2 6 14.1 101.5 6 19.5 100 6 33 91 136

Activity of soybean LO was continuously monitored in the absence or presence of cytosolic fractions obtained from the cells indicated. The effect on lag phase was expressed in units of ‘‘inhibitory activity’’ (1/mg protein to increase the lag phase to fivefold the control value) and effect on reaction rate as a percentage of the control slope. AEC, alveolar epithelial cell; *p , 0.05 compared to buffer.

effective as those as high as 10 mM. This suggests that the mechanism by which MNP cytosol modulated soybean LO activity involved decreasing the quantities of lipid hydroperoxides. The next logical step was to determine if the inhibition of 5-LO activity in AM and PM cytosol could be reversed by the addition of excess HpOD. The standard cell-free 5-LO activity utilizes 2.6 mM HpOD. When we added 10 mM HpOD we noted an increase in macrophage cytosol cell-free 5-LO activity: AM cytosol

FIGURE 6. Reversal of the rat AM cytosol-induced prolongation of soybean LO reaction curves by hydroperoxide. Control soybean LO reaction mixture was incubated without (a) or with (b) 35 mg of rat AM cytosol. At the indicated time following the addition of AM cytosol, 10 mM of 13 (S)-HpOD was added.

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. TABLE 2. Characteristics of rat AM cytosol ‘‘inhibitory activity’’

Experiment 1 Control LO Untreated AM cytosol NEM-treated AM cytosol Experiment 2 Control LO Untreated AM cytosol Boiled AM cytosol Experiment 3 Control LO Untreated AM cytosol Proteinase K-treated AM cytosol

Lag phase (min)

Inhibitory activity (units)

2.6 11.4 14

— 75.8 61.7

2.9 8.7 1.9

— 161.3 0

3.4 11.4 3.3

— 98 0

Activity of soybean LO was continuously monitored in the absence (control LO) or presence of rat AM cytosol subjected to various treatments. In separate experiments AM cytosol was 1) incubated with NEM (25 mM) for 30 min 2) boiled for 3 min, and 3) incubated with proteinase K (50 mg/mL) for 1 h, prior to addition to the reaction mixture. Comparison is made between the effects of addition of untreated AM cytosol and AM cytosol treated as indicated.

(5.3-fold increase), and PM cytosol (4.4-fold increase). There was no significant change in PMN cytosol cell-free 5-LO activity (1.2-fold increase). This indicates that inhibition of soybean LO and 5-LO activity by macrophage cytosol is likely due to the same mechanism namely the regulation of peroxide levels. One possible candidate to mediate this action is glutathione peroxidase, a cytosolic enzyme which uses reduced glutathione in order to reduce lipid hydroperoxides (11). To evaluate this possibility, we preincubated MNP cytosol for 30 min with the sulfhydryl reactant N-ethylmaleimide at 25 mM, a concentration known to rapidly deplete reduced glutathione via conjugation in intact macrophages (12). There was no effect of such pretreatment on the “inhibitory” activity of PBM cytosol (Table 2), ruling out the possibility that a glutathione-dependent enzyme such as glutathione peroxidase was responsible for prolongation of the soybean LO lag phase by MNP cytosol. Characteristics of MNP Cytosol Inhibitory Factor Boiling of MNP cytosol for 3 min completely abolished any “inhibitory” activity (Table 2). Moreover, preincubation of MNP cytosol with proteinase K (50 mg/mL) for 1 h destroyed its ability to prolong the soybean LO lag phase. Freezing and thawing MNP cytosol reduced its potency. Taken

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. together, these observations strongly suggest that the factor responsible for prolongation of the soybean LO lag phase is a protein. It is not a non-specific effect of protein, however, since the addition of albumin at concentrations as high as 100 mg/mL had no effect on the soybean LO lag phase (data not shown).

Discussion Despite the considerable ability of MNPs to elaborate LT, little is known about the regulation of 5-LO in these cells. Although cytosol fractions of AMs and PMs contained substantial amounts of 5-LO protein, they contained little or no detectable 5-LO activity. Our current findings indicate that this discrepancy is accounted for by the presence of a protein factor(s) in MNP cytosols with “inhibitory” activity against 5-LO. The inhibitory factor also regulates soybean LO and appears to scavenge hydroperoxides which are necessary for initiation of the lipoxygenation reaction. A clue to the mechanism of action of MNP cytosol was suspected when it was incubated with soybean LO in a continuous spectrophotometric assay. MNP cytosol prolonged the lag phase of soybean LO in a dose-dependent fashion, without reducing the rate of product formation, once the reaction commenced. Since the normal lag phase in this and other LO reactions reflects the time required for the accumulation of hydroperoxides which are necessary to activate the enzyme, we suspected that the factor(s) in MNP cytosol was acting by scavenging hydroperoxides. Immediate reversal of the prolongation of the lag phase by the addition of exogenous hydroperoxides in concentrations known to activate LO (1–10 mM) (13) supports this hypothesis. Increased exogenous hydroperoxide levels in MNP cytosols also augmented cell-free 5-LO activity, sugesting that the same mechanism of scavenging hydroperoxides reduces both soybean and 5-LO activity. Recently, a novel compound termed YT-18 (2,3-dihydro-2,4,6,7-tetramethyl-2-[(4-phenyl-l-piperazinyl) methyl]-5-benzofuranamine) was shown to inhibit 5-LO activity by a similar mechanism of scavenging peroxide activators (14). Glutathione peroxidase is the major enzymatic mechanism for decreasing the concentrations of lipid hydroperoxides (15). However, the distribution of LO “inhibitory” activity which we observed does not correlate with the known distribution of this enzyme in various cell types; the glutathione peroxidase content of AMs has been reported to be about twice that of PMNs and three-fold that of PMs (16). Our findings that the sulfhydryl reactant N-ethylmaleimide failed to abrogate “inhibitory” activity provided additional experimental evidence against the involvement of glutathione peroxidase as well as another glutathionedependent enzyme, 5-HpETE peroxidase (17,18). DTT, present in our homogenization buffer, is another reducing agent which might impair

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. TABLE 3. Relationship between discrepant levels of 5-LO activity and protein and the presence of ‘‘inhibitory’’ activity against 5-LO/LO. Source of cytosol

5-LO activity

5-LO protein

5-LO inhibition

Soybean LO prolonged lag

Human PMN Human PBM Rat AM Rat PM Rat type II AEC Human U937

111 1 1 1 2

111 11 111 111 2

2 1 11 11 ND

2 1 11 11 2

2

2

ND

2

In mixing experiments aliquots of the different cellular cytosols were added to both the cell-free 5-LO HPLC assay and the soybean LO spectrophotometric assay, as described in Material and Methods. The ‘‘inhibitory’’ activity is compared to the relative amounts of 5-LO activity and protein in the cytosolic subcellular fracitons, as seen in Figure 1. AEC, alveolar epithelial cell; ND, not done.

5-LO activity (19). However, its final concentration in soybean LO reaction mixtures was only 30 mM, and it was present not only in MNP cytosols but also in PMN cytosol which exhibited no demonstrable effect on the soybean LO lag phase. An alternative hypothesis for the cytosolic inhibitory activity might be explained by a protein containing an anti-oxidant moiety which maintains 5-LO in a reduced state, preventing its activity (20 –22). If this is the case, 5-LO activity occurs when the anti-oxidant is exhausted, which occurs when it is fully oxidized through the pseudoperoxidase activity of 5-LO using the lipid hydroperoxide as substrate and oxidant. Only cells which demonstrated greater relative amounts of 5-LO protein than activity in cytosolic fractions displayed “inhibitory” activity (Table 3). This was true for rat AMs and PMs and human PBMs. PMs exhibited the greatest discrepancy and had the most potent inhibitory effect on 5-LO and soybean LO activity. PBMs exhibited the least discrepancy between 5-LO protein and activity and had substantially less inhibitory effect. Cell types which had considerable amounts of both cytosolic 5-LO protein and activity, such as human PMNs, DMSO-differentiated HL-60 cells, and RBL-1 cells, contained no inhibitory activity. Recently, other investigators have described a discrepancy between 5-LO protein and 5-LO enzyme activity in a monocyte cell line differentiated with 1,25-dihydroxyvitamin D3 or transforming growth factor b (23). Cytosolic components of these cells with low LT synthetic capacity inhibited purified 5-LO activity in a cell-free assay (24). Another important finding from our data are that cells which lack 5-LO and are unable to elaborate LT, like type II AEC and the U937 monocytic cell line, demonstrated no “inhibitory” activity in their cytosol (Table 3).

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Cytosolic Inhibitor of 5-Lipoxygenase Activity: Coffey et al. Homeostasis is served by the existence of endogenous counter-regulatory mechanisms to check the activities or functions of pro-inflammatory pathways. Examples of this phenomenon include anti-oxidants, protease inhibitors antagonists to occupancy of the interleukin-1 (IL-1) receptor, and endogenous inhibitors of cyclooxygenase. No definitive endogenous regulatory mechanism has been described for the 5-LO pathway, despite the intense pro-inflammatory actions of its products. It is attractive to speculate that the protein factor(s) in MNP cytosol identified in the present investigation might serve such a purpose. Elucidating its significance will require its purification and the development of more sensitive approaches to its detection.

Acknowledgments This work was supported by a grant from the American Lung Association of Michigan. M.J.C. is the recipient of National Institutes of Health Clinical Investigator Development Award. M.P.-G. was supported by the NIH (R01-HL471), and a Career Investigator Award from the American Lung Association.

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Editor: Dr. A. Ford-Hutchinson Received: 03-19-98 Accepted: 05-28-98

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