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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1954-1961.)
© 1997 American Heart Association, Inc.

Articles

Cytokine Modulation of LDL Oxidation by Activated Human Monocytes

Virginia A. Folcik; Rozina Aamir; ; Martha K. Cathcart

From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to Martha Cathcart, PhD, Department of Cell Biology, Building NC1, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail cathcam{at}cesmtp.ccf.org

	Abstract

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Abstract There is considerable evidence to suggest that cytokines modulate the pathological cellular events that occur in human atherosclerosis. We sought to determine the effects of T-helper-lymphocyte (T_H)-1- and T_H2-type cytokines on the ability of human monocytes to oxidize LDL, one of the pathological processes believed to occur in atherosclerosis. The ability of opsonized zymosan (ZOP)-activated human monocytes to oxidize LDL in a 24-hour period was significantly enhanced by pretreatment of the monocytes with the T_H2 cytokines, interleukin (IL)-4, or IL-13 compared with untreated monocytes. In contrast, interferon (IFN)-, a T_H1 cytokine, inhibited LDL oxidation by activated monocytes. Treatment with IFN- also prevented the IL-4- and IL-13-mediated enhancement of LDL oxidation by ZOP-activated monocytes. Untreated or cytokine-treated unactivated monocytes did not oxidize LDL. The enhancement of LDL oxidation mediated by IL-4 or IL-13 treatment was not due to a mitogenic effect of the cytokines on the monocytes, nor to modulation of superoxide anion (O₂^-) production. The cytokine regulation of 15-lipoxygenase (LO) in the monocytes was also examined. IL-4 and IL-13 induction of 15-LO mRNA and 15-LO activity in the monocytes was confirmed, as was the previously reported inhibition of induction by IFN-. In summary, IL-4 and IL-13 enhance the ability of activated human monocytes to oxidize LDL, whereas IFN- inhibits the cell-mediated oxidation. The up- and downregulation of activated monocyte-mediated LDL oxidation by these cytokines correlates with the expression of 15-LO activity. Considerable evidence suggests that the progression of atherosclerosis includes events that are immunologically mediated, lending potential physiological relevance to these in vitro observations.

Key Words: cellular activation • cytokines • human • monocytes • macrophages • LDL

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Atherosclerosis is a disease that involves a state of chronic inflammation. The presence of T lymphocytes, likely a source of cytokines, in atherosclerotic lesions has long been established.^{1 2 3 4 5} T-lymphocyte responses have been categorized as pro-inflammatory (T_H1 type) or anti-inflammatory (T_H2 type). T lymphocytes of theT_H1 type produce cytokines such as IFN-, IL-2, and lymphotoxin, whereas those of the T_H2 type produce cytokines such as IL-4, IL-5, IL-6, and IL-13 (reviewed in Mosmann and Coffman⁶ ). mRNA for cytokines of both types has been detected in human atherosclerotic lesions.⁷ IFN- has been detected in lesions in areas surrounding T lymphocytes as well as intracellularly.^{3 7} Both IFN-- and IL-4-producing T lymphocytes have been cloned from human atherosclerotic lesions.⁸ The expression of major histocompatibility complex class II by smooth muscle cells, macrophages, and endothelial cells^{9 10 11} and the expression of 15-LO in human lesions¹² provide further evidence that cytokines such as IFN-, IL-4, and IL-13 may be modulating an inflammatory response in the environment of the lesion. Additionally, the presence of 15-LO and IL-4 have been demonstrated in transplant coronary artery disease.^{13 14}

One of the major pathological processes that occurs in atherosclerotic lesions that may be immunologically modulated is the oxidation of LDL (reviewed in Chisolm and Penn¹⁵ ). We have studied an in vitro model of LDL oxidation using activated human monocytes, one of the cell types thought to be responsible for mediating LDL oxidation in vivo. We found that in medium without added metal ions, monocyte activation is necessary for LDL oxidation to occur. In this culture system, LDL oxidation is completely inhibited by SOD, indicating a role for O₂^-.¹⁶ Additionally, general antioxidants¹⁷ and general inhibitors ofLO, but not inhibitors of cyclooxygenase¹⁸ or specific inhibitors of 5-LO,¹⁹ inhibit the oxidation of LDL by activated human monocytes.

The enzyme 15-LO is believed to be one of the potential initiators of lipid oxidation in vivo. 15-LO protein, mRNA, and evidence of activity have been detected in vascular lesions of rabbits and humans in proximity with macrophages and oxidized LDL.^{12 20 21} Purified 15-LO is capable of oxidizing human LDL in vitro.^{22 23} Murine peritoneal macrophages, cells that constitutively express 12/15-LO, oxidize LDL to a significantly greater extent when activated with ZOP than activated peritoneal macrophages from mice in which the macrophage 12/15-LO gene has been abolished.²⁴ Others have found that murine fibroblasts exhibit an enhanced capacity to cause the formation of lipid peroxides in LDL after transfection with the human 15-LO gene.^{25 26} Two T_H2 cytokines, IL-4 and IL-13, induce the enzyme 15-LO in human monocytes.^{27 28} Because of the potential physiological significance of this observation, we examined the effect of IL-4 and IL-13 on the ability of ZOP-activated human monocytes to oxidize LDL. Additionally, it has been shown that IFN- prevents the induction of 15-LO by IL-4 or IL-13 in human monocyte/macrophages.^{27 28} IFN-, a proinflammatory cytokine, has been shown to inhibit the oxidation of LDL by mouse peritoneal macrophages²⁹ and human monocyte-derived macrophages in Ham's F-10 medium (a medium containing free metal ions) supplemented with l-tryptophan.³⁰ Here, the effect of IFN- on the ability of ZOP-activated monocytes to oxidize LDL was examined as well.

Because cytokines may affect more than one cellular function that could modulate the ability of the monocytes to oxidize LDL, the effect of each of the aforementioned cytokines on O₂^- production was examined, and any potential mitogenic effect on the monocytes was assessed.^{31 32} IL-4 has additionally been shown to upregulate mannose receptor expression in murine macrophages³³ and human monocytes,³⁴ an effect that might influence the level of ZOP activation.³⁵ The contribution of this effect was examined as well. By using cytokines with effects relevant to inflammatory processes and by studying their ability to modulate monocyte-mediated LDL oxidation, we hope to gain a better understanding of the oxidative mechanisms that are thought to contribute to the pathology of vascular disease.

	Methods

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LDL Isolation
LDL (density, 1.019 to 1.063) was isolated from human plasma (from freshly drawn blood) as previously described.^{36 37 38} The distilled, deionized water used for preparation of the density solutions was additionally treated with chelating resin. All dialyses were performed in the dark in bottles filled completely to minimize unnecessary oxidative stress on the LDL. LDL was dialyzed against Dulbecco's PBS with approximately 1.0 g/L chelating resin using fast-dialyzing Spectra/Por molecular-porous membrane tubing (molecular weight cut off, 12 000 to 14 000) for approximately 2 hours immediately before use to remove EDTA, which was used as an antioxidant and preservative during preparation. Unless otherwise indicated, LDL was added to yield a final concentration of 0.43 mg cholesterol/mL.

Cell Isolation and Culture
Monocytes were isolated from whole blood as previously described.^{18 19} Within 1 day of isolation, the monocytes were plated at 0.5x10⁶ cells/mL in 60 µL of DMEM with 10% bovine calf serum per well in Costar 96-well tissue culture plates. On the day cells were plated or on subsequent days (as indicated), the cytokines were added to the wells using RPMI 1640 or PBS with 0.5 to 1.0% BSABSA (essentially fatty acid free) as a vehicle. Control (vehicle-treated) wells received the same quantity of RPMI 1640 or PBS with BSA as those treated with cytokine. Human recombinant IL-4 and IFN- were purchased from Upstate Biotechnology Inc. Human recombinant IL-13 was purchased from R&D Systems. In accordance with the manufacturer's instructions, cytokines were reconstituted and stored at -20 or -70°C in PBS with 0.5 to 1% BSA. The doses of cytokine chosen were those previously shown to affect 15-LO activity in human monocytes.^{27 28}

Cultures were continued at 37°C in a humidified atmosphere with 10% CO₂ for the length of time indicated. When preincubation with cytokines was completed, the medium in the wells was changed to RPMI 1640 with phenol red. At this time, LDL and the activator, ZOP (final concentration, approximately 1.7 mg/mL),³⁹ were added to the wells where indicated. In all experiments the incubation was continued for 20 to 24 hours. At the end of the incubation, 10 µL of BHT (1 mmol/L) and 10 µL of EDTA (10 mg/mL) were added to prevent any further oxidation.

O₂^- Production
O₂^- production by the monocytes was measured as SOD-inhibitable cytochrome C reduction in 96-well tissue culture plates following the method of Pick and Mizel⁴⁰ with minor modifications. After 3 days of incubation in the presence or absence of cytokines, the medium in the wells was changed to RPMI 1640 without phenol red. Cytochrome C (from horse heart) was added to yield a final concentration of 160 µmol/L in 100 µL of RPMI 1640 per well, with and without 300 U/mL SOD (from bovine erythrocytes). ZOP was added to activate the cells (final concentration, 1.2 mg/mL). After incubation for 1 hour at 37°C, the plates were read in a Molecular Devices ThermoMax microplate reader at 550 nm. The nanomoles of O₂^- produced per milliliter (per 0.5x10⁶ monocytes) were calculated using the extinction coefficient 158.73.⁴⁰

Assay for TBARS
The assay for TBARS was performed in 96-well tissue culture plates, following a method kindly provided by Elliott Sigal and Craig Laughton (Syntex, Palo Alto, Calif.) with minor modifications. Standards were prepared with malonaldehyde bis(dimethyl acetal) and included on every plate. The standards were added in a volume equal to the starting volume of all the samples, usually 70 µL. At the end of the 24-hour incubation, BHT and EDTA were added to all wells as described above. The same additions were made to all samples, standards, and blanks. Fifty microliters of trichloroacetic acid (50% wt/vol) and 75 µL of 1% thiobarbituric acid in 0.3% NaOH were added to all wells. The plates were wrapped in plastic and incubated on a rack in a water bath for 40 minutes at 60 to 65°C. At the end of the incubation, the plates were cooled, centrifuged, then read on a Cytofluor II microwell fluorescence reader with excitation at 530 nm and emission at 590 nm. Calculations were made from the standard curve using Cytocalc II software to determine nanomole equivalents.

Cell Number Determinations
Comparison of cell numbers was accomplished by quantitation of DNA present in the culture wells at the end of the incubation with or without cytokines for 3 days. The DNA was quantified with a Cytoprobe Hoechst DNA assay kit. The assay is based on the binding of Hoechst 33258 to DNA.^{41 42} The assay was performed in 96-well flat-bottom and V-bottom plates (to prevent the loss of any nonadherent cells) with similar results.

Detection of 15-LO Activity in Human Monocytes Pretreated With Cytokines
Human monocytes were isolated and plated in Costar six-well tissue culture plates at 1x10⁶ cells/mL in 3 mL of DMEM with 10% bovine calf serum and incubated as described above for 3 days with 700 pmol/L IL-4, 700 pmol/L IL-4, plus 100 pmol/L IFN- or the same volume of vehicle for the cytokines, 0.5% BSA in PBS. At the end of the preincubation, the medium in the wells was changed to PBS with 5 mmol/L glucose. A control well without cells was prepared and treated in the same manner. Linoleic acid (final concentration, 160 µmol/L) was added as substrate, and the incubation was continued for another 20 to 25 minutes. At the end of the incubation, BHT (approximately 500 µmols/L) and an internal standard, [³H]-12- hydroxyeicosatetraenoic acid, were added, and the lipids were extracted from the wells and processed as previously described for analysis of linoleic acid oxidation products by reversed-phase HPLC.³⁸ The reduced linoleic acid oxidation products (HODEs) were collected and further analyzed by chiral-phase HPLC with a Chiralcel OD HPLC column and a mobile phase of n-hexane//isopropyl alcohol/acetic acid at a ratio of 100:5:0.1.⁴³ The flow rate for the mobile phase was 0.9 mL/min. The HODEs were detected by absorbance at 236 nm, and the internal standard was detected in the chiral-phase system using an online Radiomatic Instruments Flow-One Beta radioactive flow detector and Radiomatic Flo-Scint I at a flow rate of 1.5 mL/min. The retention times of the HODEs were determined using racemic and stereospecifically pure 13- and 9-HODE standards.

Determination of Stereospecificity of Oxidation Using LDL as Substrate
LDL (0.5 mg LDL cholesterol/mL) was incubated with ZOP-activated human monocytes under the same conditions that we previously used to study LDL oxidation in vitro.³⁸ Cytokine pretreatment of monocytes was for 3 days with 700 pmol/L IL-4 or 500 pmol/L IL-13 as described above. LDL was dialyzed as described above, then added to culture wells containing 1x10⁶ monocytes in RPMI or PBS with 5 mmol/L glucose. The incubation was continued for 6 to 24 hours. LDL was also oxidized by incubation with soybean 15-LO (type 1 from soybean) at 5000 U/mL in sodium borate buffer, pH 9.²² Oxidation was stopped by the addition of BHT (final concentration, 500 µmols/L). The lipids were extracted and saponified, and the fatty acids were resolved by reversed-phase HPLC.³⁸ Chiral-phase HPLC was performed on these samples using previously described methods.²¹

	Results

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To determine how the cytokines IL-4, IL-13, and IFN- might affect the ability of human monocytes to oxidize LDL, human monocytes were isolated and cultured with the cytokines, then their ability to oxidize LDL was examined. The monocytes were preincubated with the cytokines for 1 to 3 days. For groups incubated with cytokine for 1 or 2 days, addition to the cells was delayed for 2 days or 1 day, respectively, so that all groups would remain in culture for the same number of days. The results from a representative experiment including preincubation of the monocytes with IL-4 are shown in Fig 1, A. Monocytes, pretreated with IL-4 at 700 or 1400 pmol/L or with vehicle, were then activated with ZOP. In all experiments, regardless of cytokine pretreatment, activation of the monocytes with ZOP was still required for detectable LDL oxidation to occur. The IL-4-treated monocytes oxidized LDL to a significantly greater extent than controls pretreated with vehicle. Cell-free controls ruled out any effect of the cytokines used herein on lipid oxidation in the absence of cells (data not shown). IL-4 at 700 pmol/L appeared to have a nearly maximal effect on monocyte-mediated oxidation of LDL. The degree of enhancement appeared to exhibit some donor variation, and in general, enhancement of oxidation was greater when oxidation by untreated monocytes was lower. It was also noted that monocytes, kept in culture for 3 days without cytokine treatment, generally tended to oxidize LDL to a lesser extent than freshly isolated monocytes. This effect was not due to treatment with vehicle, because control monocytes incubated without any pretreatment for 3 days exhibited essentially the same capacity to oxidize LDL as cells incubated with vehicle (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of time of pretreatment and cytokine dose on monocyte-mediated LDL oxidation. Human monocytes were incubated with the indicated quantity of vehicle, IL-4 or IL-13, for the number of days indicated. Addition of cytokine to monocytes pretreated for 1 or 2 days was delayed by 2 days or 1 day, respectively, so that all groups spent a total of 3 days in culture before activation and incubation with LDL. After the cytokine pretreatment, the medium in the wells was changed to RPMI 1640, and LDL and the activator, ZOP, were added. After 20 to 24 hours of incubation, LDL oxidation was determined as TBARS. A, Monocytes were pretreated with the vehicle (BSA) or IL-4 at 700 or 1400 pmol/L. Background oxidation in the absence of monocytes is shown. B, In the same experiment shown in panel A, monocytes were pretreated with IL-13 at 100 pmol/L, 500 pmol/L, and 1000 pmol/L. The error bars represent the SD of samples performed in triplicate in an experiment representative of five performed.

In some experiments, the level of monocyte-mediated LDL oxidation was additionally determined using an assay for lipid peroxides.⁴⁴ When LDL oxidation was assessed using assays for both lipid peroxides and TBARS, the results obtained from the assay for lipid peroxides were similar to those obtained using the TBARS assay (data not shown). In the 12 experiments performed, IL-4-treated (700 pmol/L) monocytes oxidized LDL, resulting in 4.46±1.43 nmols TBARS/0.5 mg LDL cholesterol, which was significantly more than vehicle-treated monocytes, which resulted in 1.78±0.92 nmols TBARS/0.5 mg LDL cholesterol (P<.0001).

Enhancement of LDL oxidation by activated monocytes was also evident in cells pretreated with IL-13. In Fig 1, B, data are presented from a representative experiment in which cells were preincubated for 1 to 3 days with IL-13 at a range of doses. The enhancement of LDL oxidation was dose dependent and increased with the number of days of pretreatment. The IL-13-mediated enhancement appeared to be maximal with 3 days of pretreatment at 500 pmol/L. IL-13 treatment alone did not induce monocytes to oxidize LDL without activation by ZOP. Data compiled from five experiments performed with activated monocytes pretreated with the optimal dose of IL-13 showed significantly more LDL oxidation than monocytes pretreated with vehicle (4.15±1.60 versus 1.32±0.16 nmols TBARS/0.5 mg LDL cholesterol, respectively; P<.02).

IFN- is a T_H1-type cytokine with effects that generally oppose those of the T_H2-type cytokines IL-4 and IL-13.^{6 27 28} For this reason, we examined the effect of IFN- on the ability of human monocytes to oxidize LDL both alone and in combination with IL-4 and IL-13. The results shown in Fig 2, A, are from a representative experiment in which cells were preincubated for 3 days with varying doses of IL-4 in the presence or absence of IFN- at 100 pmol/L. IFN- did not induce unactivated monocytes to oxidize LDL (data not shown). There was considerable inhibition of LDL oxidation by 100 pmol/L IFN- in combination with IL-4 at all doses as compared with cultures using IL-4 and no IFN- (Fig 2, A). IFN- treatment also inhibited LDL oxidation by vehicle-treated activated monocytes (Fig 2, A and B). The IFN--mediated inhibition of LDL oxidation by IL-4- or vehicle-treated, activated monocytes was detectable at 10 pmol/L (Fig 2, B). The inhibition of LDL oxidation by cells pretreated with the combination of cytokines was also dose dependent (Fig 2, B). Similarly, IFN- also inhibited LDL oxidation mediated by activated monocytes pretreated with IL-13 (Fig 3). This inhibition was dose dependent as well.

View larger version (21K): [in this window] [in a new window]   Figure 2. LDL oxidation by activated human monocytes pretreated with a combination of IL-4 and IFN- at various doses. A, Human monocytes were preincubated for 3 days with the vehicle or IL-4 at the indicated concentrations or IL-4 plus IFN- at 100 pmol/L before incubation for 24 hours with LDL and the activator, ZOP. B, Monocytes were also pretreated with 700 pmol/L IL-4 plus the indicated doses of IFN- or with the vehicle and the indicated doses of IFN-. Background oxidation in the absence of cells was 0.33±0.02 nmoles TBARS/0.5 mg LDL cholesterol (mean±SD, n=6). The error bars represent the SD of three replicates from an experiment representative of six performed or the SD of six replicates (*) from the same experiment.

View larger version (14K): [in this window] [in a new window]   Figure 3. LDL oxidation by activated human monocytes pretreated with a combination of IL-13 and IFN- at various doses. Human monocytes were preincubated for 3 days with the indicated doses of the vehicle or IL-13 in the absence or presence of IFN- at 50 or 100 pmol/L. Background oxidation in the absence of cells was 0.25±0.01 nmoles TBARS/0.5 mg LDL cholesterol (mean±SD, n=6). The error bars represent the SD of three replicates from an experiment (filled squares and triangles) or the SD of six replicates from the same experiment (open squares). The experiment shown is representative of six performed.

Cytokines affect many cellular functions. For example, IL-4 is a known mitogen for lymphocytes.^{31 32} To examine the possibility that a cytokine-dependent increase in cell number was responsible for the enhancement of activated monocyte-mediated LDL oxidation, the number of monocytes present at the end of a 3-day preincubation with or without cytokines (700 pmol/L IL-4 or 500 pmol/L IL-13) was quantified and compared. Relative cell number was determined by quantifying double-stranded DNA.^{41 42} Neither of the cytokine-treated groups had more DNA than the vehicle-treated group; in fact, the IL-4 treated group had significantly less DNA (P=.033, Mann-Whitney two-sample test; data not shown). These results rule out a cytokine-induced increase in monocyte number as an explanation for the observed enhancement of oxidation.

Another effect that IL-4 has on human monocytes is upregulated expression of mannose receptors.³⁴ This effect may be relevant to activated monocyte-mediated LDL oxidation in that the activator, ZOP, may interact with mannose receptors on the monocytes.³⁵ To examine whether upregulated expression of mannose receptors by IL-4 was responsible for the enhanced ability of the monocytes to oxidize LDL, -mannan was used to pretreat the monocytes (before ZOP activation) at concentrations previously shown to block mannose receptors on monocyte/macrophages (100 or 200 µg/mL).^{33 34 35} Blocking of mannose receptors by -mannan did not inhibit the ability of vehicle- or IL-4-pretreated monocytes to oxidize LDL (data not shown).

O₂^- production by activated human monocytes has previously been found to be necessary for monocyte-mediated LDL oxidation to occur.¹⁶ The effects of IL-4, IL-13, and IFN- on the respiratory burst were therefore examined. As expected, ZOP activation of the monocytes resulted in significantly more O₂^- production as compared with that detected in the absence of activator (Table 1). O₂^- production by activated human monocytes that were pretreated with 700 pmol/L IL-4 was slightly lower than that detected with monocytes pretreated in the absence of cytokines (Table 1, set A; P=.07, Student's t test). Additionally, none of the doses of IL-13 caused any significant effect on the respiratory burst of ZOP-activated human monocytes, separately comparing each dose to the group without cytokine (Table 1, set B). IFN- has previously been shown to enhance the respiratory burst of macrophages.^{45 46} IFN- treatment of the monocytes slightly increased the levels of O₂^- production with and without ZOP activation (Table 1, set C), but the increased levels were not significantly greater than their respective cytokine-free controls (Table 1, set C; IFN- with ZOP versus no cytokine with ZOP, P=.34; IFN- without ZOP versus no cytokine without ZOP, P=.14). Furthermore, neither IL-4 nor IL-13 increased the amount of O₂^- released at later time points after the initial burst of O₂^- production (data not shown). Modulation of the respiratory burst by pretreatment of the monocytes with IL-4, IL-13, or IFN- did not positively correlate with the enhanced or inhibited ability of activated human monocytes to oxidize LDL.

View this table: [in this window] [in a new window]   Table 1. Effect of Cytokine Pretreatment on O2- Production by Human Monocytes1

IL-4 has been shown to induce the presence of 15-LO in human monocytes, and this induction was prevented by IFN-.²⁷ To verify that 15-LO enzymatic activity was present in the IL-4-pretreated monocytes, but not in those treated with IFN-, the stereospecificity of linoleic acid oxidation by cytokine pretreated monocytes was assessed.^{43 47 48} Monocytes were isolated and incubated in the presence or absence of 700 pmol/L IL-4, with and without IFN- (100 pmol/L), for 3 days. The stereospecific oxidation of linoleic acid was then quantitatively assessed to detect 15-LO activity as described in "Methods." Monocytes pretreated with the vehicle alone had no detectable 15-LO activity (Fig 4, A). Monocytes pretreated with IL-4 clearly exhibited 15-LO activity, as evidenced by a predominance of 13-(S)-HODE as compared to 13-(R)-HODE (Fig 4, B), and this effect of IL-4 was prevented by cotreatment with IFN- (Fig 4, C). Pretreatment of the monocytes with IFN- alone resulted in no detection of 15-LO activity (Fig 4, D). Negligible quantities of linoleic acid oxidation products were detected in the cell-free control (data not shown). In a separate experiment, linoleic acid was also incubated with monocytes that were kept in culture for 3 days with and without IL-4, then activated with ZOP. Without IL-4 pretreatment, no 15-LO activity was detectable in the ZOP-activated monocytes (data not shown). IL-4-pretreated monocytes that were then activated with ZOP did have detectable 15-LO activity (data not shown). The induction of 15-LO mRNA in monocytes pretreated with IL-4 and IL-13 was also verified by Northern blot analysis (data not shown). These results are consistent with the results of Conrad et al²⁷ and Nassar et al.²⁸

View larger version (23K): [in this window] [in a new window]   Figure 4. 15-LO activity in monocytes pretreated with IL-4. Monocytes were pretreated for 3 days in the presence or absence of 700 pmol/L IL-4 or 100 pmol/L IFN-, then the medium was changed, and the cells were given 160 µmol/L linoleic acid as substrate for oxidation. After a 25-minute incubation, the lipids were extracted, and the oxidized fatty acids were separated by reversed-phase HPLC. The reduced oxidation products of linoleate oxidation (13- and 9-HODE) were collected and further analyzed by chiral-phase HPLC (a method that resolves the S and R stereoisomers). The figure shows chiral-phase HPLC analysis of HODEs from linoleic acid incubated with monocytes pretreated with the vehicle (BSA) (A) (S/R ratio=1.28), with monocytes that had been pretreated with 700 pmol/L IL-4 (B) (S/R ratio=7.45), with monocytes that had been pretreated with 700 pmol/L IL-4 and 100 pmol/L IFN- (C) (S/R ratio=2.71), or with monocytes that had been pretreated with 100 pmol/L IFN- (D) (S/R ratio=1.07). The absorbance peak areas were adjusted for the recovery of the internal standard.

Stereospecific oxidation of linoleic acid is a clear indicator of 15-LO activity.^{47 48} Esterified linoleic acid is the most abundant fatty acid substrate for oxidation in LDL, so the ratio of 13-(S)-HODE to 13-(R)-HODE (the primary oxidation product of linoleic acid) was assessed in monocyte oxidized LDL (Table 2). There was no significant difference in the S/R ratio of 13-HODE from LDL oxidized by IL-4-pretreated activated monocytes compared with 13-HODE from LDL oxidized by vehicle-pretreated activated monocytes. Similar results were obtained with IL-13 pretreatment. 13-HODE from LDL oxidized by soybean 15-LO was also formed without stereospecificity (Table 2), indicating that 15-LO can promote the oxidation of esterified linoleic acid in LDL by a nonstereospecific mechanism. Under the same in vitro conditions, free linoleic acid was oxidized by soybean 15-LO to form 13-(S)-HODE almost exclusively (data not shown).

View this table: [in this window] [in a new window]   Table 2. S/R Ratio of 13-HODE Derived From Oxidized LDL1

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Because of the evidence for an inflammatory response involving T lymphocytes in the environment of atherosclerotic lesions^{1 2 3 4 5 7 8 9 10 11} and the presence in lesions of LDL oxidation products,^{12 20 21} we examined how several cytokines with relevant, previously defined effects on monocytes would affect the ability of activated human monocytes to oxidize LDL. IL-4 and IL-13 were chosen to represent the T_H2 subset of cytokines, generally regarded as being anti-inflammatory in nature. IFN- was chosen to represent the T_H1 set of cytokines, generally regarded as proinflammatory and abundant in human atherosclerotic lesions.^{3 7} If one considers oxidation processes to be proinflammatory events, then the cytokine effects observed here might seem contrary to their previously understood and categorized effects in states of inflammation. That is, IFN- inhibits cell-mediated oxidation, and IL-4 and IL-13 enhance it. The inhibitory effect on cell-mediated LDL oxidation that we (Figs 2 and 4) and others^{29 30} have observed for IFN- has several implications. First, IFN- may actually be providing a net protective effect in the lesion environment by slowing the process of cell-mediated oxidation and lesion progression. Second, the presence of 15-LO and oxidation products in lesions^{12 21} despite the abundant presence of IFN-⁷ may also indicate that long-term exposure of the cells in the lesion to IFN- results in a refractory state.

IFN- has the proinflammatory effect on monocyte/ macrophages of increasing the respiratory burst,^{45 46} whereas IL-4 has the anti-inflammatory effect of decreasing the release of O₂^- by human monocytes.^{45 49} Our laboratory has defined a role for O₂^- in LDL oxidation by activated human monocytes in that the process is completely inhibited by SOD, although LDL need not be present during the respiratory burst to become oxidized. Our laboratory has determined that, under these conditions, after the peak of the respiratory burst has subsided, O₂^- continues to be produced at low levels and LDL oxidation remains sensitive to SOD inhibition.¹⁶ Consequently, minor changes in the magnitude of the respiratory burst will not necessarily affect LDL oxidation by activated monocytes. Consistent with all previous observations, the slight decrease in the peak levels of O₂^- production during the respiratory burst due to IL-4 pretreatment (Table 1) had no inhibitory effect on the ability of monocytes to oxidize LDL. O₂^- production by activated monocytes at later times (up to 24 hours) after the respiratory burst was also not altered by pretreatment with IL-4 or IL-13 (data not shown). Monocytes pretreated with IFN- but not activated with ZOP produced slightly elevated basal levels of O₂^- (Table 1) but did not oxidize LDL. It has previously been shown in cell-free systems that O₂^- alone causes minimal lipid peroxidation on fatty acids in LDL⁵⁰ (Zendedel-Haghighi A, Li Q, Cathcart MK, unpublished data, 1993) and other biological membranes.^{51 52 53} Thus, it appears that O₂^- production by monocytes is not sufficient for monocytes to oxidize LDL and that additional factors are required.

Another oxidation mechanism that monocyte/macrophages possess that is regulated by cytokines is the enzyme 15-LO. 15-LO activity becomes detectable in monocytes only after pretreatment with IL-4 or IL-13^{27 28} (Fig 4). We have previously reported evidence for the involvement of an enzyme, presumably 15-LO, in forming stereospecific lipid oxidation products in human atherosclerotic lesions.²¹ Our results from human tissue were consistent with those of Kuhn et al,²⁰ showing that stereospecifically formed oxidation products could be detected in atherosclerotic lesions from rabbits. Because 15-LO mRNA, protein, and activity had been detected in atherosclerotic lesions,^{12 20 21} it seemed relevant to determine what effect cytokines that induce 15-LO would have on the ability of monocytes to oxidize LDL in vitro. What we found was a marked dose- and time-dependent enhancement of the ability of activated monocytes to oxidize LDL after IL-4 and IL-13 pretreatment using doses that induce 15-LO in human monocytes (Figs 1 through 4). Pretreatment of the monocytes with IL-5 (100 or 200 pmol/L), another T_H2 cytokine that does not induce 15-LO in monocytes,²⁷ had no effect on the ability of activated monocytes to oxidize LDL (data not shown).

The enhanced ability to oxidize LDL that we have found in human monocytes correlates with other reports of cell-mediated oxidation systems in which 15-LO was introduced or eliminated and significant changes were detected in the cells' ability to oxidize LDL. In one case, the transfection of a fibroblast cell line with 15-LO conferred upon it an enhanced capacity to oxidize LDL.^{25 26} In the other case, the peritoneal macrophages from mice in which the constitutively expressed 12/15-LO gene was eliminated by homologous recombination were compared with wild-type peritoneal macrophages for their ability to oxidize LDL.²⁴ The peritoneal macrophages from the wild-type mice oxidized LDL to a significantly greater extent upon activation with ZOP than the macrophages from the 12/15-LO-deficient mice. The murine macrophage 12/15-LO enzyme is thought to be the murine counterpart to human 15-LO.²⁴

Another effect of IL-4 on monocyte/macrophages that could affect their ability to oxidize LDL in our system is the upregulation of mannose receptor expression^{33 34} because ZOP could be interacting with mannose receptors on the monocytes.³⁵ We addressed this by blocking the mannose receptors on the cytokine- or vehicle-treated monocytes with -mannan before activation with ZOP. There was no inhibition of the ability of the monocytes to oxidize LDL with or without cytokine pretreatment (data not shown), indicating that mannose receptor upregulation is unlikely to be responsible for the enhanced oxidation of LDL by activated monocytes. In agreement with these findings, others have assessed the ability of monocytes to phagocytose zymosan after IL-4 or IFN- treatment and have found it to be unaltered.⁴⁵

Because IL-4, IL-13, and IFN- undoubtedly exert a multitude of effects upon monocytes, we cannot conclude that the enhanced ability of the activated monocytes to oxidize LDL was necessarily due to the induction of 15-LO in these cells. Although 15-LO activity was clearly induced in monocytes by pretreatment with IL-4 (Fig 4, B), when the oxidized fatty acid products from LDL oxidized by monocytes pretreated with IL-4 were analyzed, stereospecific oxidation (a fingerprint of 15-LO activity) was not detected (Table 2). There are several possible explanations for this observation. It is possible that the contribution to LDL oxidation from monocyte LO activity was not detectable because it primarily contributed to initiation events, which then were followed and obscured by oxidation products from nonenzymatic reactions, or that racemization occurs under the in vitro conditions and also prevents detection of any initial stereospecificity. Belkner et al^{23 54} have shown that purified LO oxidizes complex substrates such as LDL with consistently lower levels of stereospecificity, compared with nearly complete stereospecific oxidation of pure fatty acid substrate. We have found a similar lack of stereospecificity with soybean 15-LO oxidation of LDL (Table 2), whereas linoleic acid was oxidized by soybean 15-LO with nearly complete stereospecificity (data not shown). The study of mechanisms of oxidation of LDL is also complicated by the presence of endogenous antioxidants in LDL.⁵⁵ Another possible explanation for this finding is that 15-LO products play a role in cellular signaling mechanisms rather than direct oxidation of LDL substrate in vitro. At the other end of the spectrum, it is possible that 15-LO induction is merely coincidental with some other cellular mechanism that is upregulated and significantly enhances the ability of the activated monocytes to oxidize LDL. The current lack of a genuinely specific means to inhibit 15-LO prevents us from definitively demonstrating or refuting this mechanism.

In summary, we have found that the ability of activated human monocytes to oxidize LDL is significantly enhanced by pretreatment of the monocytes with the anti-inflammatory cytokines IL-4 and IL-13 under the same conditions that induce the enzyme 15-LO in these cells.^{27 28} This enhancement is not explained by a mitogenic effect, altered interaction with ZOP, or increased O₂^- production by the monocytes. The enhanced capacity to oxidize LDL is inhibited by cotreatment with proinflammatory IFN- at concentrations that prevent 15-LO induction, and the ability of activated monocytes to oxidize LDL in the absence of IL-4 or IL-13 is inhibited by similar concentrations of IFN-. Inhibition of LDL oxidation by IFN- treatment alone indicates that IFN--mediated inhibition may entail more than counteraction of IL-4- and IL-13-induced effects. Alone, cytokine pretreatment that causes the induction of 15-LO in monocytes is not sufficient to render the monocytes capable of oxidizing LDL lipids to detectable levels in our culture system; additional activation is required.

Substantial evidence from studies of atherosclerotic lesions suggests that atherosclerosis is an inflammatory process involving various components, including activated lymphocytes, activated monocytes, oxidation of LDL, and the presence of 15-LO.^{1 2 3 4 5 7 8 9 10 11 12 13 14 15 20 21} Our studies suggest a potential contributing role of the T_H2 cytokines IL-4 and IL-13 in the pathogenesis of atherosclerosis and a countereffect by IFN-, a product of T_H1 lymphocytes. Additional studies characterizing the exact mechanisms responsible for the cytokine regulation of monocyte-mediated oxidation of LDL are warranted.

	Selected Abbreviations and Acronyms

 

BHT = butylated hydroxytoluene BSA = bovine serum albumin HODE = hydroxy-octadecadienoate HPLC = high-performance liquid chromatography IFN- = interferon- IL = interleukin LO = lipoxygenase O2- = superoxide anion SOD = superoxide dismutase TBARS = thiobarbituric acid reactive substances TH = T-helper lymphocyte ZOP = opsonized zymosan

	Acknowledgments

 
This work was supported by an Individual National Research Service Award (grant HL08904-03) (V.F.) and grant HL51068 (M.K.C.) from the National Institutes of Health, Bethesda, Md. We thank Jack Battisto, PhD, and Paul E. DiCorleto, PhD, for reviewing the manuscript and providing helpful input. We also appreciate the expert technical assistance of Laura Yoho. This article was presented in part at the 68th Scientific Sessions of the American Heart Association (Anaheim, Calif; November 1995).

Received September 30, 1996; accepted April 4, 1997.

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