Cellular and Molecular Mechanisms of the Selective Regulation of IL-12 Production by 12/15-Lipoxygenase

Melissa K. Middleton, Tanya Rubinstein and Ellen Puré
J Immunol January 1, 2006, 176 (1) 265-274; DOI: https://doi.org/10.4049/jimmunol.176.1.265

Abstract

IL-12 drives type I immune responses and can mediate chronic inflammation that leads to host defense as well as disease. Recently, we discovered a novel role for 12/15-lipoxygenase (12/15-LO) in mediating IL-12p40 expression in atherosclerotic plaque and in isolated macrophages. We now demonstrate that 12/15-LO regulates IL-12 family cytokine production in a cell-type and stimulus-restricted fashion. LPS-stimulated elicited peritoneal macrophages derived from 12/15-LO-deficient (Alox15) mice produced reduced IL-12 and IL-23 levels, but comparable amounts of several other inflammatory mediators tested. Furthermore, LPS stimulation triggered an increase in wild-type macrophage 12/15-LO activity, whereas pharmacological inhibition of 12/15-LO activity suppressed LPS-induced IL-12 production in wild-type macrophages. 12/15-LO-deficient macrophages also produced reduced levels of IL-12 in response to TLR2 stimulation, but not in response to CpG (TLR9) or CD40/CD40L-mediated activation. In contrast to our previous finding of reduced IL-12 production in the setting of atherosclerosis, we found that comparable IL-12 levels were produced in Alox15 and wild-type mice during an acute response to LPS in vivo. This paradox may be explained by normal production of IL-12 by 12/15-LO-deficient neutrophils and dendritic cells, which are major sources of IL-12 during acute inflammation. Finally, we detected selectively decreased association of the transcription factors IFN consensus sequence binding protein and NF-κB with the IL-12p40 promoter in 12/15-LO-deficient macrophages. Taken together, these findings reveal a highly selective pathway to IL-12 production that may prove a useful target in chronic inflammation while sparing the acute response to infection.

Interleukin 12 plays a critical role in determining the nature of both the innate and adaptive inflammatory responses. The importance of IL-12 in host defense against bacterial, parasitic, and fungal pathogens has been well established (1). IL-12 also contributes to chronic inflammatory disorders such as atherosclerosis and inflammatory bowel disease (2, 3). Therefore, elucidating the mechanisms by which IL-12 is regulated is key to obtaining a better understanding of the immune response and is likely to provide opportunities for developing novel therapeutic approaches for the treatment of inflammatory diseases.

IL-12 is a heterodimer, consisting of a p40 and a p35 subunit (4). IL-12p35 is constitutively expressed in many cell types and can be further up-regulated in response to inflammatory stimuli, whereas p40 expression is induced exclusively in cells of the immune system, such as macrophages, dendritic cells (DCs),3 and neutrophils, in response to stimulation (5). In addition to heterodimerizing with p35 to form biologically active IL-12p70, IL-12p40 can be secreted alone as monomers or homodimers and can heterodimerize with p19 to form the novel cytokine, IL-23 (5). Production of many inflammatory mediators, including IL-12, is induced by cell-cell interactions such as CD40/CD40L and by the binding of microbial-derived ligands to pattern recognition receptors such as TLRs. TLRs are specific for common microbial products such as LPS, a bacterial cell wall component that activates TLR4, Staphylococcus aureus Cowan strain (SAC), and its derivatives, which activate TLR2, and unmethylated CpG motifs in bacterial DNA that stimulate TLR9 (6). Endogenous ligand stimulators of IL-12 production have also been described and include minimally modified low-density lipoproteins, heat shock protein 60, fatty acids, and the association of CD44 with fragments of extracellular matrix components such as hyaluronan, which are likely to provide inflammatory stimulation in noninfectious settings such as atherosclerosis (7). The signaling cascades downstream of TLRs ultimately induce the activation of the IL-12p40 promoter by multiple transcription factors, among them NF-κB and IFN regulatory factor (IRF) family members, including NF-κBp50 and IFN consensus sequence binding protein (ICSBP), respectively (5). IL-12 and IL-23 stimulate T lymphocytes, NK cells, and NKT cells to produce IFN-γ (8). In turn, IFN-γ primes macrophages to subsequently produce markedly elevated levels of IL-12 and IL-23 (5). This reciprocal activation forms the foundation of the Th1-type cytokine response (type I inflammatory response), which is essential for cellular immunity (5). The type I axis is thought to suppress the activation of the Th2 (type 2, humoral) response, which is dominated by IL-4 and IL-10 (9). Likewise, IL-4 and IL-10 both can inhibit IL-12 production, contributing to a balance between type I and type II responses (9). In the absence of IL-12 in vivo, IFN-γ production is severely reduced, compromising the type I pathway (5). Although IL-12 has been shown to aggravate chronic inflammatory disorders such as atherosclerosis, its regulation in the context of this and other inflammatory diseases remains poorly understood.

Eicosanoids, lipid mediators that are derived from arachidonic acid, both promote and suppress inflammation (10). The major mediators of eicosanoid production include the cyclooxygenase and lipoxygenase families (11). 12/15-lipoxygenase (12/15-LO) is a pro-oxidant, eicosanoid-producing enzyme that incorporates molecular oxygen into unsaturated lipids such as linoleic and arachidonic acid (12).

Growing evidence suggests that 12/15-LO may modulate the inflammatory response. Others have reported that 12(S)-hydroxyeicosatetraenoic acid (12(S)HETE), a major product of the 12/15-LO pathway, can promote monocyte-endothelial interactions in diabetic and atherosclerotic mouse models (13, 14). Recently, it was demonstrated using short-interfering RNAs that decreased 12/15-LO expression resulted in decreased oxidative stress and reduced MCP-1 levels in a monocytic cell line (15).

Previously, while studying 12/15-LO-deficient mice on an atherosclerosis-prone background (Apobec-1/low density lipoprotein receptor double knockout), we observed a significant reduction in aortic lesion IL-12p40 as compared with 12/15-LO wild-type mice (16). Furthermore, the induction of IL-12 was defective in 12/15-LO-deficient macrophages stimulated with LPS in vitro.

In this study, we have defined cellular and molecular mechanisms by which 12/15-LO regulates IL-12 production. We demonstrate that 12/15-LO activity mediates IL-12 production in wild-type macrophages and that 12/15-LO regulates the expression of IL-12 in a cell-type and stimuli-restricted manner that can be modulated by IFN-γ. In addition, we demonstrate that 12/15-LO is involved in the recruitment of ICSBP and NF-κB to the IL-12p40 promoter. These observations provide novel insight into the regulation of type I cytokines in the inflammatory response.

Materials and Methods

Mice

Age and sex-matched C57BL/6 and Alox15 mice on a C57BL/6 background (backcrossed 11 generations) were purchased from The Jackson Laboratory, housed in the Wistar Institute animal facility (Philadelphia, PA), and used at 8–12 wk of age. For in vivo LPS studies, mice received injections i.p. with 50 μg of Escherichi coli LPS (Sigma-Aldrich), 3 nm of 1668 CpG (synthesized by Oligos Etc.), or PBS vehicle control. All animals were treated in accordance with a Wistar Institute Institutional Animal Care and Use Committee-approved protocol.

Thioglycollate-elicited peritoneal cells

C57BL/6 and Alox15 mice received injections i.p. with 1 ml of sterile 3% Brewer’s thioglycollate broth (Sigma-Aldrich). At 16–20 h (neutrophils) or 4 days (macrophages), cells were harvested by peritoneal lavage with Ca2+/Mg2+-free PBS, and cultured in RPMI 1640 supplemented with 10% FCS, 50 μM 2-ME, 1% penicillin, streptomycin, and fungizone in 5% CO2 as described previously (17). Neutrophils were found to be at least 90% pure by cytospin analysis. Macrophages were at least 95% pure by flow cytometric or cytospin analysis after adherence purification.

Bone marrow-derived cells

Bone marrow cells were cultured at 2 × 105 cells/ml in 10% RPMI 1640 supplemented with 10% FCS, 50 μM 2-ME, 1% penicillin, streptomycin, and fungizone, and 20 ng/ml GM-CSF (PeproTech). For bone marrow-derived macrophages, cells were washed at day 5 to remove all nonadherent cells and harvested using 10 mM EDTA, as described previously (18). Macrophages were confirmed to be 95–98% pure by flow cytometry for F4/80 and CD11b expression and/or cytospin analysis. DCs were prepared as described (19). Briefly, the GM-CSF concentration was halved at day 10, nonadherent cells were aspirated, and loosely adherent cells were harvested at days 11–12. The purity of CD11c+ cells was determined by flow cytometric analysis to be between 85 and 95%.

Cell stimulations

Cells were treated with 1 μg/ml E. coli LPS (Sigma-Aldrich), 2 μg/ml 1668 CpG (Oligos Etc.), peptidoglycan (InvivoGen), SAC (EMD Biosciences), or CD40L-CD8 hybridoma supernatant with 10 μg/ml anti-CD8α cross-linking Ab (20) for 24 h, and supernatants were stored at −20°C for future analysis. The 12/15-LO inhibitor, PD146176, was obtained from Sigma-Aldrich and used at the indicated doses. Non-LPS stimulants were pretreated with 25 μg/ml polymyxin B (Sigma-Aldrich) for 20 min at room temperature before being added to cells to inhibit any contaminating endotoxin. As indicated in some experiments, cells were primed with 500 U/ml IFN-γ (R&D Systems) for 16 h before stimulation. For RNA analysis, cells were stimulated with IFN-γ + LPS (or LPS alone) for 4 h before RNA extraction with TRIzol (Invitrogen Life Technologies). All cultures were normalized to viability by the metabolic MTT assay as described previously (21).

Reverse transcription reaction/quantitative real-time PCR (Q-PCR)

RNA was treated with Turbo DNase (Ambion) according to the manufacturer’s instructions to remove any contaminating genomic DNA, and the absence of appreciable genomic DNA was confirmed by real-time PCR of the treated RNA. RNA was normalized for concentration, and reverse transcription reaction was performed using a cDNA synthesis kit (Applied Biosystems) according to manufacturer’s instructions. Q-PCR analysis was performed using SYBR Green Master Mix and run in the ABI 7000 machine (Applied Biosystems). Gene-specific primers (see Table I⇓) were designed using Primer Express (Applied Biosystems), and RNA levels were normalized using β-actin as an internal control. Fold induction of RNA expression was determined by dividing all normalized values within a data set by the normalized arbitrary units of the unstimulated wild-type control.

View this table:
Table I.

Real-time primer sequences

Cytokine levels

IL-12p40, IL-12p70, TNF-α, and IL-6 OptEIA ELISA kits were purchased from BD Biosciences, and supernatant was analyzed according to the manufacturer’s instructions. Nitrite levels were measuring using a Griess Reagent Assay kit purchased from Cayman Chemicals. The levels of 12(S)HETE and 13(S)-hydroxyoctadecadienoic acid (13(S)HODE) were analyzed by ELISA (Assay Designs). The IL-23 ELISA was performed in the laboratory of Dr. Chris Hunter (University of Pennsylvania, Philadelphia, PA) as described previously (22).

Chromatin immunoprecipitation (ChIP)

Four micrograms of Ab (goat anti-ICSBP, rabbit anti-NF-κBp50, and isotype controls; Santa Cruz Biotechnology) were used to precipitate SDS lysates from 106 macrophages per condition overnight. Ab-Ag complexes were collected with protein G Sepharose rotation for 2 h before being washed under stringent conditions to remove nonspecific DNA. Ab-Ag complexes were then eluted, and subsequently the cross-links were reversed in Tris-EDTA/SDS overnight. Protein was digested with proteinase K, and DNA was recovered by phenol chloroform extraction. The percentage of DNA bound by a particular transcription factor was quantified by real-time PCR for the IL-12p40 or TNF-α promoter and normalized to input DNA. β-Actin was used as a control against nonspecific DNA contamination. Primer sequences are listed in Table I⇑.

Statistical analysis

Unpaired two-tailed student t tests were calculated using Excel software (Microsoft). One-way ANOVA was calculated in Graphpad Prism (GraphPad). A p value of <0.05 was considered statistically significant.

Results

12/15-LO regulates IL-12 family member production

We previously presented evidence that IL-12 production is mediated by a novel 12/15-LO-dependent pathway as well as by 12/15-LO-independent mechanisms (16). Specifically, we reported that the production of IL-12p40 in response to LPS stimulation was defective in Alox15 macrophages compared with wild-type macrophages in vitro and in murine atherosclerotic plaque (16). In view of the critical role of IL-12 in driving the type I inflammatory response, we sought to further characterize this novel pathway and to define the cellular and molecular mechanisms by which it impacts the production of IL-12 family cytokines. As previously reported using Alox15 mice backcrossed seven generations, we found LPS-stimulated IL-12p40 production was markedly attenuated in unprimed as well as IFN-γ-primed macrophages from Alox15 mice backcrossed 11 generations to C57BL/6 compared with wild-type littermate controls (Fig. 1⇓, A and B). Macrophages derived from 12/15-LO heterozygous mice produced intermediate levels of IL-12p40 upon stimulation (Fig. 1⇓A), revealing a gene dosage effect of 12/15-LO in IL-12p40 regulation. We next extended these observations by quantifying the levels of the functional IL-12 heterodimer. The production of the IL-12 heterodimer was also found to be largely 12/15-LO-dependent in this context (Fig. 1⇓C). Regulation of the IL-12p40 subunit was confirmed to be on the RNA level by Q-PCR (Fig. 1⇓D). In contrast, IL-12p35 mRNA levels were comparable between wild-type and 12/15-LO-deficient macrophages, indicating that the reduced IL-12p70 production is due to the reduction in IL-12p40 expression alone or in addition to posttranscriptional regulation of IL-12p35 (Fig. 1⇓E).

FIGURE 1.
FIGURE 1.

Reduced IL-12 production in 12/15-LO-deficient macrophages. Unprimed and IFN-γ-primed thioglycollate-elicited peritoneal macrophages were stimulated with 1 μg/ml E. coli LPS for 24 h before harvesting supernatant. Cytokine levels were assessed by ELISA. C57BL/6 = 12/15-LO+/+; heterozygous = 12/15-LO+/−; Alox15 = 12/15-LO−/−. IL-12p40 levels in supernatant of stimulated unprimed (A) or IFN-γ-primed macrophages (B). C, IL-12p70 levels produced by IFN-γ-primed LPS-stimulated macrophages. IL-12p40 (D) and IL-12p35 (E) RNA levels in macrophages stimulated for 4 h with IFN-γ + LPS. n ≥ 3. A student t test p value of <0.05 was considered statistically significant (∗).

To address the question of whether 12/15-LO is directly involved in IL-12 production or, alternatively, whether 12/15-LO regulates macrophage maturation or differentiation, we compared the phenotype of thioglycollate-elicited macrophages isolated from Alox15 mice and wild-type mice. We regularly obtained numbers and percentages of macrophages from Alox15 peritoneal exudates that were comparable to those obtained from wild-type mice (data not shown). Furthermore, we observed no differences in the cell surface expression of F4/80, CD11b, CD44, MHC class II, or TLR4 in thioglycollate-elicited macrophages from Alox15 and wild-type mice (data not shown), indicating the lack of a gross maturational defect in Alox15 macrophages.

12/15-LO activity regulates IL-12 production in wild-type macrophages

To investigate whether the activity of 12/15-LO regulates IL-12 production in wild-type macrophages, we first evaluated whether stimulation with LPS results in the activation of 12/15-LO by measuring the release of 12(S)HETE and 13(S)HODE, major products of the 12/15-LO pathway (23). We found that the levels of 12(S)HETE and 13(S)HODE were indeed increased upon stimulation with LPS in wild-type, but not Alox15 macrophages (Fig. 2⇓A). To determine the impact of 12/15-LO activity on IL-12 production, we used an inhibitor of 12/15-LO, PD146176. Pretreatment with PD146176 led to a dose-dependent decrease in LPS-induced IL-12p40 production by wild-type macrophages (Fig. 2⇓B). The inhibitor had no effect on the residual levels of IL-12 produced by Alox15 macrophages (data not shown). These data indicate that the activity of 12/15-LO is critical for optimal IL-12 production in mature, LPS-stimulated wild-type macrophages, and further supports our conclusion that the Alox15 defect in IL-12 production is not due to a developmental abnormality.

FIGURE 2.
FIGURE 2.

LPS enhances 12/15-LO activity, which mediates IL-12 production in wild-type macrophages. A, Elicited macrophages were stimulated with LPS for 1 h. The levels of the 12/15-LO pathway products 12(S)HETE and 13(S)HODE (B) in the conditioned media were determined using commercial kits. C, Elicited macrophages were pretreated for 45 min with the indicated concentrations of the 12/15-LO inhibitor PD146176, or vehicle control, before stimulation with LPS. Conditioned media were assayed for cytokine production by ELISA. n ≥ 3. A student t test p value of <0.05 was considered statistically significant (∗).

Selective regulation of inflammatory mediators by 12/15-LO

We explored the selectivity of the effect of 12/15-LO on IL-12 production by measuring the production of other inflammatory mediators. The 12/15-LO inhibitor PD146176 had no effect on TNF-α production by wild-type macrophages (Fig. 2⇑B). Moreover, LPS-induced production of TNF-α, NO, and IL-6 was not affected in Alox15 macrophages as compared with wild-type macrophages with or without IFN-γ priming (Fig. 3⇓, A–C). Similarly, the mRNA levels of IL-18 and IL-10 produced by LPS-stimulated Alox15 macrophages were comparable to wild-type as well (Fig. 3⇓, D–E). The reduction in IL-12p40 production in Alox15 macrophages raised the question of whether the production of IL-23, a second member of the IL-12 family of cytokines that shares the p40 subunit, was also diminished in Alox15 macrophages compared with wild-type. Although levels of IL-23 were undetectable after stimulation with LPS alone, significant levels of IL-23 were produced by LPS-stimulated wild-type macrophages primed by IFN-γ, but were markedly reduced in Alox15-derived macrophages in comparison (Fig. 3⇓F). Like IL-12p70, the reduction in IL-23 production appears to be due to the regulation of the p40 subunit alone because the levels of the subunit unique to IL-23, p19, were comparable between 12/15-LO-deficient and wild-type macrophages (Fig. 3⇓G). Thus, regulation of cytokine production by 12/15-LO appears to be selective for IL-12 family members.

FIGURE 3.
FIGURE 3.

Selective regulation of macrophage inflammatory mediator production by 12/15-LO. Unprimed (A–C, left panels) and IFN-γ-primed (A–C, right panels) thioglycollate-elicited peritoneal macrophages were stimulated with 1 μg/ml E. coli LPS for 24 h (or IFN-γ + LPS for 4 h; D, E, and G). Conditioned media were assayed for the following: TNF-α (A), nitrite (B), IL-6 (C), IL-18 mRNA (D), IL-10 mRNA (E), and IL-23 (F) levels in IFN-γ-primed LPS-stimulated macrophages. G, p19 mRNA levels in macrophages stimulated with LPS and IFN-γ. Nitrite levels were measured by Griess reagent. Cytokine levels were measured by ELISA and mRNA levels by Q-PCR. n ≥ 3. A student t test p value of <0.05 was considered statistically significant (∗).

12/15-LO-dependent expression of IL-12 is stimuli-restricted, and IFN-γ can prime for enhanced IL-12 production via a 12/15-LO-dependent pathway

LPS primarily stimulates via TLR4, and our results indicate that the TLR4 signaling that leads to IL-12 production occurs via both 12/15-LO-dependent and -independent pathways. To determine whether 12/15-LO specifically regulates IL-12 production triggered by the TLR4 receptor or whether the defect extends to other TLR pathways, we stimulated wild-type and Alox15 macrophages with other TLR agonists. When Alox15 macrophages were stimulated with SAC, which activates via TLR2 as well as by undefined TLR2-independent mechanisms, we detected dramatically lower levels of IL-12p40 compared with wild-type macrophages, although TNF-α levels were again akin to the levels produced by wild-type macrophages (Fig. 4⇓A) (24). Similar results were obtained using the selective TLR2 agonist S. aureus-derived peptidoglycan (Fig. 4⇓B). In contrast, Alox15 macrophages stimulated through the TLR9 receptor using bacterial CpG or through the CD40 pathway using CD40L produced levels of IL-12 comparable to wild-type macrophages (Fig. 4⇓, C and D). However, whereas IFN-γ-priming of wild-type macrophages markedly enhanced CpG-induced IL-12 production as expected, priming with IFN-γ did not enhance IL-12 production by CpG-stimulated Alox15 macrophages (Fig. 4⇓D). This striking observation indicates that not only is 12/15-LO regulation of IL-12 stimuli-restricted, but also indicates that IFN-γ can prime macrophages to produce enhanced levels of IL-12 in a 12/15-LO-dependent manner.

FIGURE 4.
FIGURE 4.

Stimulus-restricted regulation of IL-12 production by 12/15-LO. Unprimed (left panels) and IFN-γ primed (right panels) thioglycollate-elicited peritoneal macrophages were stimulated with 1 μl/ml Pansorbin cells (SAC; A), 10 μg/ml peptidoglycan (B), 1 μM CpG 1668 (C), or cross-linked by CD40L-CD8 hybridoma supernatant (D) for 24 h before harvesting conditioned media. Cytokine levels were assessed by ELISA. n ≥ 3. A student t test p value of <0.05 was considered statistically significant (∗).

Cell-type-restricted regulation of IL-12 production by 12/15-LO

Given the marked decrease in IL-12 production by LPS-stimulated Alox15 macrophages in vitro, we tested whether Alox15 mice have a reduced acute response to LPS or CpG stimulation in vivo. Surprisingly, the levels of IL-12p40 we detected in the plasma of Alox15 mice 3 h following administration of LPS or CpG were comparable to those detected in wild-type mice (Fig. 5⇓A). These data presented an apparent paradox with respect to two of our other findings. The first is the apparent discrepancy between the results we obtained with LPS stimulation in vivo vs in vitro. Second, there is the apparent discrepancy comparing the in vivo results assaying IL-12 production in atherosclerotic lesions (16) with those obtained during the acute response to administration of LPS (Fig. 5⇓A). We considered the possibility that the defect in IL-12 production of isolated macrophages in vitro was not recapitulated in the acute response to LPS in vivo due to 12/15-LO-independent production of IL-12 by other cell types. Multiple cells produce IL-12 in vivo during an acute response to LPS (5). Production of IL-12 in these other cell types, if predominantly mediated by a 12/15-LO-independent pathway(s), may obscure the defect in macrophage production of IL-12 in Alox15 mice. DCs produce copious amounts of IL-12 early upon stimulation in vivo (25, 26). Therefore, DCs seemed a potential candidate to explain the ability of 12/15-LO-deficient mice to produce normal amounts of IL-12 in the in vivo acute response to LPS. To test this hypothesis, we investigated whether deficiency in 12/15-LO impacts IL-12 production by bone marrow-derived DCs in vitro. We found that LPS-stimulated Alox15-derived DCs produced comparable levels of IL-12p40 to wild-type DCs (Fig. 5⇓C). Given the ability of IFN-γ to influence the dependency of CpG-induced IL-12 production on 12/15-LO, we evaluated whether IFN-γ could also lead to at least a partial dependency of DCs on 12/15-LO for IL-12 production. However, priming of bone marrow-derived DCs with IFN-γ before LPS stimulation did not result in differential IL-12 production between Alox15 and wild-type cells (Fig. 5⇓C). This suggests that in contrast to peripheral macrophages, optimal IL-12 production by DCs in response to LPS is 12/15-LO-independent. To determine whether wild-type murine DCs express 12/15-LO, we performed Q-PCR analysis. Unlike peritoneal macrophages, we were unable to detect 12/15-LO expression in DCs on the RNA level (data not shown). Similar results were obtained using bone marrow-derived DCs generated with a combination of GM-CSF and IL-4 (data not shown), which is reported to yield more mature DCs (19).

FIGURE 5.
FIGURE 5.

Cell-type-restricted regulation of IL-12 production by 12/15-LO. A, Mice were stimulated with 25 μg of LPS (left), 3 nm 1826 CpG (right), or PBS vehicle control for 3 h before being sacrificed. Serum IL-12p40 levels were assessed by ELISA. Unprimed (left panel) and IFN-γ-primed (right panel) peritoneal thioglycollate-elicited macrophages (PEMs; B), bone marrow (BM)-derived DCs (C), and BM-derived macrophages (Macs; D) were stimulated with 1 μg/ml LPS for 24 h. E, Thioglycollate-elicited neutrophils were stimulated with 1 μg/ml LPS for 4 h. IL-12p40 levels in supernatants were assessed by ELISA, and mRNA levels were assessed by Q-PCR. A student t test p value of <0.05 was considered statistically significant (∗).

In addition to mature-elicited peritoneal macrophages, immature macrophages derived from bone marrow cells in vitro are another important model for studying macrophage cytokine production. However, it was previously reported that bone marrow-derived macrophages do not express 12/15-LO, even after stimulation with a myriad of cytokines and activators (23). Consistent with this finding, we found that Alox15 bone marrow-derived macrophages elaborated normal levels of IL-12p40 upon LPS stimulation that were not differentially affected by IFN-γ priming (Fig. 5⇑D). The inability to detect 12/15-LO in bone marrow-derived macrophages, despite abundant expression in mature tissue macrophages, leaves to question the mechanism by which 12/15-LO (and the 12/15-LO-dependent pathway to IL-12 production) is induced in these cells in vivo.

To better define the cell-type selectivity of 12/15-LO’s effect on IL-12 production, it was important to investigate neutrophils, which, like DCs, are involved in acute IL-12 production (5). To do this, we assayed the ability of thioglycollate-elicited Alox15-derived neutrophils to produce IL-12 in comparison to wild-type neutrophils. Similar to DCs, thioglycollate-elicited neutrophils produced comparable amounts of IL-12p40 mRNA to wild-type cells (Fig. 5⇑E), indicating that LPS-induced IL-12 production in this cell type is also not influenced by 12/15-LO expression under these circumstances.

12/15-LO regulates the association of ICSBP and NF-κB to the IL-12p40 promoter

We previously reported decreased nuclear levels of ICSBP, but comparable nuclear levels of IRF-1, PU.1, and c-Rel to wild-type in stimulated Alox15 macrophages (16). To further investigate the mechanism by which 12/15-LO regulates ICSBP and IL-12 production, we performed ChIP assays on wild-type vs Alox15 macrophages for ICSBP and NF-κBp50. Interestingly, we found that association of both these transcription factors with the IL-12p40 promoter was reduced (Fig. 6⇓, A–C). Although ICSBP is relatively selective for IL-12 production, NF-κBp50 regulates a number of other cytokines, including TNF-α. To investigate the selectivity of 12/15-LO’s effect on NF-κBp50, we tested in parallel the same ChIP DNA using real-time primers specific to the TNF-α promoter. Strikingly, these data revealed that the reduction in the promoter activity of NF-κB is at least relatively selective for the IL-12p40 promoter, because binding of this transcription factor to the TNF-α promoter in Alox15 macrophages was comparable to wild-type (Fig. 6⇓D).

FIGURE 6.
FIGURE 6.

Association of ICSBP and NF-κB with the IL-12p40 promoter is reduced in Alox15 macrophages. Macrophages were unstimulated (Unstim) or stimulated for 4 h with IFN-γ + LPS. Cells were cross-linked with 10% formaldehyde for 30 min, lysed in SDS lysis buffer, sonicated, and immunoprecipitated with an isotype control Ab (A), anti-ICSBP (B), or NF-κB (C and D). Bound promoter levels were assessed by Q-PCR for the IL-12p40 or TNF-α promoters vs β-actin control. A student t test p value of <0.05 was considered statistically significant (∗).

Discussion

These studies provide evidence for selective regulation of IL-12 family member cytokine production by 12/15-LO. We demonstrate that this novel pathway is engaged in a cell-type and stimulus-selective manner. The differential role of 12/15-LO in IL-12 production in various cell types correlates with expression of the enzyme and may, at least in part, form the basis for the dependence of IL-12 production on 12/15-LO in chronic but not acute inflammation. Furthermore, our results indicate that 12/15-LO regulates IL-12p40 gene expression by selectively promoting the association of transcription factors ICSBP and NF-κB to the IL-12p40 promoter.

IL-12 and IL-23 have been shown to be important for the inflammatory response. For instance, atherosclerosis-prone mice lacking the p40 or p35 subunits of IL-12 have significantly reduced lesion formation, whereas mice deficient in the p19 subunit of IL-23 are protected against experimental autoimmune encephalitis (27, 28). Both cytokines were shown to play a role in determining T cell function (29). A greater understanding of the pathways that mediate IL-12 and IL-23 production may therefore provide potential targets for developing novel therapeutic approaches to treating inflammatory diseases.

12/15-LO activity is induced by stimulation with the cytokine IL-4 as well as by glutathione depletion (30, 31). The major products of murine 12/15-LO are the oxidized derivatives of linoleic and arachidonic acid, 13-hydroperoxyoctadecadienoic acid (13(S)-HpODE) and 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12(S)-HpETE), respectively, which are then reduced to 12(S)HETE and 13(S)HODE (14). Additionally, both cyclooxygenase-2 and 12/15-LO produce small amounts of 15(S)HETE (12, 32). Each of these lipid mediators has been shown to affect cellular function. For instance, 12(S)HETE induces the activation of MAPK family members, c-Jun, and NF-κB (10, 33). In contrast, 12(S)HETE, 13(S)HODE, and 15(S)HETE can activate the nuclear transcription factor peroxisome proliferator-activated receptor, which may lead to a decrease in inflammatory cytokine production (34). Despite the ability of these lipids to regulate other functions in other cell types, we were not able to bypass the requirement for 12/15-LO for IL-12 production by adding exogenous 12HpETE, 12-HETE, 15-HpETE, 15-HETE, 13HpODE, or 13-HODE, nor was IL-12 production by wild-type macrophages impaired by pretreatment with a neutralizing Ab to 13(S)HODE (M. K. Middleton and E. Puré, unpublished results). These observations make it unlikely that 12/15-LO regulates IL-12 production through these mediators. An alternative explanation for the impact of 12/15-LO on IL-12 production may be offered by the fact that 12/15-LO produces reactive oxygen species in the process of lipid oxidation that can contribute to the oxidative state of the cell (15, 35, 36, 37). Coupled with the burgeoning evidence for the role of reactive oxygen species in the regulation of IL-12 and other cytokines, the release of superoxide and other radicals by 12/15-LO may provide another possible mechanism by which this enzyme regulates IL-12 production (38, 39, 40).

In this study, we focused on the role of 12/15-LO in regulating several potent pathways to IL-12 expression in macrophages. Our results establish that the dependence on 12/15-LO for IL-12 production is in part determined by the stimulant. For example, TLR4, TLR2, and HA/CD44 (C. Cuff and E. Pure, unpublished results), but not TLR9 or CD40-mediated activation of IL-12 production proved to be dependent on 12/15-LO in macrophages. Our findings thus add to the growing body of evidence that signaling pathways to IL-12 do not converge as consistently as originally thought; different “danger signals” being able to selectively stimulate different components of the inflammatory response through distinct signaling pathways. Furthermore, our data show that TLR signaling can be differentially regulated in different cell types. Understanding the mechanisms by which 12/15-LO regulates IL-12 production should therefore provide insight into the various signaling pathways leading to IL-12 production.

T cell and NK cell priming of macrophage IL-12 production by IFN-γ is critical for the clearance of many pathogens and is also involved in the exacerbation of inflammation. The priming effect of IFN-γ is on a transcriptional level and dependent on the promoter segment between −222 and −204, where the ets consensus sequence lies (41, 42). This is a region to which the transcription factor ICSBP binds, as well as other IRF family members (42). However, the signaling pathway by which IFN-γ primes IL-12 production has not been well defined. Based on the defect we observed in IFN-γ priming in 12/15-LO-deficient macrophages for enhanced IL-12 production, we propose that at least one of the mechanisms by which IFN-γ primes for IL-12 production proceeds via a 12/15-LO-dependent pathway. Interestingly, in this study, we have noted a significant reduction in the amount of ICSBP binding to the IL-12p40 promoter in stimulated Alox15 macrophages. It has previously been reported that ICSBP-deficient macrophages produce reduced amounts of IL-12 in response to stimulation with LPS, but normal amounts of IL-12 upon stimulation with CpG (43, 44). This observation recapitulates the IL-12 phenotype in Alox15 macrophages characterized in this study. However, upon IFN-γ priming, a different picture emerges. Comparison between unprimed and primed LPS and SAC-stimulated macrophages from wild-type or Alox15 mice reveals that Alox15 macrophages exhibit a fold increase in IL-12 production similar to wild-type, indicating that 12/15-LO does not affect IFN-γ priming of IL-12 production in these instances. In contrast, IFN-γ priming of IL-12 production by CpG-stimulated macrophages may proceed via a distinct 12/15-LO-dependent mechanism because IFN-γ priming led to enhanced IL-12 production in wild-type but not Alox15 macrophages. Thus, similar to stimulation of IL-12 production, it appears that IFN-γ priming of IL-12 production can occur via both 12/15-LO-dependent and -independent pathways. Interestingly, we also did not observe an impairment in other aspects of IFN-γ signaling in Alox15 macrophages, because MHC class II levels as well as STAT1 and p38 phosphorylation were comparable to wild-type (M. K. Middleton and E. Puré, unpublished results). Our finding of decreased NF-κB binding to the IL-12p40 but not the TNF-α promoter may also account for some of the selective effects of 12/15-LO on cytokine production. This is most likely mediated by the preferential association of the different heterodimers of NF-κB family members, such as p50/p65 and p50/c-Rel to individual cytokine gene promoters, although the fact that the IL-12p40 promoter contains a unique NF-κB half site may also contribute to the susceptibility of the IL-12p40 promoter to regulation by 12/15-LO (45, 46). In this study, we have focused on characterizing the IL-12p40 promoter in cells stimulated with both IFN-γ and LPS, which induces maximal IL-12 production, to look for differences in transcription factor association. Investigating the ability of NF-κB, ICSBP, and other transcription factors to associate with the IL-12p40 promoter during stimulation with LPS alone, in the absence of IFN-γ may provide further insight into 12/15-LO-mediated regulation of IL-12 production. We are currently investigating this possibility.

The fact that 12/15-LO deficiency had no effect on acute LPS or CpG-induced IL-12 production in vivo may seem to contradict our previous data of decreased IL-12p40 in atherosclerotic plaque. Although we have not yet formally excluded the possibility that the acute response of the macrophage in vivo is unaffected by the deficiency in 12/15-LO, the differences we have observed may very well be due to the cell types involved in each case. DCs and neutrophils respond initially to stimulation with LPS, and DC subsets such as plasmacytoid DCs produce robust cytokine levels in response to stimulation with CpG in vivo (25, 47). However, these cell types are relatively rare in atherosclerotic plaque (25, 47, 48). Conversely, macrophages predominate in atherosclerotic plaque and in many other chronically inflamed tissues (7, 49, 50). These observations raise the intriguing prospect that the dependency on 12/15-LO for IL-12 production may increase during the evolution of the inflammatory response toward chronic disease as mature tissue macrophages emerge as an increasingly dominant source of IL-12, in which case the 12/15-LO pathway may be selectively implicated in chronic as opposed to acute inflammation. If this hypothesis is correct, it would suggest that 12/15-LO may be worth pursuing as a target in the treatment of chronic inflammatory diseases that may spare the acute response to infection.

Acknowledgments

We thank Dr. Christopher Hunter for invaluable help with the IL-23 ELISA and Dr. Charles Chau for technical advice with the ChIP assay and real-time PCR. We are also grateful to Drs. Colin Funk and Lei Zhao for their input and scholarly discussions, and Dr. Christopher Hunter and Jason Hall for critical reading of this manuscript. We express our appreciation of Irene Crichton for excellent laboratory management skills and Adrienne Whitmore for assistance with graphics. Finally, we acknowledge the generous support of Jeffrey Faust at the Flow Cytometry Core facility and Anthony Lucente (Information Systems) at the Wistar Institute for technical assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by Public Health Services Grants AI45813 and T32CA01940 provided by the National Institute of Health and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

  • ↵2 Address correspondence and reprint requests to Dr. Ellen Puré, Wistar Institute, 3601 Spruce Street, Room 368, Philadelphia, PA 19104. E-mail address: pure{at}wistar.org

  • ↵3 Abbreviations used in this paper: DC, dendritic cell; SAC, Staphylococcus aureus Cowan strain; IRF, IFN regulatory factor; ICSBP, IFN consensus sequence binding protein; 12/15-LO, 12/15-lipoxygenase; 12(S)HETE, 12(S)-hydroxyeicosatetraenoic acid; Q-PCR, quantitative real-time PCR; 13(S)HODE, 13(S)-hydroxyoctadecadienoic acid; ChIP, chromatin immunoprecipitation; 13(S)-HpODE, 13(S)-hydroperoxyoctadecadienoic acid; 12(S)-HpETE, 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid.

  • Received June 3, 2005.
  • Accepted October 17, 2005.

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