Lipid mediator metabolic profiling demonstrates differences in eicosanoid patterns in two phenotypically distinct mast cell populations.

Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD(2) and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC(4)), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses.

, as well as for inducing smooth muscle contraction ( 15 ) and affecting development of allergic airway disease, fi brosis, and vascular injury (16)(17)(18). Cysteinylleukotrienes (cys-LT) are mediators of human asthma, and anti-leukotrienes are effective therapeutics in the clinical management of disease ( 19 ). Mast cells are the predominant producer of PGD 2 and its metabolites [e.g., 9 ␣ , 11 ␤ -prostaglandin F 2 (9 ␣ , 11 ␤ -PGF 2 ); 2,3-dinor-9 ␣ , 11 ␤ -PGF 2 ; tetranor-PGDM] and can be used as an indicator of mast cell activation ( 20 ). Mice overproducing PGD 2 develop increased airway infl ammation as well as Th2 cytokine production following sensitization and challenge compared with wild-type mice ( 21 ), and targeted disruption of the DP receptor protects mice against allergic asthma ( 22 ). Besides products of the COX and 5-LO pathways ( Fig. 1 ), mast cells have been shown to produce products of the 12/15-LO pathway, including eoxins, 15hydroxy-eicosatetraenoic acid (HETE), and 15-oxo-eicosatetraenoic acid (KETE) ( 23,24 ). However, to date research on mast cell-derived oxylipins has focused on a small number of lipids, and limited information is available regarding the breadth of prospective oxylipins produced or the potential for phenotypic differences.
To perform an in-depth analysis of oxylipin generation in mast cells, we employed targeted quantitative lipid metabolic profi ling of supernatants from calcium ionophore-treated murine mast cells of the connective tissue-like mast cell (CTLMC) and mucosal-like mast cell (MLMC) phenotypes. Well-established protocols were used for the development of MLMCs and CTLMCs (25)(26)(27)(28), followed by stimulation with the conventional mast cell secretagogue A23187, used since the mid-70s to induce histamine and lipid mediator release from mast cells ( 29,30 ). Multivariate statistical models were then employed to investigate the time dependence of oxylipin production and the relationship between mast cell phenotype and oxylipin biosynthesis.

Mast cell cultures
CTLMCs and MLMCs were derived by culturing bone marrow cells (BMC) from C57BL/6 mice as previously described ( 26 ). All animal experiments were performed according to the guidelines of the Animal Ethics Committee in Stockholm and the PHS policy. In brief, CTLMCs were developed in medium containing recombinant murine stem cell factor (50 ng/ml) and 1 ng/ml recombinant murine interleukin (IL)-4. MLMCs were developed in medium containing 5 ng/ml IL-3, 5 ng/ml recombinant rapidly release preformed infl ammatory mediators stored in granules, produce lipid mediators de novo, and synthesize cytokines, chemokines, and growth factors. The spectra of released mediators are dictated by the stimuli; for example, IgE-receptor aggregation causes degranulation, lipid mediator secretion, and release of cytokines, whereas other stimuli, such as CD153, only lead to a specifi c release of primarily chemokines ( 3 ).
There exists a considerable heterogeneity among tissue mast cells. In humans, they are divided into two main phenotypes: those that contain the granule proteases tryptase and chymase (designated as MC TC ) and those that only express tryptase (MC T ) ( 4 ). The corresponding phenotypes in rodents are referred to as "connective tissue-like" and "mucosal-like" mast cells, respectively. Recent fi ndings have identifi ed a more substantial heterogeneity among human lung mast cells assessed by morphology and expression of different receptors, enzymes, and mediators ( 5 ). One example is the more pronounced expression of 5-lipoxygenase (5-LO) in mast cells located in the small airways and pulmonary vessels, compared with mast cells in the central airways and parenchyma ( 5 ). The consequence of this heterogeneity and differential mediator release is that the effect of mast cell activation can vary substantially, dependent on stimuli and mast cell phenotype.
Oxidized lipid mediators (e.g., eicosanoids, oxylipins) are one major and potent group of mediators released by mast cells. Oxylipins are signaling lipids formed from fatty acids via mono-or dioxygenase-catalyzed oxygenation that play prominent roles in infl ammatory diseases (6)(7)(8). These compounds include the arachidonic acid (AA)-derived prostaglandins and leukotrienes (LT) as well as analogous compounds produced from an array of polyunsaturated fatty acid substrates [e.g., linoleic acid (LA), ␣ -linolenic acid, docosahexaenoic acid]. The biosynthesis of oxylipins is initiated via three major pathways: cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) (6)(7)(8). The activity of these pathways and the binding of their products to their corresponding receptors are important drug targets for multiple infl ammatory diseases ( 9 ).
Following activation (e.g., via the high-affi nity IgE-receptor), mast cells rapidly generate prostaglandin D 2 (PGD 2 ) and the leukotrienes C 4 (LTC 4 ) and B 4 (LTB 4 ) ( 10 ). Both PGD 2 and the LTs are proinfl ammatory mediators involved in modulating and attracting immune competent cells, as well as acting on structural cells ( 10 ). For instance, LTs are important for the recruitment of neutrophils, eosinophils, monocytes, T-cells, and dendritic cells  Table I. deuterated standards (200 nM), 10 µl BHT, and EDTA solution (0.2 mg/ml) were added to the samples, which were then placed at Ϫ 20°C for 1 h, followed by centrifugation (10 min, 4°C <7000 rpm) to remove precipitate. The supernatant was removed, and 4 ml MilliQ water was added, and then the samples were applied to washed and conditioned SPE columns. Samples were washed with 3 ml of MilliQ water by gravity. The aqueous plug was pulled from the cartridges under high vacuum, and cartridges were further dried with low vacuum for ‫ف‬ 30 min. Cys-LT and eoxins were eluted with 1.5 ml of methanol into cryotubes (Corning Inc., NY) containing 6 l of 30% glycerol in methanol. Volatile solvents were removed using a GMI miVac Concentrator system (MN). Samples were reconstituted in 100 l of 2:3 methanol:water containing 176 nM of the technical standard (CUDA). The samples were vortexed for 1 min, fi ltered [Millipore centrifugal fi lters, Durapore (PVDF) 0.1 m], transferred to autosampler vials, and stored at Ϫ 20°C until analysis. A Waters (MA) Acquity UPLC separation module coupled to a Waters Xevo triple quadrupole mass spectrometer equipped with an electrospray source was used for analyses, and separation was performed via a Waters 2.1 × 50 mm Acquity UPLC BEH C18 column with a 1.7 m particle size. Cys-LT and eoxins were separated using a gradient containing (A) water and (B) acetonitrile (supplementary Table II). Gradient elution was performed at a fl ow rate of 500 l/min during a 10 min run. Cys-LT and eoxin transition and LC retention times are provided in supplementary Table III. Oxylipins were separated using a gradient containing (A) MilliQ water and 0.1% acetic acid and (B) acetonitrile:isopropanol 90:10 (supplementary Table IV). Gradient elution was performed at a fl ow rate of 500 l/min during a 21 min run. The mass spectrometer was operated in positive SRM mode for cys-LTs and eoxins and negative SRM mode for oxylipins. All compounds were quantifi ed using internal standard methods as previously described ( 31 ). Compounds detected above the limit of quantifi cation (LOQ) were quantifi ed and recalculated back to the original sample concentrations. To demonstrate the capability of the method, supplementary

Statistical methods
Univariate statistical analysis was performed using the Student t -test and Bonferroni's multiple comparison test ( 33 ). Principal component analysis (PCA) and orthogonal projections to latent structures (OPLS) were performed using SIMCA-P+12 (Umetrics, Umeå, Sweden) following log base 10 transformation, mean centering, and UV scaling ( 34 ). Model performance was reported as cumulative correlation coeffi cients for the model (R 2 ), and predictive performance was based on cross validation calculations (Q 2 ) and CV-ANOVA.

PCR array analysis of both mast cell phenotypes demonstrates the presence of transcripts for the required enzymes in oxylipin biosynthesis
Oxylipins are formed via two major metabolic pathways involving a suite of enzymes that oxidize the fatty acid substrate to form a bioactive product ( Fig. 1 ). To confi rm the presence of the necessary message of the enzymes murine IL-9, 1 ng/ml recombinant human TGF-␤ 1 , and recombinant murine stem cell factor (50 ng/ml). Media, IL-4, IL-3, IL-9, TGF-␤ 1, and stem cell factor were obtained from PeproTech Nordic, Sweden. All cells were cultured for a minimum of 2 weeks for MLMC and 4 weeks for CTLMC prior to use. The maturity and purity of the cells were examined by toluidine blue staining and fl ow cytometry analysis for expression of Kit and Fc RI, using FITC-anti-mouse CD117 (Kit) mAb 2B8 and FITC-conjugated rat IgG2b isotype control (BD Pharmingen, NJ), FITC-conjugated anti-mouse Fc RI-␣ mAb MAR-1, and FITC-conjugated Armenian hamster IgG isotype control (eBioscience, CA).

PCR array
RNA was isolated from 2 × 10 6 unstimulated mast cells as pellets in RNAlater by the RNeasy Mini Kit essentially according to the manufacturer's instructions (Qiagen, Sweden). The cells were lysed in a fi nal concentration of 40 mM DTT (Sigma Aldrich, Sweden) and disrupted using a rotor-stator homogenizer. The RNA concentration was measured using a Nanodrop 3300 (Thermo Scientifi c, DE). For each sample, 160 ng of cDNA was prepared by the QuantiTect Rev. Transcription Kit (Qiagen, Sweden) according to the instructions provided in the kit. The lowdensity arrays, containing primers for 16 mouse genes involved in the synthesis of eicosanoids and three endogenous controls, were custom made (Applied Biosystems, Sweden). cDNA from the different mast cells were fi rst titrated in a Taqman PCR assay to determine the optimal amount of cDNA to use as a template. A negative control cDNA, synthesized without the reverse transcriptase enzyme, was also included. The primers used for titration were 18S rRNA and JNK. cDNA (160 ng) in Taqman Gene Expression Master Mix (Applied Biosystems, Sweden) was added per port in the low-density array plate and run on an ABI 7900H instrument (Applied Biosystems, Sweden) according to the recommended instructions. The total number of cycles was 42. The fi nal analysis of raw data was performed using the software SDS 2.2 (Applied Biosystems, Sweden).

Oxylipin, cysteinyl-leukotriene, and eoxin extraction and quantifi cation
Calibration and deuterium-labeled oxylipin, cys-LT, and eoxin standards as well as the technical standard N -cyclohexyl-N ′dodecanoic acid urea (CUDA) were obtained from Cayman Chemical (MI) and Larodan Fine Chemicals (Sweden). All standards used, as well as full abbreviations, are provided in supplementary Table I. Oxylipins, cys-LTs, and eoxins were offl ine SPE extracted from 0.5 ml cell supernatant aliquots using Waters (MA) Oasis-HBL 60 mg cartridge columns. Oxylipins were extracted using the same procedure as previously described ( 31 ). Due to the low recoveries of cys-LTs using the standard analytical protocols ( 31,32 ), these compounds were extracted and analyzed using a separate method developed for cys-LT quantifi cation. The recovery of d 5 -LTE 4 with the general oxylipin method ( 31 ) was 7.1 ± 9.2% averaged across all 96 samples, as opposed to 35.4 ± 14.8% with the cys-LT-specifi c method (the cys-LT-specifi c method gave recoveries of 48.4 ± 17.0% and 40.4 ± 12.1% for d 5 -LTC 4 and d 5 -LTD 4 , respectively). Accordingly, cys-LT and eoxin extraction was performed as follows: 4 ml of methanol, 10 µl COX products. The sum of COX-regulated products secreted from the MLMCs was consistently ‫ف‬ 10-fold higher relative to the CTLMCs at all time points following activation ( Table 1 , Fig. 3 ). The three most abundant COX products [PGD 2 , TXB 2 , and 12-hydroxy-heptadecatrienoic acid (HHTrE)] together comprised ‫ف‬ 95% of the total amount of COX products in both cell types at 8 and 15 min. However, the portion of PGD 2 in the CTLMCs was signifi cantly larger compared with the MLMCs (15 min: 64 ± 6% versus 24 ± 3%; P = 6.0E Ϫ 08 ), whereas the portions of TXB 2 (15 min: 8 ± 2% versus 29 ± 1; P = 6.5E Ϫ 10 ) and 12-HHTrE (22 ± 6% versus 42 ± 2%; P = 1.8 E Ϫ 05 ) were signifi cantly larger in the MLMCs ( Fig. 2B ). Moreover, the ratio of TXAS-dependent responsible for oxylipin formation in both CTLMCs and MLMCs, we performed a PCR array on mRNA purifi ed from resting CTLMCs and MLMCs as well as on the BMCs that are the source for differentiation. Overall, both cell types demonstrated the presence of the necessary mRNA for oxylipin biosynthesis (supplementary Fig. II). CTLMCs expressed the highest mRNA levels of COX-1, cytosolic PGE synthase (cPGES), and microsomal PGE synthase (mPGES), as well as high levels of thromboxane A synthase (TXAS). MLMCs expressed greater levels of TXAS mRNA and low levels for COX-1 and COX-2. Compared with BMCs, both mast cell types showed increased mRNA expression of the hematopoetic PGD synthase PTGDS2. For LT synthesis, CTLMCs expressed the highest levels of LTA 4 hydrolase (LTA4H), whereas MLMCs expressed the highest levels of 5-LO and LTC 4 synthase (LTC4S). Both CTLMCs and MLMCs expressed high levels of fi ve-lipoxygenase activating protein (FLAP), but the levels were not signifi cantly greater than those of BMCs. For the 12/15-LO pathway, both CTLMCs and MLMCs evidenced higher mRNA levels than BMCs. Overall, the transcript data illustrated an induction of critical enzymes for oxylipin formation during mast cell differentiation. The data evidenced both expression similarities as well as distinct patterns between CTLMCs and MLMCs.

Activated mast cells demonstrate phenotype-specifi c oxylipin profi les
Ninety oxylipins representing three metabolic pathways (COX, LO, and CYP) were screened using LC-MS/MS analysis. Oxylipins produced by 5-LO and COX pathways ( Fig. 1 ) were the predominant species in both phenotypes (95-98%), but 8-LO, 12/15-LO, and CYP products were also observed. The distributions of COX-and 5-LO-regulated compounds were distinct, with the portion of 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs at 15 min ( Fig. 2A ). In total, 28 oxylipins were detected above the method limit of quantifi cation (LOQ) in the CTLMCs (COX, n = 4; 5-LO, n = 12; 8-LO, n = 4; 12/15-LO, n = 5; and CYP, n = 4), and 27 oxylipins were detected above the LOQ in the MLMCs (COX, n = 7; 5-LO, n = 13; 8-LO, n = 3; 12/15-LO, n = 4; and CYP, n = 0) (supplementary Table V). An additional four oxylipins (PGE 1 , PGE 2 , PGF 2 ␣ , and LTE 4 ) were present above the method LOD in the CTLMCs and an additional three oxylipins [12-HETE, 9(10)-epoxy-octadecenoic acid (EpOME), and 12 (13)-EpOME] were detected above the LOD in the MLMCs (supplementary Table V). As shown in Fig. 3 , oxylipin levels increased greatly in both phenotypes upon activation with the ionophore A23187. However, CTLMC levels increased linearly over time in contrast to MLMC levels, which increased exponentially with minor changes after 2 min. The average concentrations (nM) and the standard deviations for both cell types are shown in Table 1 , with individual values given in supplementary Table VI     Samples detected at a mixture of quantitative levels, LOD and/or ND levels (for details, see supplementary Table VI). For statistical purposes LOD were calculated as one third of the LOQ and ND as one tenth of LOQ levels (supplementary Table V).
( Fig. 2A ). The majority of these compounds were detected in signifi cantly higher levels in the CTLMCs, but the measured concentrations were close to the LOD and also detected in signifi cantly higher concentrations in the controls ( Table 1 ). The main exception was the 8-LO product 8-HETE, which was observed in signifi cantly higher concentrations in the CTLMCs 15 min following activation ( Table 1 ).
To investigate differences between CTLMCs and MLMCs at 2 and 15 min, two OPLS-DA models ( Fig. 5A , B ) were constructed, and the p(corr) values were plotted in a shared and unique structures (SUS) plot ( Fig. 5C ) ( 35,36 ). The models, both constructed with one predictive concurrently with TXA 2 from PGH 2 ( 43 ) and has been shown to have chemotactic activity in leukocytes ( 44 ), as well as serving as a substrate for NAD+-dependent 15-hydroxyprostaglandin dehydrogenase ( 45 ). Additionally, elevated levels of TXB 2 and 12-HETrE indicate extensive TXAS activity with high production and abundance of TXA 2 , an important mediator possessing prothrombotic properties ( 46 ). TXA 2 has also been implicated in the development of bronchial hyper-responsiveness ( 47 ). It remains to be established whether the observed differences, although statistically signifi cant , in COX-derived products have physiological relevance. The distribution of COXgenerated compounds in MLMCs showed that TXAS-derived TXB 2 and 12-HHTrE were signifi cantly larger in composition (%) relative to the CTLMCs, which produced PGD 2 as the main COX product ( Fig. 2 ). It should be noted that both PGD 2 and PGE 2 will be further metabolized via 15-hydroxy PGDH and 15-oxo-PG ⌬ 13-reductase to their corresponding 13,14-dihydro-15-oxo-metabolites (13,14-dihydro-15-keto PGD 2 and 13,14-dihydro-15-keto PGE 2 ) ( 48,49 ). The half-lives of these compounds are relatively short and are further metabolized to give 11 ␤hydroxy compounds that have also undergone ␤ -oxidation of one or both side chains. Accordingly, results regarding the relative contributions of the LO and COX pathways should be tempered with the knowledge that downstream metabolites were not assayed.
The response curve of the CTLMCs was linear over the measured time course, whereas the MLMC curve evidenced essentially saturated levels at 2 min. Interesting were the observed shifts in 5-LO products, which were signifi cantly higher in the CTLMCs at 15 min ( P = 0.02) compared with 2 min [higher in the MLMCs ( P = 0.008)] and 8 min [not signifi cant ( P = 0.4)]. According to the SUS plot in Fig. 4 , this shift is most prominent for the cys-LTs, as well as for 5-HETE and 5-KETE. Other compounds, such as LTB 4 and 5,6-DiHETE, also evidenced this shift from strongly correlating with MLMCs at 2 min to essentially no correlation at 15 min. It is unclear whether these shifts are further accentuated with increased time. The ratio is constant between 5-LO and COX products at 8 min and 15 min (8 min, 88 versus 6%; 15 min, 90 versus 6%, respectively), suggesting that they are increasing at the same relative rate. Considering the major role of LTB 4 and cys-LTs in mast cell biology and infl ammatory regulation ( 6,7,10 ), the phenotype-specifi c differences in concentration and relative abundance of LTC 4 versus LTB 4 will most likely have biological implications. For example, numerous studies have established the impact of LTs in recruitment of T-cells and dendritic cells and in development of allergic airway disease ( 11,12,50 ). Data also indicate that LTC 4 and LTD 4 are key players in fi brosis and vascular injury via the CysLT2 receptor, and they have been implicated in the pathogenesis of human abdominal aortic aneurysm ( 17,18,51 ). Accordingly, the observed phenotype-specifi c differences in LT production may have ramifi cations for the etiology of pathological processes in both the onset and resolution of disease. 12/15-LO-produced 15-HETE, evidenced a shift from correlating with the MLMCs at 2 min to correlating with the CTLMCs at 15 min. The responses of LTB 4 and 5,6-Di-HETE were notable, shifting from strongly correlating with the MLMCs at 2 min [p(corr): 0.81 and 0.88] to essentially no correlation with the MLMCs at 15 min [p(corr): 0.13 and Ϫ 0.14 ]. These results support those in Fig. 3A , with the 5-LO products shifting from higher concentrations in the MLMCs at 2 min to higher concentrations in the CTLMCs at 15 min. This trend was also observed for the main 8-LO products. Furthermore, the model illustrates that the CYP products correlate with the CTLMCs, and the COX products correlate with the MLMCs.

DISCUSSION
From the results presented herein, it is evident that the two main mast cell phenotypes, CTLMC and MLMC, have different oxylipin profi les, both regarding the expression of the necessary enzymes for their generation and in activated pathways following stimulation. Both univariate and multivariate analyses found that the COX-and 5-LO-regulated lipid cascades are the major pathways activated following stimulation. However, the phenotypes evidenced major differences in the distribution of 5-LO and COX products at 15 min after stimulation (CTLMCs, 90% and 6%, versus MLMCs, 58% and 40%, respectively). Generally, gene expression data did not correlate closely with lipid mediator production, with signifi cantly higher expression of 5-LO and FLAP in the MLMCs, and COX1 and COX2 levels signifi cantly higher in the CTLMCs. However, the oxylipin profi les evidenced improved correlation with gene expression when focused on the enzymes downstream in the biosynthetic cascades [i.e., COX (PTGDS2 and TXAS were signifi cantly higher in the MLMCs) and 5-LO (LTA4H was signifi cantly higher in the CTLMCs, whereas LTC4S exhibited no difference between phenotypes)]. However, it should be noted that expression values were not measured in the cells following stimulation, and thus, correlating gene expression with activated cells should be performed with caution.
The observed differences in the oxylipins produced downstream of the COX and 5-LO cascades may be of biological signifi cance. PGD 2 , which is produced by PGDS, has previously been reported as the primary COX product in mast cells ( 20 ). Effects induced by PGD 2 are dependent on two main receptors, the PGD 2 receptor1 (DP1) ( 22 ) and the chemo-attractant homologous receptor expressed on Th2 cells (CRTH2) ( 37 ). The receptors can regulate infl ammatory cell migration, control cytokine production, and mediate lipid synthesis , and there is also speculation that they may cross-talk during infl ammatory events (38)(39)(40). It is notable that CRTH2 also can be activated by 11-dehydro-TXB 2 ( 41 ), a metabolite of TXB 2 that is formed via the COX/TXAS pathway ( Fig. 1 ) ( 42 ). The levels of PGD 2 in the MLMCs were signifi cantly higher at 15 min ( P = 0.0003) compared with the CTLMCs ( Fig. 3 ). TXB 2 is a stable, nonenzymatically produced product of TXA 2 that is rapidly formed via hydrolysis ( 42 ). 12-HHTrE is formed products of DGLA (LTB 3 and 5-HETrE) and EPA (LTB 5 and 5-HEPE) were still found in signifi cantly lower amounts at 15 min in the CTLMCs.
This study is the fi rst to provide detailed information on lipid mediator production in mast cells. The results provide evidence that the oxylipin profi les of murine mast cells are phenotype specifi c, with additional differences in biosynthesis response curves following stimulation with ionophore. Further mechanistic studies are required to investigate whether the trends observed in murine mast cells are also found in the human phenotypes. The disparate oxylipin profi les in murine mast cells may affect the infl ammatory cycle from the activation to resolution phase, as well as the overall activity and regulation of the phenotypes, in a time-dependent manner, with implications for disease pathology.
The authors thank Ingrid Delin for excellent technical support. The 8-LO products 8-HETE and 8-HETrE also evidenced a shift similar to 5-HETE, 5-KETE, and the cys-LTs in the SUS plot. In mice, these compounds are regulated via the 8-LO pathway ( 52 ), but in humans, these compounds are produced nonenzymatically ( 9 ). It has previously been shown that A23187 treatment of dorsal skin on NMRI mice causes increased levels of 8-HETE ( 53 ). It should also be noted that all of the HETEs can potentially be produced via free radical oxidation. The stereochemistry of the hydroxyl moiety can indicate synthetic route ( S for LO activity versus R for monoxygenase activity); however, it is now well established that lipoxygenases can also remove a pro-R hydrogen from the substrate with antarafacial insertion of oxygen to generate products with R chirality ( 9 ). Accordingly, the enantiomeric excess is required to determine synthetic source, with a racemic mixture indicating free radical oxidation and predominantly R or S indicating a biosynthetic source. Because chiral chemistry was not employed in this study, results should be tempered with the understanding that the synthetic source of the HETEs is unclear.
Human mast cells have previously been shown to produce products of the 12/15-LO pathway, including 15-HETE, 15-KETE, and eoxins ( 23,24 ). The primary 12/15-LO products are 12-and 15-HETE, of which 15-HETE was predominantly observed in both MLMCs and CTLMCs. ( Table 1 ). The downstream oxidation products (12-and 15-KETE) were not observed, but this is potentially due to the high LOD levels for these compounds ( ‫ف‬ 10 nM). It has previously been shown that 15-KETE is the primary product in human cord blood mast cells, suggesting that there could be species-specifi c differences in mast cell eicosanoid levels ( 23 ). We also observed 5,15-DiHETE and LXA 4 , which are produced by 12/15-LO following 5-LO action ( 7,54,55 ). LXA 4 , which was primarily detected in the MLMCs (supplementary Fig. IV), is involved in the resolution phase of infl ammation ( 7,54,56 ). As such, it is of particular interest to study its potential effects in the mast cell and its surrounding microenvironment. If lipoxin levels are also specifi c for the mast cell phenotypespecifi c human equivalents, it could have implications for disease. Additionally, some other AA-and LA-mediated 12/15-LO products were found, particularly in the CTLMCs ( Figs. 4 and 5 , Table 1 ). However, the amounts were detected in quantities close to the LOQ. The lack of eoxin detection may also indicate species differences between human and murine mast cells.
Of the characterized compounds in this study, the majority were derived from AA (n = 20). Additionally a number of LA (n = 5), dihomo-␥ -linolenic acid (DGLA; n = 5), and eicosapentaenoic acid (EPA; n = 2) compounds were found in quantitative amounts in both phenotypes, demonstrating that murine mast cells have the capacity to produce these mediators if the substrate is present. As such, the ratio of AA versus EPA or DGLA products in the phenotypes could potentially be altered depending on source of nutrition and microenvironment. Notable also is that the DGLA-derived PGE 1 and PGD 1 (COX) followed the same response curve as PGE 2 and PGD 2 but that the 5-LO