Sulfur mustard (SM) has been widely used as a chemical warfare agent including most recently in Syria. Mice exposed to SM exhibit an increase in pro-inflammatory cytokines followed by immune cell infiltration in the lung, however, the mechanisms leading to these inflammatory responses has not been completely elucidated. Mast cells are one of the first responding innate immune cells found at the mucosal surfaces of the lung and have been reported to be activated by SM in the skin. Therefore, we hypothesized that nitrogen mustard (NM: a surrogate for SM) exposure promotes activation of mast cells causing chronic respiratory inflammation. To assess the role of mast cells in NM-mediated pulmonary toxicity, we compared the effects of NM exposure between C57BL/6 and B6.Cg-KitW-sh/HNihrJaeBsmJ (Kit^W-sh; mast cell deficient) mice. Lung injury was observed in C57BL/6J mice following NM exposure (0.125 mg/kg) at 72 h, which was significantly abrogated in Kit^W-sh mice. Although both strains exhibited damage from NM, C57BL/6J mice had higher inflammatory cell infiltration and more elevated prostaglandin D₂ (PGD₂) present in bronchoalveolar lavage fluid compared with Kit^W-sh mice. Additionally, we utilized murine bone marrow-derived mast cells to assess NM-inducedearly and late activation. Although NM exposure did not result in mast cell degranulation, we observed an upregulation in PGD₂ and IL-6 levels following exposure to NM. Results suggest that mast cells play a prominent role in lung injury induced by NM and may contribute to the acute and potentially long-term lung injury observed caused by SM.

Oxylipin Sample Preparation

All standards and internal standards used for LC/MS/MS analysis of arachidonic acid, docosahexaenoic acid and linoleic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan). All HPLC solvents and extraction solvents were HPLC grade or better. All analysis was done at the University of Colorado School of Pharmacy mass spectrometry facility.

Bronchoalveolar lavage fluid (BALF) samples were pretreated for solid phase extraction. Briefly, samples were diluted in a 9% methanol, 1% ethanol solution by adding 100 µl methanol and 10 µl of the internal standard solution (10 pg/µl each of 5(S)-HETE-d₈, 8-iso-PGF2a-d₄, 9(S)-HODE-d₄, LTB4-d₄, LTD4-d₅, LTE4-d₅, PGE2-d₄, PGF2a-d₉ and RvD2-d₅ in ethanol) and sufficient water to create 1 ml of solution. Samples were then homogenized by vortexing and purified by solid phase extraction (SPE).

Lipid mediators from cells were isolated and purified using SPE as follows. The reconstituted extracts were loaded on a Strata-X 33 µm 30 mg/1 ml SPE column (Phenomenex, Torrance, California) preconditioned with 2 volumes of 1.0 ml methanol. The SPE column was then washed with 10% methanol and then eluted directly into a reduced surface activity/maximum recovery glass autosampler vial with 1.0 ml of methyl formate. The methyl formate was evaporated completely from the vial with a stream of nitrogen and then the SPE cartridge was then eluted with 1.0 ml of methanol directly into the same autosampler vial. The methanol was evaporated to dryness with a stream of nitrogen and then the sample was reconstituted with 20 μl of ethanol. The samples are analyzed immediately or frozen at −70°C until analysis.

Liquid Chromatography-Mass Spectrometry

Quantitation of lipid mediators was performed using 2-dimensional reverse phase HPLC tandem mass spectrometry (LC/MS/MS). The HPLC system consisted of an Agilent 1290 autosampler (Agilent Technologies, Santa Clara, California), an Agilent 1200 binary SL loading pump (pump 1), an Agilent 1290 binary analytical pump (pump 2) and a 6 port switching valve. Pump 1 buffers consisted of 0.1% formic acid in water (solvent A) and 9:1 v: v acetonitrile: water with 0.1% formic acid (solvent B). Pump 2 buffers consisted of 0.01% formic acid in water (solvent C) and 1:1 v: v acetonitrile: isopropanol (solvent D).

About 5 μl of extracted sample was injected onto an Agilent SB-C18 2.1 × 5 mm 1.8 μm trapping column using pump 1 at 2 ml/min for 0.5 min with a solvent composition of 97% solvent A: 3% solvent B. At 0.51 min the switching valve changed the flow to the trapping column from pump 1 to pump 2. The flow was reversed, and the trapped lipid mediators were eluted onto an Agilent Eclipse Plus C-18 2.1 × 150 mm 1.8 um analytical column using the following gradient at a flow rate of 0.3 ml/min: hold at 75% solvent A : 25% solvent D from 0 to 0.5 min, then a linear gradient from 25% to 75% D over 20 min followed by an increase from 75% to 100% D from 20 to 21 min, then holding at 100% D for 2 min. During the analytical gradient pump 1 washed the injection loop with 100% B for 22.5 min at 0.2 ml/min. Both the trapping column and the analytical column were re-equilibrated at starting conditions for 5 min before the next injection.

Mass spectrometric analysis was performed on an Agilent 6490 triple quadrupole mass spectrometer in negative ionization mode. The drying gas was 250°C at a flow rate of 15 ml/min. The sheath gas was 350°C at 12 ml/min. The nebulizer pressure was 35 psi. The capillary voltage was 3500 V. Data for lipid mediators was acquired in dynamic MRM mode using experimentally optimized collision energies obtained by flow injection analysis of authentic standards. Calibration standards for each lipid mediator were analyzed over a range of concentrations from 0.25 to 250 pg on column. Calibration curves for each lipid mediator were constructed using Agilent Masshunter Quantitative Analysis software. Samples were quantitated using the calibration curves to obtain the on-column concentration, followed by multiplication of the results by the appropriate dilution factor to obtain the concentration in pg/ml.

High Dose : 0.125 mg/kg Nitrogen Mustard dose

KIT Wsh mice - Sample numbers ending in K

C57BL6J mice - samples ending in B

BALF- Bronchoaleveolar lavage fluid

Numbers represent animal ID

LOD - below the limit of detection

LOQ- below the limit of quantitation

AA- arachidonic acid pathway

DHA- docosahexaenoic acid pathway

LA- linoleic acid pathway

DGLA- Dihomo-y-linolenic acid 

EPA- eicosapentaenoic acid 

LOX - lipoxygenase

CYP - cytochrome

COX- cyclooxygenase

ROS- reactive oxygen species