Lipid body formation during maturation of human mast cells

Abstract

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

Fig. 1.

Identification of LBs in human MCs with light and with electron microscopy. Human peripheral blood-derived CD34⁺ progenitor cells from 18 donors were grown under defined culture conditions, which induced their differentiation into mature MCs, as described in Materials and Methods. A–D: The cells were stained with Oil Red O/hematoxylin at the indicated weeks in culture. Representative images of cells from a single donor are shown. Each inset shows one typical cell representative for the corresponding time point in culture. E, F: Mast cells from an early (6-week) and late (29-week) culture were analyzed by transmission electron microscopy. In each panel, one typical LB is indicated with an arrow. N, nucleus; wk, week.

Fig. 2.

Quantification of LBs in maturing MCs. The cumulative volumes of LBs in 10,000 MCs each derived from three different MC donors were analyzed by flow cytometry as a function of culture time. The results of each donor (A, B, and C) are shown as fluorescence intensities. The number of weeks of culture is indicated.

Fig. 3.

Lipid bodies in resting and degranulated MCs. A, B: Mast cells from resting and immunologically activated cells (at 14 weeks of culture) were analyzed by transmission electron microscopy. In both panels, one typical LB is indicated with an arrow. C: Magnifications of typical LBs and secretory granules of resting and activated MCs. D: Mature MCs (at 16 week of culture) were activated for 1 h via IgE cross-linking to induce their degranulation (activated cells). Control cells were incubated in the absence of anti-IgE (resting cells). Then, all cells were fixed, permeabilized, and incubated with anti-tryptase antibody, washed, and probed with phycoerythrin-conjugated goat anti-mouse IgG. Cytoplasmic LBs were stained with fluorescent BODIPY 493/503, and the cells were analyzed by flow cytometry. The values shown are mean fluorescence intensities (1 × 10⁴ cells/sample) for the stimulated cells expressed as percentages of fluorescence intensities of resting cells, and they represent the averages of values obtained with cells from two donors.

Fig. 4.

Expression of PAT family members in human MCs. Transcript levels of the PAT family members plin1, plin2, plin3, plin4, and plin5 were determined by RT-PCR using mRNA from human MCs at 14 wk of culture (MC, lane 1), LAD2 cells (LAD2, lane 2), and sets of sequence-specific primers. Human reference total RNA was used as positive control (pCtrl, lane 3), and PCR performed without the addition of template served as negative control (nCtrl, lane 4). Beta actin-specific primers were used to test the quality of cDNA templates (bottom panel). The PCR products were analyzed by agarose gel electrophoresis.

Fig. 5.

PLIN2 and PLIN3 proteins in CD34⁺-derived MCs and LAD2 MCs. Lysates of CD34⁺-derived MCs (MC; 80 μg protein per lane) and LAD2 cells (LAD2; 70 μg protein per lane) were analyzed by Western blotting with guinea pig anti-human PLIN2 antiserum (lane 1) and with guinea pig anti-human PLIN3 antiserum (lane 2), respectively. Bound primary antibodies were visualized with HRP-labeled rabbit anti-guinea pig IgG.

Fig. 6.

Intracellular localization of PLIN2 and PLIN3 in MCs. For the analysis of the intracellular distribution of PLIN2 (A) and PLIN3 (B), MCs were probed with mouse anti-human PLIN2 or with guinea pig anti-human PLIN3. The primary antibodies were probed with Alexa Fluor-594 goat anti-mouse IgG (A) or Alexa Fluor-594 goat anti-guinea pig IgG (B). LBs were stained with BODIPY 493/503, and nuclei were counterstained with DAPI. Orthogonal projections of confocal z-stacks of representative LBs are shown (A, B). The main images of both panels show an xy-section of a MC at the z-positions indicated by dark blue lines in the xz-plane (x-axis/z-axis; small top rectangular figure) and the yz-plane (y-axis/z-axis; small right rectangular figure). Insets (A, B) show magnified orthogonal views of the indicated single LBs.

Fig. 7.

Lipid composition of human MC lipid bodies. Lipid bodies were isolated from human MCs at 18 wk of culture, and their lipids were extracted and separated by thin-layer chromatography. A, B: Lane 1 contains the indicated lipid standards (Std), and lane 2 contains the lipids of the isolated LBs. A: Neutral lipids. B: Polar lipids. Arrowheads indicate site of sample application.

Fig. 8.

Induction and inhibition of lipid body formation in MCs. Mast cells were incubated in the presence of stearic acid (18:0), oleic acid (18:1), or arachidonic acid (20:4), each complexed to BSA (molar ratio 6:1) in the absence or presence of the long chain acyl-CoA synthetase inhibitor Triacsin C at 37°C for 18 h. Lipid bodies were quantified by flow cytometry. Fluorescence intensities of the treated cells are shown and expressed as percentages of the fluorescence intensities of untreated control cells. Values represent the mean ± SD of cells (1 × 10⁴ cells/sample) obtained from three donors (n = 3). Ctrl, control; FA, fatty acid.

Fig. 9.

Mass spectrometric analysis of MC lipid bodies. Lipid bodies were induced in MCs by incubating the cells in the presence of arachidonic acid, as described in Fig. 8. The bodies were then isolated, and their lipids extracted and analyzed by mass spectrometry. A: Positive ESI-MS of total lipid extract of LBs. Triacylglycerols (small rectangular box) are detected between m/z 880 and 1,050. B: Arachidonic acid-containing triacylglycerols (large rectangular box) were detected by the neutral loss scan for 321 amu during ESI-MS of neutral lipids extracted from LBs. The main arachidonic-acid-containing triacylglycerol species, denoted [sum of acyl carbons]:[sum of double bonds in the chains] with their m/z values and most likely acyl chain composition in parenthesis, were 58:9 (m/z 946; 18:1-20:4-20:4), 60:12 (m/z 968; 20:4-20:4-20:4) and 62:12 (m/z 996; 20:4-20:4-22:4). The intensity scales of the spectra were normalized to the largest signal in the selected m/z range. amu, atomic mass unit.