Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation

Abstract

The specific intracellular sites at which enzymes act to generate arachidonate-derived eicosanoid mediators of inflammation are uncertain. We evaluated the formation and function of cytoplasmic lipid bodies. Lipid body formation in eosinophils was a rapidly (<1 h) inducible response which was platelet-activating factor (PAF) receptor-mediated, involved signaling through protein kinase C, and required new protein synthesis. In intact and enucleated eosinophils, the PAF-induced increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. All principal eosinophil eicosanoid-forming enzymes, 5-lipoxygenase, leukotriene C4 synthase, and cyclooxygenase, were immunolocalized to native as well as newly induced lipid bodies in intact and enucleated eosinophils. Thus, lipid bodies are structurally distinct, inducible, nonnuclear sites for enhanced synthesis of paracrine eicosanoid mediators of inflammation.

Figure 1

PAF receptor–mediated induction of lipid body formation in eosinophils. (A) Human eosinophils, at a concentration of 10⁶ cells/ml, were treated with PAF (10⁻⁸–10⁻⁶M) (white squares) or lyso-PAF (10⁻⁸– 10⁻⁶ M) (black squares) for 1 h at 37°C. (B) Human eosinophils at a concentration of 10⁶ cells/ml were pretreated with WEB 2086 (10–100 μM), pertussis toxin (100 ng/ml), or vehicle for 1 h at 37°C. The cells were then treated with PAF (1 μM) or vehicle for 1 h at 37°C. Lipid bodies were enumerated using light microscopy after osmium staining. Each point represents the mean ± SEM of lipid bodies on 50 consecutive eosinophils from three to five independent assays using different donors. *Statistically significant difference between PAF and the vehicle. ⁺Statistically significant differences caused by pretreatment with WEB 2086 or pertussis toxin.

Figure 2

Immunolocalization of 5-LO to lipid bodies of human eosinophils. (A and C) Human eosinophils from normal volunteers were incubated with PAF (1 μM, for 1 h) to induce lipid body formation, and lipid bodies were visualized by endogenous labeling with the fluorescent fatty acid–containing diglyceride, 1-acyl-2-(7-octyl BODIPY™-1-pentanoyl)-sn-glycerol (1 μM, for 1 h). Fluorescent fatty acid–labeled lipid bodies were visualized as green punctate intracytoplasmic inclusions under FITC excitation. (B) With specific anti–5-LO rabbit antiserum and glucose-oxidase immunocytochemistry, there was 5-LO staining diffusely in the cytoplasm as well as at punctate lipid bodies (which matched those in A). (D) With control nonimmune rabbit serum there was no lipid body or cytoplasmic staining.

Figure 3

Immunogold localization of 5-LO in freshly isolated, unstimulated eosinophils from an HES donor shows gold label indicating 5-LO in lipid bodies (A and C), which was absent from lipid bodies stained with control solid phase 5-LO–absorbed anti–5-LO antisera (B) or nonimmune control serum (D). In A, cytoplasmic tubules and vesicles of smooth endoplasmic reticulum adjacent to the lipid body also have gold particles associated with them (arrows) which are absent in the 5-LO absorption control (arrow, B). The lipid bodies in A and B are not homogeneously dense and reveal numerous small, round areas which are electron lucent and nonmembrane bound. In contrast, lipid bodies in C and D appear more homogeneous in their content. In C, several gold particles reside in the perinuclear cistern (arrow); the nonimmune antibody control has one gold particle attached to electron-dense chromatin in the nucleus. A, ×29,640; B, ×36,480; C, ×44,080, D, ×45,600.

Figure 4

Immunolocalization of LTC₄ synthase and COX to lipid bodies of human eosinophils. Human eosinophils from normal volunteers were incubated with PAF (1 μM, for 1 h) to induce lipid body formation, and lipid bodies were visualized by endogenous labeling with the fluorescent fatty acid–containing diglyceride, 1-acyl-2-(7-octyl BODIPY™-1-pentanoyl)-sn-glycerol (1 μM, for 1 h). Fluorescent fatty acid–labeled lipid bodies were visualized as green punctate intracytoplasmic inclusions under FITC excitation (A, C, and E). LTC₄ synthase (B) and COX (D) were localized to lipid bodies using anti-LTC₄ and anti-COX, respectively, affinity-purified rabbit IgG, and glucose-oxidase immunocytochemistry. Rabbit IgG was used as a control (F). Lipid bodies exhibit dark punctate staining in B and D which matches fluorescent lipids in A and C, respectively. (Some fluorescent lipid bodies are out of the plane of focus and not visible.)

Figure 5

PAF-induced both lipid body formation and priming for LTC₄ (A) and PGE₂ (B) production by human eosinophils. Eosinophils (10⁶/ml) were stimulated for 1 h at 37°C with concentrations of PAF or vehicle alone. Data are means ± SEM of eicosanoids formed by eosinophils and lipid body numbers in eosinophils from six to eight independent experiments. Increasing numbers of lipid bodies correlated with increased production of each eicosanoid (r ⩾0.97, P <0.03 for both, Fisher's r to z transformation). *Statistically significant differences (P <0.05, paired t test) between PAF and the vehicle. LTC₄ and PGE₂ in supernatants were measured by ELISA after incubation with 0.5 μM A23187 for 15 min.

Figure 6

Eosinophil cytoplasts and immunolocalization of eicosanoid-forming enzymes. Freshly isolated eosinophils (A), eosinophil cytoplasts (B), and karyoplasts (C) were fixed in methanol and stained with Diff-Quik (Baxter Healthcare Corp., Miami, FL). Eosinophil cytoplasts were incubated with PAF (1 μM, for 1 h) to induce lipid body formation. 5-LO (D), LTC₄ synthase (E), and COX (F) were localized at cytoplast lipid bodies using specific anti–5-LO rabbit antiserum, affinity-purified rabbit IgG anti-LTC₄ synthase, or anti-COX, respectively, and glucose-oxidase immunocytochemistry. Substitution of the primary antibody by nonimmune rabbit serum (G) or rabbit IgG (H) were controls.

Figure 7

Dose-dependent effect of PAF (10⁻⁸–10⁻⁶M) on both lipid body formation and priming for LTC₄ (A) and PGE₂ (B) production by eosinophil cytoplasts. Cytoplasts (10⁶/ml) were stimulated for 1 h at 37°C with PAF or vehicle alone. Data are means ± SEM of eicosanoids formed by cytoplasts and lipid body numbers in cytoplasts from three to four independent assays. *Significantly > value without PAF stimulation (P <0.05). LTC₄ and PGE₂ in supernatants were measured by ELISA after incubation with 0.5 μM A23187 for 15 min.

Figure 8

Immunoblotting of 5-LO in lipid bodies and other subcellular fractions of unstimulated and PAF-stimulated eosinophils. Lipid bodies and other subcellular fractions were isolated from unstimulated and PAF-stimulated (1 μM, 1 h) eosinophils as described in Materials and Methods. Proteins (15 μg) from subcellular fractions were electrophoresed on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted with anti–5-LO serum. Purified recombinant 5-LO protein was used as a standard for the Western blot. The anti–5-LO serum recognized a 78-kD protein, which comigrated with the purified recombinant 5-LO standard, in subcellular fractions, prominently including the cytosol and buoyant lipid bodies from PAF-stimulated eosinophils.