Lysosomal phospholipase A2 and phospholipidosis

Abstract

A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2-/-) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2-/- mice was normal. Lpla2-/- mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2-/- mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2-/- mice. By 1 year of age, Lpla2-/- mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2-/- mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice.

FIG. 1.

A. Strategy for producing allelic series of mutations at the Lpla2 locus. The partial map of the murine Lpla2 locus is displayed. Horizontal lines and open boxes with numbers represent introns and Lpla2 exons, respectively. Vertical lines represent restriction sites: M, SmaI; P, SpeI; D, DraI; A, SacI. The Lpla2 double conditional targeting vector was designed. Shaded triangles represent loxP sites flanking the Lpla2 gene exon 5, and shaded half-circles represent FRT sites flanking the neomycin resistance cassette (PGK neo). The targeted allele was generated by homologous recombination. The primers used for PCR are shown as horizontal arrows with letters. The conditional allele was generated by Flp-mediated excision. The heterozygous mice carrying targeted alleles were mated with FLP1 transgenic mice to delete the neo cassette. The null allele was generated by Cre-mediated excision. The heterozygous mice carrying conditional alleles were mated with EIIa Cre transgenic mice to delete exon 5. B. Genotype analysis by PCR. Genomic DNA was extracted from mouse tail, and PCR was performed to evaluate homologous recombination. The primers a and d as well as b and c were used for upper panel and lower panel assays, respectively. TV indicates targeting vector. M indicates molecular marker. C. Reverse transcription-PCR assay. Total RNAs were isolated from various mouse organs and synthesized cDNA. PCR was performed using primers which are able to cover the Lpla2 coding region. M, molecular marker φ_174 RF DNA/HaeIII; H, heart; Li, liver; S, spleen; K, kidney; T, thymus; B, brain; Lu, lung. D. Immunoblots of Lpla2 from the alveolar macrophages obtained from Lpla2^+/+, Lpla2^+/−, and Lpla2^−/− mice. Alveolar macrophages were isolated from mice as described in Materials and Methods, and the soluble fraction was separated by gel electrophoresis as previously described (1). A rabbit anti-LPLA2 polyclonal antibody (¹⁰⁰RTSRATQFPD¹⁰⁹) was used for detection. E. Transacylase activity in the soluble fraction of alveolar macrophages obtained from Lpla2^+/+, Lpla2^+/−, and Lpla2^−/− mice. Each soluble fraction (3 μg of protein) obtained from 3-month-old Lpla2^+/+, Lpla2^+/−, and Lpla2^−/− mouse alveolar macrophages was incubated for 30 min at 37°C in citrate buffer, pH 4.5, with 40 μM NAS in liposomal form, and formation of 1-O-acyl-NAS was determined as described in Materials and Methods. The transacylase specific activities for the Lpla2^+/+, Lpla2^+/−, and Lpla2^−/− mouse alveolar macrophages were 3.9 μg/min/mg protein, 2.08 μg/min/mg protein, and undetected, respectively. w/o, without; Fr., fraction.

FIG. 2.

Lpla2 activities on unsaturated phosphatidylcholines by Lpla2^+/+ and Lpla2^−/− mice. A. Transacylase activities of Lpla2 on two different phosphatidylcholines. The soluble fraction (2 μg of protein) obtained from 3-month-old Lpla2^+/+ mice was incubated for a suitable time period at 37°C in citrate buffer, pH 4.5, with 40 μM NAS incorporated into phospholipid liposomes (DOPC or POPC/dicetyl phosphate/NAS [7:1:2 molar ratio]), and formation of 1-O-oleoyl-NAS was determined as described in Materials and Methods. The left panel shows the TLC. The time-dependent formation of 1-O-oleoyl-NAS from DOPC or POPC/dicetyl phosphate/NAS liposomes by the soluble fraction in Lpla2^+/+ and Lpla2^−/− mice. The experiment is representative of three experiments with comparable results. (B) Comparison of phospholipase A2 activities in the lungs and alveolar macrophages of Lpla2^+/+ and Lpla2^−/− mice. Phospholipase A2 activities were measured as described in Materials and Methods using 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. OA denotes oleic acid. C. Degradation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine by alveolar macrophages. Alveolar macrophages (1.3 × 10⁶ cells) obtained from 3-month-old Lpla2^+/+ and Lpla2^−/− mice were incubated with ¹⁴C-labeled POPC/dicetyl phosphate (10:1 molar ratio) liposomes for 4 h at 37°C. After the incubation, the cellular lipid was extracted as described in Materials and Methods. Lipid extract was applied to an HPTLC and developed in a solvent system consisting of chloroform/acetic acid (9:1) (left) or chloroform/methanol/water (60:35:8) (right). After the development, the plate sprayed with ENHANCE was exposed on an X-ray film at −80°C. LysoPC indicates lysophosphatidylcholine. AMs, alveolar macrophages.

FIG. 3.

Light microscopy of tissues from 1-year-old Lpla2^+/+ and Lpla2^−/− mice. Panels A and B represent PAS staining of formalin-fixed inflated lungs and of spleens from 1-year-old wild-type and Lpla2⁻/⁻ mice, respectively. The histology is noteworthy for increased numbers of enlarged alveolar macrophages, as denoted by the arrows in the air spaces of the null mice and a mononuclear infiltrate surrounding the small airways and arteries. Panels C and D represent PAS staining from the spleens of 1-year-old wild-type and Lpla2^−/− mice, respectively. PAS-positive macrophages are denoted by the arrows.

FIG. 4.

Electron micrographs of alveolar macrophages (A and B) and peritoneal macrophages (C and D) obtained from 3-month-old Lpla2^+/+ and Lpla2^−/− mice. (A and C) Lpla2^+/+ mice; (B and D) Lpla2^−/− mice.

FIG. 5.

Spleen changes in 1-year-old mice. A. Representative spleens from Lpla2^+/+ and Lpla2^−/− mice. B. Comparative weights of the wild-type and knockout spleens from 4- and 12-month-old mice are displayed. C. Phospholipid profile in the spleens obtained from Lpla2^+/+ and Lpla2^−/− mice. The spleen homogenates obtained from 12-month-old Lpla2^+/+ and Lpla2^−/− mice were dispersed in chloroform/methanol mixture, and the lipid extraction was carried out as described in Materials and Methods. The graph denotes the phospholipid profiles of PE, PC, phosphatidylserine (PS), sphingomyelin (SM), and phosphatidylinositol (PI). The error bars indicate standard deviations (n = 4).