Differential expression of platelet-activating factor acetylhydrolase in lung macrophages

Abstract

Platelet-activating factor (PAF) acetylhydrolase plays a crucial role inactivating the potent inflammatory mediator, PAF. PAF is implicated in the initiation and propagation of acute lung injury. Although PAF acetylhydrolase is a constitutively active plasma protein, increased PAF production during inflammatory events may necessitate an increase in PAF acetylhydrolase activity in the local environment. A series of experiments were conducted to determine whether the systemic administration of LPS to Sprague-Dawley rats resulted in enhanced expression of PAF acetylhydrolase in lung tissue. Ribonuclease protection assays revealed a dramatic increase in PAF acetylhydrolase mRNA, which peaked at 24 h following in vivo LPS administration. The increase in PAF acetylhydrolase mRNA was dose dependent and was detected when as little as 10 microg/kg of LPS was administered. Western blot analyses of lung tissue homogenates confirmed an increased production of PAF acetylhydrolase protein in response to LPS. In addition, Western blot analyses revealed the rat PAF acetylhydrolase protein exhibited heterogeneous molecular weights with predominant species migrating at 63 and 67 kDa. Some of the molecular weight heterogeneity likely resulted from extensive glycosylation of the secreted protein. Immunohistochemical analyses of lung tissue sections and colocalization experiments revealed a heterogenous population of cells that express the plasma-type PAF acetylhydrolase. Lung interstitial macrophages were PAF acetylhydrolase positive, but surprisingly, alveolar macrophages did not increase expression of PAF acetylhydrolase in response to systemic LPS administration. In addition, rat granulocytes consisting primarily of neutrophils were strongly positive for PAF acetylhydrolase in the LPS-exposed lung tissue. The absence of immunoreactive PAF acetylhydrolase in alveolar macrophages obtained from bronchial alveolar lavage confirmed that systemic LPS administration resulted in enhanced PAF acetylhydrolase expression only in a subset of lung macrophages.

Fig. 1.

Time course of platelet-activating factor (PAF) acetylhydrolase mRNA induction in lung tissue from LPS-exposed animals. Lung tissue was harvested from rats exposed to LPS (3 mg/kg) for 0, 1, 3, 6, 12, 24, and 48 h and immediately frozen in liquid nitrogen. Total RNA was prepared from the lung tissue, as detailed in the materials and methods. Aliquots of total RNA (80 μg) were analyzed by ribonuclease protection assays (RPA). The total RNA was hybridized in solution with ³²P-labeled antisense RNA probes for PAF acetylhydrolase (245 bp) and GAPDH (355 bp). The RNA-RNA hybrids were then digested with RNase A/T1 and separated on a 6% polyacrylamide, 8-M urea gel and exposed to a phosphorimage screen (A). Probe, undigested full-length antisense probes; tRNA, RNase digested negative control. GAPDH and PAF acetylhydrolase (PAF-AH) labels are adjacent to the respective protected fragments. The RPA shown is representative of three independent experiments. B: densitometric quantitation of the representative RPA.

Fig. 2.

Induction of lung tissue PAF acetylhydrolase mRNA in response to varying doses of LPS. Twenty-four hours after infusion of 0, 0.01, 0.1, 1.0, and 3.0 mg/kg LPS, lung tissue was harvested, and total RNA was isolated as described in the materials and methods. A: aliquots of total RNA (80 μg) were analyzed by RPA. The total RNA was hybridized in solution with ³²P-labeled antisense RNA probes for PAF acetylhydrolase (245 bp) and GAPDH (355 bp). The RNA-RNA hybrids were then digested with RNase A/T1 and separated on a 6% polyacrylamide, 8-M urea gel and exposed to a phosphorimage screen. Probe Alone, undigested full-length antisense probes; tRNA, RNase digested negative control. The arrows point to protected fragments for GAPDH and PAF acetylhydrolase (PAF-AH). The RPA shown is representative of three independent experiments. B: densitometric quantitation of the representative RPA.

Fig. 3.

Western blot analysis of PAF acetylhydrolase protein induction in lung tissue from LPS-exposed animals. Lung tissue from the indicated samples was homogenized in RIPA, and aliquots (20 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. PAF acetylhydrolase protein was detected using a rabbit anti-rat PAF acetylhydrolase antibody (1/1,000) and a horseradish peroxidase labeled goat anti-rabbit secondary antibody (1/1,000). The peroxidase-labeled proteins were visualized using an enhanced chemiluminescence detection system. A: LPS (3 mg/kg) was administered and at 0, 1, 3, 6, 12, 24, and 48 h later the lung tissue was harvested and tissue homogenates were subjected to Western blot analysis. Rat serum (1/100) or cultured rat Kupffer cell lysates (20 μg) were used as a positive control. B: line graph of the quantitation of the 63 and 67 kDa PAF acetylhydrolase immunoreactive bands. C: Western blot of tissue homogenates prepared from lung tissue either removed directly after 24 h of LPS exposure (nonperfused) or after the lung vasculature was perfused for 10 min with PBS (perfused). Rat serum (1/100) was run in parallel and used as a positive control.

Fig. 4.

Digestion with N-glycosidase alters the mobility of PAF acetylhydrolase proteins. Samples of cultured Kupffer cell lysates, lung tissue homogenates, and rat serum were incubated with N-glycosidase F, as detailed in the materials and methods. Control digestions were incubated in the appropriate buffer lacking N-glycosidase F for an equal amount of time. Western blot analysis to detect PAF acetylhydrolase was performed as described previously. (-, no N-glycosidase F; +, N glycosidase F).

Fig. 5.

Immunohistochemical localization of lung PAF acetylhydrolase and rat ED1 in saline- and LPS-infused animals. Lung tissue sections (5 μm) from saline (A and C) and LPS (B and D) -treated animals 24 h after exposure were cryopreserved and fixed in a solution of methanol:acetone (1:1). The lung sections were incubated with affinity-purified rabbit anti rat PAF acetylhydrolase (1/200) and mouse anti-rat ED1 (1/700) antibodies. Localization of the anti-PAF acetylhydrolase antibody was detected using a Cy3-conjugated goat anti-rabbit secondary antibody (red). Localization of the anti-ED1 antibody was detected using a goat anti-mouse FITC-conjugated secondary antibody (green). Scale bar represents 50 μm: Original magnification: ×100. A and B: anti-PAF acetylhydrolase (red). C and D: anti-ED1 (green).

Fig. 6.

Colocalization of PAF acetylhydrolase and ED1 in LPS-exposed lung tissue. Lung tissue sections isolated from animals exposed to LPS (3 mg/kg) for 24 h were cryopreserved and fixed in a solution of methanol:acetone (1:1). The lung sections were incubated with affinity-purified rabbit anti-rat PAF acetylhydrolase (1/200) and mouse anti-rat ED1 (1/700) antibodies. Localization of the anti-PAF acetylhydrolase antibody was detected using a Cy3-conjugated goat anti-rabbit secondary antibody (red). Localization of the anti-ED1 antibody was detected using a goat anti-mouse FITC-conjugated secondary antibody (green). A: original magnification: ×400. A, A: anti-PAF acetylhydrolase. A, B: anti-ED1. A, C: merged image. Scale bar shown represents 50 μm. B: merged images at higher magnification. Arrowhead points to an ED1-positive, PAF acetylhydrolase-negative cell. Arrow points to a PAF acetylhydrolase-positive, ED1-negative cell. Scale bar shown represents 50 μm (B, subpart A) and 10 μm (B, subpart B).

Fig. 7.

Immunohistochemical analyses of PAF acetylhydrolase and HIS48 in saline- and LPS-exposed lung tissue. Lung tissue sections (5 μm) from saline (A and C)- and LPS (B and D–F)-treated animals 24 h after exposure were cryopreserved and fixed in a solution of methanol:acetone (1:1). The lung sections were incubated with affinity-purified rabbit anti-rat PAF acetylhydrolase (1/200) and mouse anti-rat HIS48 (1/200) antibodies. Localization of the anti-PAF acetylhydrolase antibody was detected using a Cy3-conjugated goat anti-rabbit secondary antibody (red). Localization of the anti-HIS48 antibody was detected using a goat anti-mouse FITC-conjugated secondary antibody (green). Scale bar represent 50 μm. A and B: anti-HIS48. C and D: anti-PAF acetylhydrolase. E and F: merged images.

Fig. 8.

PAF acetylhydrolase cytochemistry on cells obtained from bronchial alveolar lavage (BAL) fluid isolated from LPS-exposed animals. Rats received LPS (3 mg/kg), and BAL was collected at 24 h after the administration. BAL was collected by instilling and collecting four successive 5-ml volumes of PBS. The lavage fluid was centrifuged, and the cell pellet was resuspended in 1 ml PBS. Immunocytochemistry was performed on cytospin-prepared slides. Arrow points to a large PAF acetylhydrolase-negative alveolar macrophage. Arrowhead points to small PAF acetylhydrolase-positive BAL cell.