Secretory PLA2-IIA: a new inflammatory factor for Alzheimer's disease

Abstract

Secretory phospholipase A2-IIA (sPLA2-IIA) is an inflammatory protein known to play a role in the pathogenesis of many inflammatory diseases. Although this enzyme has also been implicated in the pathogenesis of neurodegenerative diseases, there has not been a direct demonstration of its expression in diseased human brain. In this study, we show that sPLA2-IIA mRNA is up-regulated in Alzheimer's disease (AD) brains as compared to non-demented elderly brains (ND). We also report a higher percentage of sPLA2-IIA-immunoreactive astrocytes present in AD hippocampus and inferior temporal gyrus (ITG). In ITG, the majority of sPLA2-IIA-positive astrocytes were associated with amyloid beta (Abeta)-containing plaques. Studies with human astrocytes in culture demonstrated the ability of oligomeric Abeta1-42 and interleukin-1beta (IL-1beta) to induce sPLA2-IIA mRNA expression, indicating that this gene is among those induced by inflammatory cytokines. Since exogenous sPLA2-IIA has been shown to cause neuronal injury, understanding the mechanism(s) and physiological consequences of sPLA2-IIA upregulation in AD brain may facilitate the development of novel therapeutic strategies to inhibit the inflammatory responses and to retard the progression of the disease.

Figure 1

sPLA₂-IIA immunoreactivity in human postmortem brain tissues. Double immunostaining depicting sPLA₂-IIA immunoreactivity in dark blue color and GFAP immunoreactivity in brown color is shown in panels A-D (using 20X and 40X objective lenses). Panel A demonstrates that little sPLA₂-IIA immunoreactivity is present in a cluster of GFAP immunoreactive astrocytes in ND hippocampus. Panel B shows many GFAP-positive astrocytes (white arrow) labeled with intense immunoreactivity for sPLA₂-IIA (dark immunoreactive products, red arrow) in AD hippocampus. At higher magnification (Panel C), sPLA₂-IIA immunoreactivity is shown in an astrocyte cell body in granular-like structures (red arrow). Panel D shows that immunoreactivity for sPLA₂-IIA (red arrows) is also present in GFAP-positive astrcoytes (white arrows) surrounding microvessels in AD hippocampus. We also detected sPLA₂ immunoreactivity in hippocampal neurons (black arrows) in ND (Panel A) and AD (Panel D) hippocampus. In Panel E, several sPLA₂-IIA immunoreactivitve profiles (red arrows) are co-localized with an amyloid plaque (brown immunoreactive area) detected by immunohistochemistry with an antibody to Aβ.

Figure 2

Co-localization of sPLA₂-IIA-positive astrocytes with thioflavin S-positive plaques. Double immunostaining of sPLA₂-IIA and GFAP combined with thioflavin S staining shows the presence of sPLA₂-IIA (red arrows) in GFAP-positive astrocytes (panels A and B) and their association with thioflavin S-positive amyloid plaques (green fluorescent area in panel A) in an ITG section from an AD case.

Figure 3

Induction of sPLA₂-IIA mRNA expression by cytokines and Aβ _1–42 in cultured human astrocytes. Phase contrast micrographs show human astrocytes in control (panel A) and IL-1β-stimulated cultures (panel B) for 24 hours. Human postmortem astrocytes were used for the sPLA2-IIA RNA study. Experiments were performed using cultures derived from 3 neuropathologically confirmed AD cases. A representative gel depicting PCR-amplified fragments for sPLA2-IIA and β-actin is shown in panel C. Gel lanes 1–5 represent the following treatments used in the astrocyte cultures: 1. control; 2. IFNγ (100 ng/ml); 3. Aβ_1–42 (2.5 μM); 4. IL-1β (20 ng/ml); 5. IL-1β and Aβ_1–42. Twenty-four hours after treatment, RNA was extracted from cells, reverse transcribed, and RT-PCR was carried out as described in methods. Panel D shows a bar graph depicting relative units of sPLA₂-IIA expression after normalization with β-actin. Significant differences (*) comparing treatment groups with controls were obtained by one-way ANOVA followed by Tukey multiple comparison post hoc test.