Immunohistochemical study of the beta-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains

Abstract

Chemokines belong to an expanding family of cytokines the primary function of which is recruitment of leukocytes to inflammatory sites. Recent evidence has shown their presence in the central nervous system. Because inflammatory responses have been implicated in the pathogenesis of Alzheimer's disease (AD), we studied the expression of CCR3, CCR5, and their ligands in normal and AD brains by immunohistochemistry. CCR3 and CCR5 are present on microglia of both control and AD brains, with increased expression on some reactive microglia in AD. Immunohistochemistry for MIP-1beta, MIP-1alpha, RANTES, eotaxin, and MCP-3 (ligands for CCR5 and/or CCR3) revealed the presence of MIP-1beta predominantly in a subpopulation of reactive astrocytes, which were more widespread in AD than control brains, and MIP-1alpha predominantly in neurons and weakly in some microglia in both AD and controls. Many of the CCR3+ or CCR5+ reactive microglia and MIP-1beta+ reactive astrocytes were found associated with amyloid deposits. Immunoreactivity for eotaxin, RANTES, and MCP-3 were not detected. Detection of these beta-chemokine receptors on microglia and some of their ligands in reactive astrocytes and neurons as well as microglia suggests a role for this system in glial-glial and glial-neuronal interactions, potentially influencing the progression of AD.

Figure 1.

CCR3 (7B11) and CCR5 (3A9) immunoreactivity in the inferior temporal lobes of a 58-year-old control patient and an 81-year-old AD patient with duration of illness for 12 years (PMIs less than 8 hours). CCR3 (a and b) and CCR5 (c and d) immunoreactivities are clearly seen on microglia of both cases. In the control case, the majority of cells stained are resting microglia, whereas in the AD brain both resting and reactive microglia cells are clearly stained, and some reactive microglia appear in clusters. All images have the same scale of magnification. Scale bar, 200 μm.

Figure 2.

Confocal images of double immunofluorescent staining. a and b: CCR3 (7B11, Cy3-red) versus Aβ (R1282, bodipy-green); c and d: CCR5 (3A9, Cy3-red) versus Aβ (R1282, bodipy-green); e and f: MIP-1β (1F12, Cy3-red) versus Aβ (R1282, bodipy-green). In a to d, many of the CCR3⁺ and CCR5⁺ reactive microglia can be seen associated with Aβ deposits. In e to f, some of the MIP-1β⁺ reactive astrocytes can also been seen in the vicinity of Aβ deposits. Images a to d are from the same AD case shown in Figure 1 ▶ ; images e to f are from an 81-year-old AD patient with duration of illness for 8 years (PMI, 4 hours). Each of the images is a projection of five Z series images 0.7 μm apart. Scale bars, 50 μm.

Figure 3.

a to d: MIP-1β (1F12) immunoreactivity in the inferior temporal lobes of a 63-year-old control patient and a 58-year-old AD patient (PMIs less than 8 hours). In the control case, weak MIP-1β staining can be found on a small population of resting and some occasional reactive astrocytes. However, in the AD case, much more widespread and stronger astrocyte staining is observed, and most of the astrocytes appear to be reactive. Insets are high-power images (inverted images) of double staining of MIP-1β (M, Cy3) versus GFAP (G, bodipy), showing a MIP-1β-positive cell is also clearly positive for GFAP. e to f: MIP-1α (11A3) immunoreactivity in the hippocampal formation of an 84-year-old AD patient (PMI, 7 hours). A diffused pattern of neuronal staining can be seen. Both neurons and neuropil were stained. Some neurons showed increased expression of MIP-1α. This pattern of immunoreactivity can be completely blocked by preabsorption with MIP-1α protein. a, c, and e: Low-power images (scale bar, 200 μm); b, d, and f are higher-power images (scale bar, 100 μm).