Neurons and astrocytes respond to prion infection by inducing microglia recruitment - PubMed (original) (raw)

Neurons and astrocytes respond to prion infection by inducing microglia recruitment

Mathieu Marella et al. J Neurosci. 2004.

Abstract

The accumulation and activation of microglial cells at sites of amyloid prion deposits or plaques have been documented extensively. Here, we investigate the in vivo recruitment of microglial cells soon after intraocular injection of scrapie-infected cell homogenate (hgtsc+) using immunohistochemistry on retinal sections. A population of CD11b/CD45-positive microglia was specifically detected within the ganglion and internal plexiform retinal cell layers by 2 d after intravitreal injection of hgtsc+. Whereas no chemotactism properties were ascribed to hgtsc+ alone, a massive migration of microglial cells was observed by incubating primary cultured neurons and astrocytes with hgtsc+ in a time- and concentration-dependent manner. hgtsc+ triggered the recruitment of microglial cells by interacting with both neurons and astrocytes by upregulation of the expression levels of a broad spectrum of neuronal and glial chemokines. We show that, in vitro and in vivo, the microglia migration is at least partly under the control of chemokine receptor-5 (CCR-5) activation, because highly specific CCR-5 antagonist TAK-779 significantly reduced the migration rate of microglia. Activated microglia recruited in the vicinity of prion may, in turn, cause neuronal cell damage by inducing apoptosis. These findings provide insight into the understanding of the cell-cell communication that takes place during the development of prion diseases.

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Figures

Figure 1.

Figure 1.

In vivo microglial cell recruitment after intraocular injection of hgtsc+. Immunohistochemical staining of retinal microglia (brown) expressing CD11b/CD45 epitopes 2 d after injection of PBS (a), hgtsc- (b), and hgtsc+ (c-e) into mouse eyes. A typically ramified microglia cell 2 d after hgtsc+ injection (d) and a representative retinal section through the optic nerve (e, arrow) are shown. Cell layers: g, ganglion cell; ip, internal plexiform; inl, inner nuclear; ep, external plexiform; onl, outer nuclear. Immunopositive microglial cells are brown; the counterstain is cresyl violet. Magnification: a-c, e, 10×; d, f, 40×. Quantification of the number of microglial cells per retinal section as a function of time after intravitreal inoculation of hgtsc- (gray bars) or hgtsc+ (black bars). The number of immunopositive cells was obtained by counting 90 independent retina sections corresponding to the whole eye from three treated mice in two separate experiments. Statistical significance as calculated by Student's t test: **p < 0.01 and not significant (n.s.) between day and d 1 in hgtsc+- treated animals, except for d 1, for which calculations were performed between hgtsc+- and hgtsc-- treated mice. g, Inhibitory effect of TAK-779 on the in vivo microglia migration 2 d after hgtsc+ injection. One microliter of hgtsc+ (black bars) or vehicle (white bars) was injected intravitreally in the presence or absence of TAK-779 (40 μ

m

). Retinal sections were processed through the immunohistology methods described in Materials and Methods. Statistical analysis (**p < 0.01) between hgtsc+ treatment conditions with and without TAK-779 is shown. Histograms show the mean ± SD of two independent experiments (n = 2) in which each condition represents three mice injected unilaterally.

Figure 2.

Figure 2.

In vitro characterization of the directional microglial cell migration on hgtsc+ stimulation of primary cultures of neurons and astrocytes. Incubations of 5 μl of vehicle (white bar), hgtsc- (gray bars), and hgtsc+ (black bars) on neurons (a) and astrocytes (c) were performed at 37°C for 18, 24, or 48 hr in the presence of the N11 microglial cell line in the top chamber. Microglia were challenged for migration through an 8 μm pore size insert for 6 hr at 37°C, then migrated cells were fixed, stained, and counted on an inverted microscope (5 fields counted/filter). Dose-responses were performed in the presence of a 24 hr stimulation time with the indicated volume of hgtsc- (gray bars) or hgtsc+ (black bars) on neurons (b) and astrocytes (d). The microglial migration assay and cell counts were done as described above. Migration assays performed with mouse primary cultures of microglia in the presence of 4-d-old neurons (e) or astrocytes (f) in the bottom chamber were stimulated for 24 hr with 5 μl of vehicle (white bar), hgtsc- (gray bar), and hgtsc+ (black bar). g, Migration assays performed with 5 μl of vehicle (white bar), hgtsc- (gray bar), and hgtsc+ (black bar) in the absence of plated cells in the bottom chamber. Histograms represent the mean of five independent experiments ± SD with two determinations each. Statistical analyses calculated with Student's t test [**p < 0.01; * p < 0.05 and not significant (n.s.)] compared hgtsc+ with hgtsc- incubation. For kinetics and dose-response experiments, statistical calculations were performed compared with the preceding concentration or incubation time.

Figure 3.

Figure 3.

Effect of PK treatment of hgtsc- and hgtsc+ on microglial cell migration. a, Untreated or PK-digested hgtsc- and hgtsc+ (5 μl), as described in Materials and Methods, were analyzed by SDS-PAGE and silver stain. Molecular mass markers are indicated on the left (kDa). The bracket indicates PK-resistant PrP (PrPres). b, Untreated or PK-digested hgtsc- (gray bars) and hgtsc+ (black bars) (5 μl) were incubated for 24 hr at 37°C on mouse primary cultured neurons seeded in the bottom chamber. Then, N11 cells were plated on the top chamber and challenged for migration for 6 hr. At least five fields of migrated cells/condition were counted. Histograms are means of three independent experiments ± SD. Statistical analyses calculated with Student's t test (**p < 0.01) compared hgtsc+ with hgtsc- incubation with or without PK digestion.

Figure 4.

Figure 4.

Chemokine expression profile in both hgtsc-- and hgtsc+-stimulated primary cultures of mouse neurons and astrocytes. a, Representative reverse transcription (RT)-PCR profile of chemokine activation in neurons or astrocytes. Mouse primary cultures of neurons or astrocytes were incubated in the presence of 5 μl of hgtsc- or hgtsc+. After 6 hr of incubation, mRNAs of the different conditions were purified and reverse transcribed. RT-PCR experiments were performed as described in Table 1 on 1 μg of total cDNA in the presence of appropriate primers for chemokines and GAPDH in the same assay. PCR products were analyzed on 2% agarose gels stained with SYBR Green I. Sizes of PCR products are, respectively, 431 bp for RANTES, 480 bp for MCP-1, 237 bp for MIP-1α, 367 bp for MIP-1β, 320 bp for MIP-2, and 1174 bp for GAPDH. Arrows indicate the PCR products of GAPDH mRNA. b, Semiquantitative profile of mRNA expression in neurons or astrocytes. RT-PCR gels for the genes indicated were quantified by densitometry using NIH Image software, with normalization to GAPDH mRNA levels. Results are expressed in terms of fold increase from hgtsc+-stimulated compared with hgtsc--stimulated cells and represent the means ± SD of data from four independent experiments. The systematic names of chemokines are in parentheses.

Figure 5.

Figure 5.

Anti-RANTES antibody and CCR-5 antagonist TAK-779 inhibit microglia migration. a, Four-day-old cortical neurons were incubated with 5 μl of vehicle (white bar), hgtsc- (gray bar), or hgtsc+ (black bars) for 24 hr at 37°C. Migration experiments were initiated by plating N11 cells on a 8 μm pore size insert for 6 hr in the presence of the indicated concentrations of anti-RANTES monoclonal antibody or unrelated rat IgG. b, The experiment was performed as described above in the presence of the indicated concentrations of TAK-779 instead of antibodies. Results are expressed as the percentage of migration rate obtained with 5 μl of hgtsc+ alone (0). Histograms represent the means of data ± SD from three independent experiments. For each histogram, statistical significance was calculated compared with the preceding concentration of anti-RANTES antibody (a) or TAK-779 (b) (**p < 0.01; *p < 0.05; n.s., not significant).

Figure 6.

Figure 6.

Microglia activation and neurotoxicity induced by PrPsc preconditioned microglia medium. a, Confluent N11 microglial cells were incubated with 5 μl of PBS (white bars), hgtsc- (gray bar), or hgtsc+ (black bar) for 12 hr. Then, nitrite concentration was measured using the Griess reagent and compared with the standard curve. Each histogram is the mean of triplicate determinations from six independent experiments ± SD. b, Representative gel analysis of 10 μg of neuronal DNA from untreated (C) or neurons treated with preconditioned microglia medium as described above. Sizes of the markers are expressed in base pairs (bp) on the left. C, DAPI staining of 4-d-old neurons treated with hgtsc- or hgtsc+ preconditioned microglia culture medium. Phase-contrast micrographs of representative microscopic fields are shown. Magnification, 40×. Arrows indicate apoptotic nuclei.

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