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Comparative Study

doi: 10.1186/1471-2377-6-12.

COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord

Affiliations

PMID: 16512913
PMCID: PMC1413551
DOI: 10.1186/1471-2377-vi-12

Complimentary PMC article

Comparative Study

COX-ii, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal string

Yiangos Yiangou et al. BMC Neurol. 2006 .

Free PMC article

Abstract

Background: While multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, in that location is increasing bear witness for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids improve progression of clinical illness in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three primal molecules known to exist expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord.

Methods: Frozen human post mortem spinal string specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were bachelor for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In improver, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a mark of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified past computerized image and optical density analysis respectively.

Results: In command spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, by and large bipolar with curt processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, past image assay. Inflammatory jail cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-ii past activated microglial cells/macrophages. An expected 70-kDa ring was seen past Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-ii immunostaining and optical density of the COX-2 lxx-kDa band in the MS grouping (r = 0.89, P = 0.0011, north = 10). MS and ALS specimens too had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions.

Conclusion: It is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in plow induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-ii and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS.

Figures

**Figure 1**
Microglial cells/macrophages in control spinal cord immunostained with antibodies to A) COX-ii, B) CB2, C) P2X₇, and D) CD68. Scale bar = l μm.

**Figure 2**
COX-2 immunoreactivity in MS spinal cord is in microglial cells/macrophages. (A) Autoradiographic localisation of ³[H] PK11195 in a spinal cord from a patient with MS, co-located with CD68 (D) (Scale bars = m μm). The square indicates the expanse where subsequent CD68 and COX-2 images were taken from. B) And Eastward) Microglial prison cell/macrophage-similar immunostaining with CD68 antibody (Scale bars = 200 μm and 100 μm respectively). C) And F) Microglial cell/macrophage-like immunostaining with COX-2 (Scale bars = 200 μm and 100 μm respectively).

**Figure 3**
COX-2 immunoreactivity is increased significantly in MS spinal string. A horizontal line indicates the median value from each grouping. * P = 0.025

**Figure four**
CB2 staining in MS and ALS spinal string is in microglial cells/macrophages. A) CB2 staining in MS spinal string. Inset: CB2 staining in parenchymal microglial cells/macrophages. (Scale bar = 50 μm). B) Microglia-similar immunostaining with CD68 in the same expanse equally in A (Calibration bar = 50 μm). C and D) CB2 staining in ALS spinal string (Calibration bar = 500 μm and fifty μm, respectively). E and F) Microglia-like immunostaining with CD68 (Scale bar = 500 μm and 50 μm, respectively).

**Figure 5**
CB2-immunoreactivity is increased in MS and ALS spinal cords. A) Mean % area of CB2 in spinal cord taken from MS (with and without lesion, filled squares and circles respectively) and control spinal string (open circles) using computerized image assay. A horizontal line indicates the median value from each group. B) Hateful % area of CB2 in ALS spinal cord and CD68 immunoreactivity taken from control and ALS spinal string using computerized image analysis. A horizontal line indicates the median value from each group, WM, white matter; VH, ventral horn; DC, dorsal column; NAWM, non affected white matter.

**Figure 6**
Peak console, representative Western blots of the COX-2 lxx kDa band in control (lanes 1–6) and MS (lanes seven – xi), and lane 12 mouse macrophage positive control. Bottom panel, optical density assay of COX-2 Western blots. Relative optical density readings of COX-2, seventy-kDa bands in the spinal cord of controls compared to MS specimens.

**Effigy 7**
COX-2 Western blots correlate with immunohistochemistry studies. Spearman plots of COX-2, seventy kDa optical density readings compared to COX-2 epitome analysis from MS spinal cords. Number of XY pairs 10.

**Effigy eight**
P2X_sevenimmunoreactivity is found in microglial cells/macrophages of MS spinal cord. (A) Autoradiographic localisation of ³[H] PK11195 in a spinal cord from a patient with MS co-located with CD68 (D) (Scale bars = 1000 μm). The square indicates the surface area where subsequent CD68 and P2X_viiimages were taken from. B) And E) Microglial cells/macrophage -like immunostaining with CD68 antibody (Scale bars = 200 μm and 100 μm respectively). C) And F) Microglial cells/macrophage -like immunostaining with P2X_seven(Calibration bars = 200 μm and 100 μm respectively).

**Figure 9**
P2X₇immunoreactivity is increased in MS spinal cord. Mean % expanse of P2X₇immunoreactivity taken from control and MS spinal cord using computerized image analysis. A horizontal line indicates the median value for each group.

**Figure 10**
Co-localisation studies of COX-ii and P2X₇in MS spinal cord. Double staining of COX-2 or P2X_viicells with the microglia marker ferritin in MS spinal cord. Sections were incubated with mixture of A) Ferritin (black) and COX-2 (red) antibodies or B) ferritin (blackness) and P2X₇(red). The bulk of COX-two or P2X₇immunoreactive cells announced to exist microglial cells/macrophages. Scale bar = 100 μm.

**Figure xi**
COX-2 immunoreactivity in ALS spinal cord found in microglial cells/macrophages predominantly in white matter. (A) Autoradiographic localisation of ³[H] PK11195 in a spinal cord from a patient with ALS co-located with CD68 (D) (Calibration confined = chiliad μm). The square indicates the expanse where subsequent CD68 and COX-2 images were taken from. B) And E) Microglial cells/macrophage -like immunostaining with CD68 antibody (Scale bars = 200 μm and 100 μm respectively). C) And F) Microglial cells/macrophage-like immunostaining with COX-2 (Calibration bars = 200 μm and 100 μm respectively).

**Figure 12**
COX-ii is increased in the dorsolateral white matter of ALS spinal cord. Top, Hateful % expanse of COX-2 immunoreactivity in spinal cord (open up box, control, shaded ALS) and bottom, Mean % area of CD68 immunoreactivity in spinal cord. Mean ± SEM * P = 0.0012, ** P = 0.0047.

**Figure 13**
P2X₇immunoreactivity localised to microglial prison cell/macrophage-similar cells in ALS spinal cord. (A) Autoradiographic localisation of ^three[H] PK11195 in a spinal cord from a patient with ALS co-located with CD68 (D) (Scale bars = one thousand μm). The foursquare indicates the area where subsequent CD68 and P2X₇images were taken from. B) And East) Microglial-similar immunostaining with CD68 antibody (Scale bars = 200 μm and 100 μm respectively). C) And F) microglial jail cell/macrophage-like immunostaining with P2X₇(Scale bars = 200 μm and 100 μm respectively).

**Figure 14**
P2X_sevenis increased in the dorsolateral white matter of ALS spinal cord. Mean % area of P2X_sevenimmunoreactivity in spinal cord (open box, control, shaded ALS) * P = 0.0001.

**Effigy 15**
Co-localisation studies of COX-2 and P2X₇in ALS spinal cord. Double staining of COX-2 or P2X₇cells with ferritin (microglia marker) in ALS spinal cord. Section incubated with mixture of A) ferritin (black) and COX-2 (red) antibodies or B) ferritin (black) and P2X_vii(crimson). All COX-2 or P2X_sevenimmunoreactive cells are microglial cells/macrophages, scale bars = 100 μm.

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