Cyclooxygenases-1 and -2 differentially modulate leukocyte recruitment into the inflamed brain

Abstract

Peripheral leukocyte recruitment in neuroinflammatory conditions can exacerbate brain tissue damage by releasing cytotoxic mediators and by increasing vascular permeability. Cyclooxygenase (COX)-derived prostaglandins promote the migration of several immune cells in vitro, however, the specific roles of COX-1 and -2 on leukocyte recruitment in vivo have not been investigated. To examine the specific effects of COX-1 or COX-2 deficiency on neuroinflammation-induced leukocyte infiltration, we used a model of intracerebroventricular lipopolysaccharide (LPS)-induced neuroinflammation in COX-1(-/-), COX-2(-/-), and their respective wild-type (WT) ((+/+)) mice. After LPS, leukocyte infiltration and inflammatory response were attenuated in COX-1(-/-) and increased in COX-2(-/-) mice, compared with their respective WT controls. This influx of leukocytes was accompanied by a marked disruption of blood-brain barrier and differential expression of chemokines. These results indicate that COX-1 and COX-2 deletion differentially modulate leukocyte recruitment during neuroinflammation, and suggest that inhibition of COX-1 activity is beneficial, whereas COX-2 inhibition is detrimental, during a primary neuroinflammatory response.

Figure 1

I.c.v. LPS induces microglial activation and leukocyte infiltration. (a, b) Iba-1 immunohistochemistry revealed highly ramified microglia in vehicle-injected mice (a) compared with increased microglial activation in LPS-injected mice whose cells have shorter and thicker processes and bigger cell bodies (b). (c–f) MPO and 7/4 immunostaining showed numerous neutrophils in LPS-injected mice (d, f), but not in vehicle-injected mice (c, e). (g–i) LPS led to the appearance of numerous 7/4⁺ neutrophils around meninges (g), parenchymal vessel (h), and choroid plexus (i). Scale bar, 100 μm.

Figure 2

Neutrophil infiltration in COX-1^−/−, COX-2^−/−, and their WT mice. (a–d) Immunohistochemistry of brain sections with 7/4 antibody. Scale bar, 100 μm. (e) Quantitation of 7/4⁺ cells showed a significant increase of infiltrated neutrophils in COX-2^−/− mice, but not in COX-1^−/− mice compared with their respective WT mice. (f) Brain MPO levels were decreased in COX-1^−/− mice and increased in COX-2^−/− mice compared with their WT controls. Mean±s.e.m. (n = 5–6 per group); *P<0.05, **P<0.01.

Figure 3

COX-2 deficiency exacerbates neutrophil infiltration. (a, b) Representative dot plots of flow cytometry of brain CD11b⁺ and CD45⁺ cells (a), and CD45⁺ and Gr-1⁺ cells (b) from WT and COX-2^−/− mice injected with LPS or vehicle. (c, d) Quantitation of the percentage of brain CD11b⁺CD45^high (c) and CD45^highGr-1^high cells (d) from COX-1^−/− and WT mice. (e, f) Quantitation of the percentage of brain CD11b⁺CD45^high (e) and CD45^highGr-1^high cells (f) from COX-2^−/− and WT mice. n = 3–6 per group; **P<0.01 versus vehicle-injected WT mice; ^#P<0.05, ^##P<0.01 versus LPS-injected WT mice.

Figure 4

PMN depletion reduces neutrophil infiltration, MPO, and IL-1β levels. (a, b) Representative dot plots of flow cytometry of brain CD11b⁺ and CD45⁺ cells (a), and CD45⁺ and Gr-1⁺ cells (b) from IgG-treated or PMN-depleted mice injected with LPS or vehicle. (c, d) Quantitation of the percentage of brain CD11b⁺CD45^high (c) and CD45^highGr-1^high cells (d) from PMN-depleted or IgG-treated mice. (e, f) Brain MPO (e) and IL-1β levels (f) by ELISA revealed a significant decrease in PMN-depleted mice compared with IgG-treated mice. Mean±s.e.m. (n = 5–6 per group); *P<0.05, **P<0.01 versus vehicle-injected mice with IgG treatment; ^#P<0.05, ^##P<0.01 versus LPS-injected mice with IgG treatment.

Figure 5

COX-2 deletion exacerbates BBB permeability and chemokine levels. (a–h) Representative brain sections from COX-1^−/−, COX-2^−/−, and their WT mice injected with LPS or vehicle and immunostained with IgG antibody. COX-1 and COX-2 deficiency differentially modulated the intensity and distribution of IgG immunoreactivity after LPS injection. Scale bar, 100 μm. (i–l) Plasma CCL2 (i), CXCL2 (j), brain CCL2 (k), and CXCL2 levels (l) by ELISA revealed a significant increase in COX-2^−/− mice compared with WT mice. Mean±s.e.m. (n = 5–6 per group); *P<0.05, **P<0.01 versus vehicle-injected WT mice; ^#P<0.05, ^##P<0.01 versus LPS-injected WT mice.