Cyclooxygenase-1 and -2 differentially modulate lipopolysaccharide-induced blood-brain barrier disruption through matrix metalloproteinase activity

Abstract

Cyclooxygenases (COX) -1 and -2 are key regulators of innate immune responses. We recently demonstrated that the expression of proinflammatory cytokines and chemokines is reduced in COX-1 null ((-/-)), and increased in COX-2(-/-) mice compared with their respective wild type controls during lipopolysaccharide (LPS)-induced innate immune activation. As chemokines are involved in leukocyte recruitment into the inflamed brain, we hypothesized that COX-1 and COX-2 deletion will differentially modulate blood-brain barrier (BBB) permeability in response to LPS. In the present study, using quantitative magnetic resonance imaging, we found that LPS-induced BBB disruption was exacerbated in COX-2(-/-) versus COX-2(+/+) mice. In the hippocampus and cortex of LPS-treated mice, matrix metalloproteinase (MMP)-3 activity was significantly decreased in COX-1(-/-) mice, whereas in COX-2(-/-) mice the activity of both MMP-9 and MMP-3, known to mediate BBB breakdown, was increased. Brain mRNA expression of the leukocyte attracting chemokine Cxcl10, the intercellular interaction molecule Icam-1, the pan-leukocyte marker Cd45 was increased in COX-2(-/-) versus COX-2(+/+) mice, whereas Cxcl10 and Cd45 mRNA expression was decreased in COX-1(-/-) versus COX-1(+/+) mice after LPS. Altogether, these results indicate that COX-2 activity modulates MMP-9 and-3 activities and is necessary to maintain BBB integrity during toll-like receptor 4-dependent innate immune activation.

Figure 1

Effects of COX-2 and COX-1 deficiency on LPS-induced increase in BBB permeability, using gadolinium-enhanced magnetic resonance imaging. (A) Representative color-coded permeability coefficient map: LPS-injected COX-2^+/+ mice (top two rows) and LPS-injected COX-2^−/− mice (bottom two rows). Four consecutive 1 mm-thick coronal brain sections are shown for each mouse. (B) Plot of mean permeability coefficient estimates K_i (mL/100g/min) in the whole brain and in the hippocampus obtained by Patlak Plot analysis in LPS-injected COX-2^+/+ (white bars) and LPS-injected COX-2^−/− mice (black bars). (C) Representative color-coded permeability coefficient map: LPS-injected COX-1^+/+ mice (top two rows) and LPS-injected COX-1^−/− mice (bottom two rows). (D) Plot of mean permeability coefficient estimates K_i (mL/100g/min) in the whole brain and in the hippocampus obtained by Patlak Plot analysis in LPS-injected COX-1^+/+ (white bars) and LPS-injected COX-1^−/− mice (black bars). Data are presented as mean±s.e.m. (n=5 to 6). ^***P<0.001, ^**P<0.01 versus LPS-injected respective wild-type mice.

Figure 2

MMP-9 activity in hippocampus and cerebral cortex of COX-1^+/+, COX-1^−/−, COX-2^+/+, and COX-2^−/− mice 24 h after LPS. (A) Representative gelatin zymogram showing the absence of MMP-9 activity in naïve COX-2-deficient and wild-type mice after 48 h incubation of the gels. Only a band around 72 kDa corresponding at MMP-2 was detected. (B) Representative gelatin zymogram showing the effects of COX-1 and COX-2 deletion on MMP-9 activity. Mice were injected intracerebroventricularly with 5 μg LPS. (C–F) Quantitative analysis of gelatinolytic activity of MMP-9 by zymography in hippocampus and cerebral cortex of LPS-injected COX-2^+/+ (n=8), COX-2^−/− (n=5), COX-1^+/+ (n=5), and COX-1^−/− (n=6) mice. Densitometric analysis of lytic zones at 72 kDa, corresponding to pro-MMP-2 activity, showed no significant change among treatments (data not shown). Data are presented as mean±s.e.m. of the 98-kDa band in arbitrary densitometric units. ^*P<0.05 versus LPS-injected respective wild type.

Figure 3

MMP-3 activity in hippocampus and cerebral cortex of COX-1^+/+, COX-1^−/−, COX-2^+/+, and COX-2^−/− mice 24 h after LPS. Hippocampal and cortical MMP-3 activity were similar in vehicle-injected mice for both COX genotypes. LPS-induced increase in MMP-3 activity was higher in COX-2^−/− mice (A, B) whereas it was reduced in COX-1^−/− mice (C, D) as compared with their respective wild-type controls. Data are presented as mean velocity (relative fluorescence unit)±s.e.m. (Vehicle-injected mice n=3 to 4; LPS-injected mice, n=5 to 8). ^***P<0.001 versus vehicle-injected mice; ^##P<0.01, ^###P<0.001 versus LPS-injected wild-type mice.

Figure 4

Effects of COX-2 and COX-1 deficiency on LPS-induced expression of mediators of BBB disruption and regulators of brain infiltration by immune cells. Quantitative real-time-PCR analysis of whole brain Timp-1, Cxcl10, Cd45, Icam-1 for COX-2^+/+, COX-2^−/−, COX-1^+/+, and COX-1^−/− mice 24 h after intracerebroventricular injection of LPS or vehicle. Data are presented as mean±s.e.m. (n=4 to 6). ^**P<0.01 ^***P<0.001 versus vehicle-injected mice; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus LPS-injected wild-type mice.

Figure 5

Distinct effects of COX-1 and -2 deletion on LPS-induced BBB disruption. Lipopolysaccharide (LPS) activates primarily microglia, which releases cytokines, chemokines, MMP-9, and MMP-3. The neuroinflammatory response, through chemokines and cytokines, also propagates to the surrounding cells such as astrocytes and endothelial cells. Chemokines initiate signals that lead to leukocyte arrest, adhesion, and extravasation through the endothelium and perivascular space. The action of MMP-9 and MMP-3, released by activated microglia and leukocytes, allow leukocyte entry into the brain parenchyma by attacking the basement membrane and glia limitans. Although present in all cell types, COX-1 is mainly found in microglia, and COX-2 is predominant in neurons and endothelial cells. COX-1 deletion decreases chemokine expression and MMP-3 activity, which most likely will attenuate LPS-induced increase in BBB permeability. However, COX-2 deletion exacerbates LPS-induced BBB disruption, through an increase in chemokine release and MMP-9 and MMP-3 activities.