Contributions
Abstract: EP1489
Type: ePoster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Recruitment of myelin-reactive CD4+ T cells to the CNS across the blood-brain barrier (BBB) is considered critical in the pathophysiology of multiple sclerosis (MS). There is emerging evidence, however, that also innate immune cells contribute to MS pathophysiology. Levels of the neutrophil-attracting chemokines CXCL5 and CXCL8 are elevated in plasma and cerebrospinal fluid of patients with MS and CXCL5 levels positively correlate with disease activity. Interestingly, CXCR2, a high affinity receptor for CXCL5 and CXCL8, is not only expressed on leucocytes, but also on non-hematopoietic cells including endothelium. This prompted us to evaluate the expression and function of CXCR2 in human brain endothelium.
Methods and results: Here we demonstrate that resting human brain endothelial cells (hCMEC/D3 cell line) express low levels of CXCR2. Both in response to inflammatory stimulation with TNFα, IL1β or TNFα + IFNγ as well as after hypoxia (1% O2), CXCR2 protein expression is up-regulated, as demonstrated by Western blot analysis. This is preceded by a corresponding increase in CXCR2 mRNA levels. For label-free real-time assessment of transendothelial resistance, cells were cultivated in an ACEA xCELLigence DP system, until they formed a confluent monolayer with tight junction formation, as indicated by a stable plateau of the cell index. Physiologically relevant, low nanomolar concentrations of recombinant CXCL5 and CXCL8 caused a dose-dependent and long-lasting decrease in transendothelial resistance of resting cells. This barrier-perturbing effect was further pronounced under inflammatory conditions and attenuated by pre-incubation with a specific CXCR2 inhibitor, suggesting a CXCR2-dependent mechanism.
Conclusions: CXCR2 is expressed on human brain endothelium and its expression is highly inducible by inflammatory stimuli or hypoxia. The neutrophil-attracting CXC chemokines CXCL5 and CXCL8 perturb barrier function of human brain endothelial cells via CXCR2. This mechanism may contribute to BBB breakdown and lesion formation in MS and represent a novel therapeutic target.
Disclosure:
Axel Haarmann, Michael Schuhmann, Guido Stoll and Mathias Buttmann have nothing to disclose.
Abstract: EP1489
Type: ePoster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Recruitment of myelin-reactive CD4+ T cells to the CNS across the blood-brain barrier (BBB) is considered critical in the pathophysiology of multiple sclerosis (MS). There is emerging evidence, however, that also innate immune cells contribute to MS pathophysiology. Levels of the neutrophil-attracting chemokines CXCL5 and CXCL8 are elevated in plasma and cerebrospinal fluid of patients with MS and CXCL5 levels positively correlate with disease activity. Interestingly, CXCR2, a high affinity receptor for CXCL5 and CXCL8, is not only expressed on leucocytes, but also on non-hematopoietic cells including endothelium. This prompted us to evaluate the expression and function of CXCR2 in human brain endothelium.
Methods and results: Here we demonstrate that resting human brain endothelial cells (hCMEC/D3 cell line) express low levels of CXCR2. Both in response to inflammatory stimulation with TNFα, IL1β or TNFα + IFNγ as well as after hypoxia (1% O2), CXCR2 protein expression is up-regulated, as demonstrated by Western blot analysis. This is preceded by a corresponding increase in CXCR2 mRNA levels. For label-free real-time assessment of transendothelial resistance, cells were cultivated in an ACEA xCELLigence DP system, until they formed a confluent monolayer with tight junction formation, as indicated by a stable plateau of the cell index. Physiologically relevant, low nanomolar concentrations of recombinant CXCL5 and CXCL8 caused a dose-dependent and long-lasting decrease in transendothelial resistance of resting cells. This barrier-perturbing effect was further pronounced under inflammatory conditions and attenuated by pre-incubation with a specific CXCR2 inhibitor, suggesting a CXCR2-dependent mechanism.
Conclusions: CXCR2 is expressed on human brain endothelium and its expression is highly inducible by inflammatory stimuli or hypoxia. The neutrophil-attracting CXC chemokines CXCL5 and CXCL8 perturb barrier function of human brain endothelial cells via CXCR2. This mechanism may contribute to BBB breakdown and lesion formation in MS and represent a novel therapeutic target.
Disclosure:
Axel Haarmann, Michael Schuhmann, Guido Stoll and Mathias Buttmann have nothing to disclose.