Ultra structure analysis of cell-cell interactions between pericytes and neutrophils in vitro.

Neutrophils' adhesion to the endothelium during inflammatory is a well-known processes. In contrast the interaction of neutrophils with cells of the neurovascular unit after they have been transmigrated into the brain is less clear. Recently, lymphocyte function-associated antigen-1 (LFA-1) dependent subendothelial crawling of neutrophils has been observed in vivo. This is mediated by intracellular adhesion molecule-1 (ICAM-1), which is expressed on the cell surface of pericytes. In our work we demonstrated in vitro a cell-cell interaction between porcine brain capillary pericytes (PBCPs) and neutrophils, with further characterization of the initial contact between these cells. PBCPs increase ICAM-1 protein expression in response to the cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, an increase in neutrophil adhesion to PBCPs was determined by immunofluorescence staining. By means of scanning force microscopy (SFM), we could additionally show that pericytes as well as neutrophils form cell extensions towards the neighboring cell. Interestingly, these extensions differ for different cell types.

Brain capillary pericytes contribute to the immune defense in response to cytokines or LPS in vitro.

The prevention of an inflammation in the brain is one of the most important goals the body has to achieve. As pericytes are located on the abluminal side of the capillaries in the brain, their role in fighting against invading pathogens has been investigated in some points, mostly in their ability to behave like macrophages. Here we studied the potential of pericytes to react as immune cells under inflammatory conditions, especially regarding the expression of the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), major histocompatibility complex II (MHC II) molecules, CD68, as well as the generation of reactive oxygen and nitrogen species (RONS), and their ability in phagocytosis. Quantitative real time PCR and western blot analysis showed that pericytes are able to increase the expression of typical inflammatory marker proteins after the stimulation with tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), interferon-gamma (IFN-γ), or lipopolysaccharides (LPS). Depending on the different specific pro-inflammatory factors pericytes changed the expression of alpha smooth muscle actin (αSMA), the most predominant pericyte marker. We conclude that the role of the pericytes within the immune system is regulated and fine-tuned by different cytokines strongly depending on the time when the cytokines are released and their concentration. The present results will help to understand the pericyte mediated defense mechanisms in the brain.

Pericytes support neutrophil transmigration via interleukin-8 across a porcine co-culture model of the blood-brain barrier.

Transmigration of neutrophils across the blood-brain barrier (BBB) to an inflamed brain tissue is an important process during neuronal inflammation. The process of neutrophil activation as well as their way of rolling along the endothelium and their transmigration is quite well understood. Nevertheless, relatively little is known about the fate of neutrophils after they have transmigrated through the endothelium. The role of the other cells of the neurovascular unit in this process is also poorly understood. Here we studied the potential of pericytes to chemo-attract neutrophils under inflammatory conditions. Quantitative real time PCR, western blot analysis, and a chemotaxis assay showed that pericytes are able to chemo-attract neutrophils by interleukin-8 (IL-8) after stimulation with lipopolysaccharides (LPS), tumor necrosis factor-alpha (TNF-α), or interleukin-1beta (IL-1ß). Then, a co-culture model consisting of primary porcine brain capillary endothelial cells (PBCECs) and primary porcine brain capillary pericytes (PBCPs) was used to analyze neutrophil transmigration across the BBB. As a model for inflammation, LPS was used and the effects of the cytokines TNF-α, IL-1ß, and interferon-gamma (IFN-γ) were analyzed. In general, all stimulants apart from IFN-γ were able to increase transendothelial neutrophil migration. This effect was significantly reduced by a specific inhibitor of matrix metalloproteinases (MMPs)-2 and -9. MMP-2/-9 secretion is expected to decrease adhesion to pericytes and thus support the transmigration of neutrophils. Additionally, in an adhesion experiment, we showed that MMP-2/-9 inhibition significantly enhances the adhesion of neutrophils to pericytes.

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage.

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFß treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFß expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFß induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFß pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFß decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria.

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-gamma and lymphotoxin alpha, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4(+) and CD8(+) T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4(+) T cell responses by helminth coinfection amplified CD4(+) T cell-mediated parasite sequestration, whereas vaccination could generate CD4(+) T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Regulation of the blood-brain barrier integrity by pericytes via matrix metalloproteinases mediated activation of vascular endothelial growth factor in vitro.

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes, and neurons. In this study, we analyzed the influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells in a species-consistent in vitro coculture model. We were able to show a barrier integrity-decreasing impact of pericytes by transendothelial electrical resistance (TEER) and (14)C-sucrose permeability measurements. The morphology analysis revealed serrated cell borders and a shift of the endothelial morphology towards a cobblestone shape under the influence of pericytes. The analysis of the two major barrier integrity modulators vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) displayed higher MMP activity and higher levels VEGF, MMP-2, and MMP-9 in the coculture, whereas VEGF levels were decreased by the MMP inhibitor GM6001, indicating a complex interplay of both. Inhibition experiments with neutralizing VEGF antibody and GM6001 increased the TEER, which proves the involvement of VEGF and MMPs in the barrier-decreasing process. Analysis of occludin yielded decreased protein content and discontinuous expression at the endothelial cell borders under the influence of pericytes. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity via MMPs and VEGF.

Metalloproteinase mediated occludin cleavage in the cerebral microcapillary endothelium under pathological conditions.

Reactive oxygen species (ROS) as well as matrix metalloproteinases (MMPs) induce modifications in the tight junction (TJ) protein occludin which is crucial for the blood-brain barrier (BBB) function. We investigated the role of ROS and MMPs in endothelial autoregulatory response on oxidative stress with respect to occludin and the BBB integrity. The ROS hydrogen peroxide (H(2)O(2)) was applied to our well-established BBB cell culture model based on primary porcine brain capillary endothelial cells (PBCEC). At low concentrations (2.5 mM), H(2)O(2)-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H(2)O(2) concentrations (>or=10 mM) occludin cleavage occurred and elevated levels of active MMP-2 were detected. Under those conditions intercellular gaps were formed within the monolayer visualizing the barrier breakdown also determined by impedance analysis. A primary structure sequence analysis revealed potential type IV collagenase sensitive motives in the first extracellular loop, thus providing evidence that occludin might be an MMP-2 substrate. MMP inhibition by the metalloproteinase inhibitor GM6001 prevented occludin degradation and reduced the intercellular gap formation. However, the barrier function quantified by impedance measurement could not be maintained despite MMP inhibition. When we applied an enzymatic activity level which caused occludin cleavage in injured PBCEC to intact PBCEC, neither occludin cleavage nor barrier impairment was observed. Thus, in our model occludin cleavage is not an autoregulatory mechanism of microcapillary endothelium in barrier modulation under oxidative stress, but only occurs upon endothelial damage.