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Diapedesis of monocytes is associated with MMP-mediated occludin disappearance in brain endothelial cells

Neuroimmunology Research Group, Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

¹Correspondence: Neuroimmunology Research Group, Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: a.reijerkerk{at}vumc.nl

SPECIFIC AIM

The blood-brain barrier (BBB), a selective barrier formed by endothelial cells and dependent on the presence of intercellular tight junctions, is compromised during neuroinflammation. Immune cell trafficking from blood into the brain may depend on mechanisms that regulate tight junction opening. A detailed study of tight junction dynamics during immune cell diapedesis has been lacking. Therefore, we have generated a brain endothelial cell line expressing green fluorescent protein-labeled occludin, a regulatory tight junction protein. The behavior and regulation of occludin during monocyte diapedesis was studied by means of live imaging.

PRINCIPAL FINDINGS

1. Occludin is localized at cell junctions when expressed in rat brain endothelial cells
To gain insight into tight junction dynamics involved in monocyte diapedesis, we generated a brain endothelial cell line that stably expressed occludin N-terminally tagged to enhanced green fluorescence protein (enhanced GFP). We used the brain endothelial cell line GP8/3.9, which normally expresses low levels of occludin. Immunoblotting analysis using anti-occludin and anti-GFP antibodies showed that GFP-occludin migrates as a single molecular mass with the expected size of 90 kDa (Fig. 1 B). Confocal laser scanning microscopy (CLSM) analyses revealed that GFP-occludin was mainly localized at the plasma membrane, associated with intercellular junctions and absent at cell borders lacking apposing cells (Fig. 1A, C-E ). As shown in Fig. 1F-H , immunostaining with anti-ZO-1 polyclonal antibodies demonstrated that GFP-occludin colocalized with the intracellular tight junction protein ZO-1 at cell-cell contacts. To investigate the effect of GFP-occludin expression on barrier function, we determined the paracellular permeability of endothelial monolayers to FITC-dextran, with an average molecular mass of 150 kDa. The GFP-occludin expressing cells showed significantly decreased permeability vs. control cells (Fig. 1I ). Mutant cells retained endothelial properties including cell morphology, proliferation rate, and adhesive capacity. Flow cytometric analysis further indicated normal expression of proteins that are involved in cell-cell interaction and/or transendothelial migration. Expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), the cell activation marker major histocompatibility complex class II (MHC class II), and the endothelial marker von Willebrand factor (vWF) were unaffected by GFP-occludin expression compared with GP8/3.9 brain endothelial cells (Fig. 1J ). In addition, firm adhesion kinetics of NR8383 monocytes to rat brain endothelial cells expressing GFP-occludin was similar to nontransduced control GP8/3.9 cells (Fig. 1K ).

View larger version (41K): [in this window] [in a new window]   Figure 1. Expression and localization of GFP-occludin fusion protein in GP8/3.9 rat brain endothelial cells. A–E) GFP-occludin (in green) preferentially localized at the plasma membrane and was recognized by anti-occludin monoclonal antibody (mAb) (in red). Immunoblotting shows that GFP-occludin migrates as a single molecular mass with the expected size of 90 kDa. GFP-occludin localized at cell-cell contacts and was absent at cell borders lacking apposing cells. F–H) Immunostaining with anti-ZO-1 polyclonal antibody (pAb) (in red) shows that GFP-occludin exactly overlapped with endogenous ZO-1 at cell-cell junctions. I) Expression of ICAM-1, VCAM-1, MHC class II, and vWF as measured by flow cytometry (filled graph: negative control, dashed line: GP8/3.9, solid line: GP8-GFP-occludin) and J) firm adhesion of monocytes were unaffected by GFP-occludin (n=4). Bars: 25 µm

2. Diapedesis of monocytes is a paracellular event and is associated with occludin disappearance
We used the GFP-occludin expressing endothelial cell line for live observation of occludin dynamics during transendothelial monocyte migration. Endothelial cells were grown on glass coverslips until confluence, incubated with fluorescently labeled monocytes, and migration was followed by CLSM. A comparison of collected images at different time points revealed that adhered monocytes first migrate toward a continuous junction of GFP-occludin and within 30 min induce the formation of a gap between two adjacent endothelial cells. We noted that gap formation is associated with local disappearance of occludin from the plasma membrane of apposing endothelial cells (Fig. 2 A). Our results do not provide evidence for monocyte migration through endothelial cells, as 3-dimensional analysis of x,y sections indicated that monocytes exclusively transmigrated the endothelial cell monolayer through cell-cell junctions. Immunoblotting analysis revealed that monocytes decreased endothelial GFP-occludin levels, which highly correlated with the number of cells added.

View larger version (58K): [in this window] [in a new window]   Figure 2. Transjunctional migration of monocytes is associated with occludin disappearance and regulated by MMPs. A) Time-lapse observation of monocytes (red) migrating through an occludin junction (green) revealed the complete local disappearance of junctional occludin. Time is indicated in minutes. B) Broad-spectrum MMP inhibitor 10 µM BB-3103 prevented monocyte-induced endothelial gap formation and occludin disappearance (time in minutes). Bars: 10 µm. C) Quantification of transjunctional migration. Data are expressed relative to the number of occludin-associated monocytes at 10 min (n=3, *P<0.05 vs. control). D) Inhibition of MMP activity abolished NR8383 monocyte-induced paracellular permeability to FITC-dextran without affecting basal permeability levels (n=4, *P<0.05).

3. Monocyte migration through occludin junctions is regulated by MMPs
The loss of occludin band intensity was indicative of occludin degradation. As a first step to understand the regulatory mechanism underlying occludin disappearance, we examined the role of MMPs in gap formation and monocyte diapedesis. We performed live cell analyses of monocyte migration through occludin junctions in endothelial monolayers in the presence or absence of 10 µM BB-3103, a broad-spectrum inhibitor of MMPs. Our results showed that BB-3103 did not affect monocyte adhesion or the approach of monocytes toward junctional occludin. Strikingly, addition of BB-3103 potently suppressed monocyte-induced loss of occludin, endothelial gap formation, and diapedesis through occludin junctions (Fig. 2B, C ). Analyses of monocyte-induced gap formation by permeability studies pointed to an important role for MMPs in this process. Inhibition of MMP activity abolished monocyte-induced paracellular permeability to FITC-dextran without affecting basal permeability levels (Fig. 2D ). BB-3103 did not block monocyte-induced occludin degradation, suggesting that endothelial gap formation, which is associated with local disappearance of occludin (regulated by MMPs), and occludin degradation (not regulated by MMPs) may be independent processes.

CONCLUSIONS AND SIGNIFICANCE

An important feature of the BBB is the presence of specialized endothelial cells connected by tight junctions. Tight junctions form a primary barrier to prevent cerebral entry of blood-borne macromolecules and immune cells via the paracellular route, and tight junction alterations have been demonstrated in several neuroinflammatory diseases. Occludin is a regulatory component of tight junctions. Here we studied the behavior of occludin during transendothelial migration of monocytes. For this purpose, we generated a rat brain endothelial cell line expressing a GFP-occludin fusion protein, which localized at intercellular junctions along with endogenous ZO-1, another tight junction protein member. Real-time observation of GFP-occludin during monocyte diapedesis revealed that this process is associated with endothelial gap formation and local disappearance of occludin. Moreover, inhibitor studies showed that gap formation and loss of occludin depend on the activity of matrix metalloproteinases.

Abnormal expression of occludin and ZO-1 was seen in inflamed tissue in brains of patients with multiple sclerosis and HIV-1-mediated encephalitis. During experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, claudin-3, is lost in inflamed vessels. Similar results are reported from studies of tight junction function in the blood-retinal barrier. During experimental autoimmune uveoretinitis, disrupted claudin-1/3, occludin, and ZO-1 in retinal venules correlate with sites of leukocyte extravasation. Our results strongly suggest that loss of vascular tight junction proteins observed in animals and patients is mainly due to cellular infiltration into the brain.

Although the process of transendothelial migration has been investigated intensely, the pathway by which inflammatory cells cross the endothelium is still subject to debate. Recently, in vitro and in vivo experiments challenged the idea that leukocytes only extravasate through endothelial cell junctions (the paracellular route) and have shown that leukocytes can also traverse by a route through endothelial cells (the transcellular route). Our studies using brain endothelial cells in an in vitro system did not provide evidence of transcellular migration, but showed that monocytes transmigrated exclusively through cell-cell junctions. Real-time imaging studies of the adherence junction protein vascular endothelial-cadherin by Luskinskas’ group also reported paracellular trafficking of leukocytes across peripheral endothelium. Transcellular diapedesis through human umbilical vein endothelial cells by monocytes, neutrophils, and lymphocytes was only described through tumor necrosis factor -stimulated endothelial cells and treatment with monocyte chemoattractant protein-1, platelet-activating factor, and stromal cell-derived factor-1. We are the first to report on live analyses of monocyte diapedesis through occludin junctions in cultured brain endothelium. Different vascular beds may have distinct preferences for paracellular or transcellular passage of leukocytes.

In the brain, transendothelial migration through the paracellular route must require mechanisms that regulate opening of tight junctions. Several studies point to a crucial role of MMPs in tight junction dynamics. Occludin contains a putative MMP cleavage site in its first extracellular loop, and peptides comprising this loop block occludin-dependent fibroblast aggregation. No direct role of MMPs in monocyte-associated occludin disappearance and endothelial gap formation has been reported before.

Immunoblotting analysis suggested that monocyte-induced disappearance of occludin was due to protein degradation. Data published by Harkness and co-workers show that dexamethasone-induced GP8/3.9 endothelial barrier function is due to decreased MMP-9 activity and mediated by elevated junctional ZO-1 expression in endothelial cells. These results could implicate that occludin disappearance is caused by ZO-1 redistrubution, which is dependent on MMP activity. It is also possible that intracellular signaling processes may have a role in occludin degradation. Evidence suggests that signaling activities may affect tight junctions (in particular, occludin) directly by inducing phosphorylation. Since others have reported that MMPs regulate disruption of cell-cell contacts and proteolysis of occludin in porcine brain capillary endothelial cells treated with phenylarsine oxide and in apoptotic epithelial cells, it will be interesting to investigate whether MMPs play a general role in occludin redistribution.

The described data substantially extend our current knowledge on the pathophysiology of leukocyte diapedesis and the behavior of junctional proteins during this process, and illustrate that compounds that strengthen the tight junction or protect tight junction proteins from enzymatic cleavage are attractive therapeutical candidates and may be beneficial in neurological disorders to resist an inflammatory attack.

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram of monocyte diapedesis through brain endothelium. A) Upon adhesion, monocytes approach endothelial cell-to-cell junctions, and B) induce local redistribution of occludin and C) formation of an endothelial gap. Occludin redistribution and endothelial gap formation involve proteolytic activity of a matrix metalloproteinase.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6099fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6099fjev1 20/14/2550    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Reijerkerk, A. Articles by de Vries, H. E. Search for Related Content PubMed PubMed Citation Articles by Reijerkerk, A. Articles by de Vries, H. E.