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. 2010 Jan 4:7:1.
doi: 10.1186/1742-2094-7-1.

Regulation of CCL2 and CCL3 expression in human brain endothelial cells by cytokines and lipopolysaccharide

Ray Chui  1 , Katerina Dorovini-Zis
Affiliations

Regulation of CCL2 and CCL3 expression in human brain endothelial cells by cytokines and lipopolysaccharide

Ray Chui et al. J Neuroinflammation. .

Abstract

Background: Chemokines are emerging as important mediators of CNS inflammation capable of activating leukocyte integrins and directing the migration of leukocyte subsets to sites of antigenic challenge. In this study we investigated the expression, release and binding of CCL2 (MCP-1) and CCL3 (MIP-1alpha) in an in vitro model of the human blood-brain barrier.

Methods: The kinetics of expression and cytokine upregulation and release of the beta-chemokines CCL2 and CCL3 were studied by immunocytochemistry and enzyme-linked immunosorbent assay in primary cultures of human brain microvessel endothelial cells (HBMEC). In addition, the differential binding of these chemokines to the basal and apical endothelial cell surfaces was assessed by immunoelectron microscopy.

Results: Untreated HBMEC synthesize and release low levels of CCL2. CCL3 is minimally expressed, but not released by resting HBMEC. Treatment with TNF-alpha, IL-1beta, LPS and a combination of TNF-alpha and IFN-gamma, but not IFN-gamma alone, significantly upregulated the expression and release of both chemokines in a time-dependent manner. The released CCL2 and CCL3 bound to the apical and basal endothelial surfaces, respectively. This distribution was reversed in cytokine-activated HBMEC resulting in a predominantly basal localization of CCL2 and apical distribution of CCL3.

Conclusions: Since cerebral endothelial cells are the first resident CNS cells to contact circulating leukocytes, expression, release and presentation of CCL2 and CCL3 on cerebral endothelium suggests an important role for these chemokines in regulating the trafficking of inflammatory cells across the BBB in CNS inflammation.

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Figures

Figure 1
Figure 1
RNA expression of CCL2 (A) and CCL3 (B) by HBMEC determined by semi-quantitative RT-PCR. Increasing amounts of RNA (not shown) were used to detect differential expression in unstimulated HBMEC and cells treated with 100 U/ml TNF-α and 500 U/ml IFN-γ for 24 hrs. After amplification with gene-specific primers, CCL2 or CCL3 and GAPDH PCR samples were run on a 2% agarose gel after 35 and 25 cycles respectively, under the following conditions: pre-PCR step (94°C for 8 min, 55 - 60°C for 30 sec and 72°C for 3 min) and cycling (94°C for 1 min, 55 - 60°C for 30 sec and 72°C for 45 sec). The data shown are from one of three experiments for each chemokine with similar results.
Figure 2
Figure 2
Intracellular localization of CCL2 in HBMEC by immunogold silver staining. (A) Unstimulated HBMEC constitutively express CCL2 as shown by the positive cytoplasmic staining in the form of fine, granular black deposits of silver-enhanced gold particles. Nuclei are counterstained with Giemsa. (B)-(D) The intensity of staining is markedly increased following incubation with (B) TNF-α (10 U/ml) for 48 hrs, (C) IL-1β (10 U/ml) for 72 hrs and (D) LPS (5 μg/ml) for 48 hrs. (E) Control cultures incubated with secondary antibody only show no staining. Scale bars = 50 μm.
Figure 3
Figure 3
Intracellular localization of CCL3 in HBMEC by immunogold silver staining. (A) In untreated cultures, cytoplasmic staining is faint indicating minimal constitutive expression. Nuclei are counterstained with Giemsa. (B)-(D) The density of the granular black cytoplasmic deposits is increased in cultures treated with (B) TNF-α (100 U/ml)+ IFN-γ (200 U/ml) for 72 hrs, (C) IL-1β (10 U/ml) for 72 hrs and (D) LPS (5 μg/ml) for 48 hrs. (E) Control cultures incubated with secondary antibody only show no staining. Scale bars = 50 μm.
Figure 4
Figure 4
Quantitation of CCL2 and CCL3 release by resting and cytokine stimulated HBMEC by ELISA. (A) Detection of CCL2 protein in supernatants of HBMEC cultures under resting conditions and following incubation with cytokines and LPS. Chemokine release was determined by sandwich ELISA at the indicated time intervals. Values represent mean release (pg/ml) ± SEM (n = 3). Tukey test p ≤ 0.001; * p < 0.05 as compared to unstimulated HBMEC. Values shown represent one of two independent, representative experiments. (B) Detection of CCL3 protein in supernatants of resting HBMEC cultures and monolayers treated with cytokines and LPS. Chemokine release was determined by sandwich ELISA at the indicated time intervals. Values represent mean release (pg/ml) ± SEM (n = 3). ANOVA p ≤ 0.001; * p < 0.05 as compared to unstimulated HBMEC. Values shown are the results of one of two independent, representative experiments.
Figure 5
Figure 5
Surface localization of CCL2 and CCL3 by immunoelectron microscopy. (D, E) Surface localization of CCL2 on HBMEC by immunoelectron microscopy. (A) In resting monolayers, gold particles indicating the presence of CCL2 are bound mostly to the apical cell surface (arrow). (B) In cultures treated with TNF-α (100 U/ml) + IFN-γ (500 U/ml) for 24 hrs gold particles are preferentially bound to the subendothelial basal lamina-like material (arrows). Arrowheads in (A) and (B) indicate the basal cell surface. Scale bars = 0.25 μm. (C) Quantification of the number of gold particles bound to the apical and basal cell surface of unstimulated HBMEC shows no significant difference between apical and basal binding (p ≥ 0.1). In cytokine-treated HBMEC cultures, there is a significant increase in the number of gold particles bound to the basal cell surface and the basal lamina-like material versus the apical surface (p ≤ 0.01). The number of gold particles at the basal cell surface is significantly greater in activated versus resting HBMEC (p ≤ 0.01). (D, E) Surface localization of CCL3 on HBMEC by immunoelectron microscopy. (A) In resting monolayers occasional gold particles are bound to the basal cell surface only (arrow). (B) Following treatment with TNF-α (100 U/ml) + IFNγ (500 U/ml) for 24 hrs gold particles are preferentially bound to the apical surface (arrow). Arrowheads in (A) and (B) indicate the basal cell surface. Scale bars = 0.25 μm. (F) Quantification of the number of gold particles bound to the apical and basal cell surfaces of unstimulated and cytokine-treated HBMEC cultures shows no statistically significant differences between the different groups (p ≥ 0.15).
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