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Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro.

Furtado JM, Bharadwaj AS, Chipps TJ, Pan Y, Ashander LM, Smith JR - Immunol. Cell Biol. (2012)

Bottom Line: Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains.Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye.In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT
Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

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ICAM-1 blockade significantly inhibits Toxoplasma gondii tachyzoite migration across simulated human retinal, but not choroidal, vascular endotheliumGraphs showing number of tachyzoites recovered from lower chambers of transwells divided by (A and B) human retinal endothelial cell monolayers or (C) human choroidal endothelial cell monolayerscultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = A, 55%; B, 52%;C, 95%). Monolayers were pre-incubated for 2 hours with (A and C) mouseanti-human ICAM-1 IgG1 (α-ICAM-1) or (B) mouse anti-human VCAM-1 IgG1 (α-VCAM-1), or control mouse IgG1 (control). There was a significant reduction in tachyzoite migration across the retinal endothelial monolayer in the presence of anti-ICAM-1 antibody (***p < 0.001), but not anti-VCAM-1 antibody (p > 0.05) (Student’s t-test). Anti-ICAM-1 antibody did not reduce tachyzoite migration across the choroidal endothelial monolayer (p > 0.05) (Student’s t-test). Each graph presents results that are representative of two independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
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Figure 2: ICAM-1 blockade significantly inhibits Toxoplasma gondii tachyzoite migration across simulated human retinal, but not choroidal, vascular endotheliumGraphs showing number of tachyzoites recovered from lower chambers of transwells divided by (A and B) human retinal endothelial cell monolayers or (C) human choroidal endothelial cell monolayerscultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = A, 55%; B, 52%;C, 95%). Monolayers were pre-incubated for 2 hours with (A and C) mouseanti-human ICAM-1 IgG1 (α-ICAM-1) or (B) mouse anti-human VCAM-1 IgG1 (α-VCAM-1), or control mouse IgG1 (control). There was a significant reduction in tachyzoite migration across the retinal endothelial monolayer in the presence of anti-ICAM-1 antibody (***p < 0.001), but not anti-VCAM-1 antibody (p > 0.05) (Student’s t-test). Anti-ICAM-1 antibody did not reduce tachyzoite migration across the choroidal endothelial monolayer (p > 0.05) (Student’s t-test). Each graph presents results that are representative of two independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.

Mentions: To investigate a possible role for the cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, in migration of T. gondii tachyzoites across human retinal vascular endothelium, we performed endothelial transmigration assays after pre-incubating endothelial monolayers with anti-human ICAM-1 antibody or control immunoglobulin G1 (IgG1). ICAM-1 blockade significantly reduced tachyzoite movement across the endothelium by approximately 50% (Figure 2A, p < 0.001). In contrast, specific antibody blockade of the related cell adhesion molecule, vascular cell adhesion molecule (VCAM)-1, did not significantly impact tachyzoite migration (Figure 2B, p > 0.05). To determine whether ICAM-1-mediated endothelial transmigration within the eye was specific to retina, we performed the same experiment, but seeded the transwell membrane with endothelial cells that were isolated from choroid, in place of the retina. Choroid is the ocular tissue that encircles the retina. Consistent with specificity of the retinal endothelial interaction, ICAM-1 antibody blockade did not impact tachyzoite migration across simulated human choroidal vascular endothelium (Figure 2C, p > 0.05). When studies were repeated, we observed variation in absolute numbers of migrated parasites, but relationships between experimental conditions remained consistent.


Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro.

Furtado JM, Bharadwaj AS, Chipps TJ, Pan Y, Ashander LM, Smith JR - Immunol. Cell Biol. (2012)

ICAM-1 blockade significantly inhibits Toxoplasma gondii tachyzoite migration across simulated human retinal, but not choroidal, vascular endotheliumGraphs showing number of tachyzoites recovered from lower chambers of transwells divided by (A and B) human retinal endothelial cell monolayers or (C) human choroidal endothelial cell monolayerscultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = A, 55%; B, 52%;C, 95%). Monolayers were pre-incubated for 2 hours with (A and C) mouseanti-human ICAM-1 IgG1 (α-ICAM-1) or (B) mouse anti-human VCAM-1 IgG1 (α-VCAM-1), or control mouse IgG1 (control). There was a significant reduction in tachyzoite migration across the retinal endothelial monolayer in the presence of anti-ICAM-1 antibody (***p < 0.001), but not anti-VCAM-1 antibody (p > 0.05) (Student’s t-test). Anti-ICAM-1 antibody did not reduce tachyzoite migration across the choroidal endothelial monolayer (p > 0.05) (Student’s t-test). Each graph presents results that are representative of two independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
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Related In: Results  -  Collection

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Figure 2: ICAM-1 blockade significantly inhibits Toxoplasma gondii tachyzoite migration across simulated human retinal, but not choroidal, vascular endotheliumGraphs showing number of tachyzoites recovered from lower chambers of transwells divided by (A and B) human retinal endothelial cell monolayers or (C) human choroidal endothelial cell monolayerscultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = A, 55%; B, 52%;C, 95%). Monolayers were pre-incubated for 2 hours with (A and C) mouseanti-human ICAM-1 IgG1 (α-ICAM-1) or (B) mouse anti-human VCAM-1 IgG1 (α-VCAM-1), or control mouse IgG1 (control). There was a significant reduction in tachyzoite migration across the retinal endothelial monolayer in the presence of anti-ICAM-1 antibody (***p < 0.001), but not anti-VCAM-1 antibody (p > 0.05) (Student’s t-test). Anti-ICAM-1 antibody did not reduce tachyzoite migration across the choroidal endothelial monolayer (p > 0.05) (Student’s t-test). Each graph presents results that are representative of two independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
Mentions: To investigate a possible role for the cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, in migration of T. gondii tachyzoites across human retinal vascular endothelium, we performed endothelial transmigration assays after pre-incubating endothelial monolayers with anti-human ICAM-1 antibody or control immunoglobulin G1 (IgG1). ICAM-1 blockade significantly reduced tachyzoite movement across the endothelium by approximately 50% (Figure 2A, p < 0.001). In contrast, specific antibody blockade of the related cell adhesion molecule, vascular cell adhesion molecule (VCAM)-1, did not significantly impact tachyzoite migration (Figure 2B, p > 0.05). To determine whether ICAM-1-mediated endothelial transmigration within the eye was specific to retina, we performed the same experiment, but seeded the transwell membrane with endothelial cells that were isolated from choroid, in place of the retina. Choroid is the ocular tissue that encircles the retina. Consistent with specificity of the retinal endothelial interaction, ICAM-1 antibody blockade did not impact tachyzoite migration across simulated human choroidal vascular endothelium (Figure 2C, p > 0.05). When studies were repeated, we observed variation in absolute numbers of migrated parasites, but relationships between experimental conditions remained consistent.

Bottom Line: Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains.Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye.In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT
Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

Show MeSH
Related in: MedlinePlus