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Expression of toll-like receptor 4 in uvea-resident tissue macrophages during endotoxin-induced uveitis.

Chen W, Hu X, Zhao L, Li S, Lu H - Mol. Vis. (2009)

Bottom Line: During the inflammatory response phase (0-48 h after injection), the proportion of CD163+ tissue macrophages having a round morphology increased (p<0.001 by one-way ANOVA) concurrently with a decrease in the proportion of dendritiform CD163+ cells.The results of the present study indicate that TLR4 expression increased in the iris and iris tissue macrophages expressed TLR4 during EIU.This has significant implications for the understanding of ocular inflammation and for interpreting the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.

ABSTRACT

Purpose: To investigate the dynamics and distribution of toll-like receptor 4 (TLR4)-positive cells and resident tissue macrophages in the uvea during endotoxin-induced uveitis (EIU) in Wistar rats.

Methods: Wistar rats (n=40) received a footpad injection of 200 microg of Vibrio cholera lipopolysaccharide (LPS), and the intensity of anterior segment inflammation was evaluated after the LPS injection. Ten rats each were killed 6, 12, 24 and 48 h after injection. Ten normal Wistar rats were killed as controls (0 h). The iris-ciliary body complex and choroids from each eye were removed and subdivided into segments. Immunohistochemical localization of TLR4 and a resident tissue macrophage marker, cluster of differentiation 163 (CD163), was performed on whole mount isolated iris-ciliary body complexes and choroids. TLR4+ and CD163+ cells in the iris were manually counted, and the cell density (cells/mm(2)) was calculated. The distribution patterns and phenotypes of cells expressing these two proteins were further characterized by double-labeled immunofluorescence studies.

Results: The iris-ciliary body complex did not express TLR4 in normal rats. TLR4+ cells were detectable in the iris stroma 6 h after injection, and the number significantly increased (p<0.001 by one-way ANOVA) 12, 24, and 48 h after injection. The morphology of TLR4+ cells hardly changed 12-48 h after injection. CD163 was expressed in the uvea in all rats. During the inflammatory response phase (0-48 h after injection), the proportion of CD163+ tissue macrophages having a round morphology increased (p<0.001 by one-way ANOVA) concurrently with a decrease in the proportion of dendritiform CD163+ cells. These changes occurred mainly in the macrophages located in the stroma bordering the iris endothelial layer. Double-labeling immunofluorescence demonstrated the co-expression of TLR4 and CD163 in round stroma cells with TLR4 located at the cell membrane and CD163 in the cytoplasm. TLR4+ cells could not be detected in choroids in any of the rats.

Conclusions: The results of the present study indicate that TLR4 expression increased in the iris and iris tissue macrophages expressed TLR4 during EIU. This has significant implications for the understanding of ocular inflammation and for interpreting the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis.

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Immunohistochemical studies for TLR4 in iris whole mounts at different times after LPS injection. The dynamics of TLR4+ cells in the iris during EIU are shown (A–D). A: Positive cells could not be detected in the control rats. Arrows represent blood vessels. B: The TLR4+ cells possessed pleiomorphic morphology at 6 h. Note the positive cells (arrowhead) that are located adjacent to blood vessels (arrow). C: Most of the TLR4+ cells possessed round-ovoid morphology at 24 h (arrowhead). Note the positive cells that are located adjacent to blood vessels (arrow). D: The TLR4+ cells at 48 h are shown. The morphology and distribution of the positive cells were similar to that at 24 h. E: No staining was seen in the iris when under identical experimental conditions when replacing of the primary antibody with normal rabbit IgG at the same concentration (negative control). The arrows indicate blood vessels. F–H: Double immunofluorescence by confocal microscopy revealed co-expression (arrow) of TLR4 (green) and CD163 (red) by resident stromal cells in the iris. I: Positive tissue control shows positive staining for TLR4 by a subpopulation of macrophage-like cells in the normal rat spleen. J: A higher power view is shown where the arrow and arrowheads represent the same cells as in panel I. K: No staining was seen in the spleen when using identical experimental conditions but with the replacement of the primary antibody with normal rabbit IgG at the same concentration (negative control). Original magnification: B–D,F–H,J 400X; A,E,I,K 200X.
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f3: Immunohistochemical studies for TLR4 in iris whole mounts at different times after LPS injection. The dynamics of TLR4+ cells in the iris during EIU are shown (A–D). A: Positive cells could not be detected in the control rats. Arrows represent blood vessels. B: The TLR4+ cells possessed pleiomorphic morphology at 6 h. Note the positive cells (arrowhead) that are located adjacent to blood vessels (arrow). C: Most of the TLR4+ cells possessed round-ovoid morphology at 24 h (arrowhead). Note the positive cells that are located adjacent to blood vessels (arrow). D: The TLR4+ cells at 48 h are shown. The morphology and distribution of the positive cells were similar to that at 24 h. E: No staining was seen in the iris when under identical experimental conditions when replacing of the primary antibody with normal rabbit IgG at the same concentration (negative control). The arrows indicate blood vessels. F–H: Double immunofluorescence by confocal microscopy revealed co-expression (arrow) of TLR4 (green) and CD163 (red) by resident stromal cells in the iris. I: Positive tissue control shows positive staining for TLR4 by a subpopulation of macrophage-like cells in the normal rat spleen. J: A higher power view is shown where the arrow and arrowheads represent the same cells as in panel I. K: No staining was seen in the spleen when using identical experimental conditions but with the replacement of the primary antibody with normal rabbit IgG at the same concentration (negative control). Original magnification: B–D,F–H,J 400X; A,E,I,K 200X.

Mentions: Lipopolysaccharides can upregulate the expression of TLR4. TLR4 could not be detected in the iris–ciliary body complex in the control group (0 h). At 6 h, a small number of TLR4+ cells were detected in the irises of two rats. The weakly positive cells exhibited pleiomorphic morphology and were located near blood vessels (Figure 3B). The number of TLR4+ cells increased significantly (p<0.001) in the iris and ciliary body of all rats at 12 h with a density of 506±39 cells/mm2 (Figure 2B), and the immunopositive cells were predominantly round-ovoid cells (Figure 3F) whose morphology was hardly altered between 12 and 48 h after the LPS injection (Figure 3C,D). The TLR4+ cells were mainly distributed in the stroma of the iris and in the perivascular location. TLR4 could not be detected in the choroid of any Wistar rats.


Expression of toll-like receptor 4 in uvea-resident tissue macrophages during endotoxin-induced uveitis.

Chen W, Hu X, Zhao L, Li S, Lu H - Mol. Vis. (2009)

Immunohistochemical studies for TLR4 in iris whole mounts at different times after LPS injection. The dynamics of TLR4+ cells in the iris during EIU are shown (A–D). A: Positive cells could not be detected in the control rats. Arrows represent blood vessels. B: The TLR4+ cells possessed pleiomorphic morphology at 6 h. Note the positive cells (arrowhead) that are located adjacent to blood vessels (arrow). C: Most of the TLR4+ cells possessed round-ovoid morphology at 24 h (arrowhead). Note the positive cells that are located adjacent to blood vessels (arrow). D: The TLR4+ cells at 48 h are shown. The morphology and distribution of the positive cells were similar to that at 24 h. E: No staining was seen in the iris when under identical experimental conditions when replacing of the primary antibody with normal rabbit IgG at the same concentration (negative control). The arrows indicate blood vessels. F–H: Double immunofluorescence by confocal microscopy revealed co-expression (arrow) of TLR4 (green) and CD163 (red) by resident stromal cells in the iris. I: Positive tissue control shows positive staining for TLR4 by a subpopulation of macrophage-like cells in the normal rat spleen. J: A higher power view is shown where the arrow and arrowheads represent the same cells as in panel I. K: No staining was seen in the spleen when using identical experimental conditions but with the replacement of the primary antibody with normal rabbit IgG at the same concentration (negative control). Original magnification: B–D,F–H,J 400X; A,E,I,K 200X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2664840&req=5

f3: Immunohistochemical studies for TLR4 in iris whole mounts at different times after LPS injection. The dynamics of TLR4+ cells in the iris during EIU are shown (A–D). A: Positive cells could not be detected in the control rats. Arrows represent blood vessels. B: The TLR4+ cells possessed pleiomorphic morphology at 6 h. Note the positive cells (arrowhead) that are located adjacent to blood vessels (arrow). C: Most of the TLR4+ cells possessed round-ovoid morphology at 24 h (arrowhead). Note the positive cells that are located adjacent to blood vessels (arrow). D: The TLR4+ cells at 48 h are shown. The morphology and distribution of the positive cells were similar to that at 24 h. E: No staining was seen in the iris when under identical experimental conditions when replacing of the primary antibody with normal rabbit IgG at the same concentration (negative control). The arrows indicate blood vessels. F–H: Double immunofluorescence by confocal microscopy revealed co-expression (arrow) of TLR4 (green) and CD163 (red) by resident stromal cells in the iris. I: Positive tissue control shows positive staining for TLR4 by a subpopulation of macrophage-like cells in the normal rat spleen. J: A higher power view is shown where the arrow and arrowheads represent the same cells as in panel I. K: No staining was seen in the spleen when using identical experimental conditions but with the replacement of the primary antibody with normal rabbit IgG at the same concentration (negative control). Original magnification: B–D,F–H,J 400X; A,E,I,K 200X.
Mentions: Lipopolysaccharides can upregulate the expression of TLR4. TLR4 could not be detected in the iris–ciliary body complex in the control group (0 h). At 6 h, a small number of TLR4+ cells were detected in the irises of two rats. The weakly positive cells exhibited pleiomorphic morphology and were located near blood vessels (Figure 3B). The number of TLR4+ cells increased significantly (p<0.001) in the iris and ciliary body of all rats at 12 h with a density of 506±39 cells/mm2 (Figure 2B), and the immunopositive cells were predominantly round-ovoid cells (Figure 3F) whose morphology was hardly altered between 12 and 48 h after the LPS injection (Figure 3C,D). The TLR4+ cells were mainly distributed in the stroma of the iris and in the perivascular location. TLR4 could not be detected in the choroid of any Wistar rats.

Bottom Line: During the inflammatory response phase (0-48 h after injection), the proportion of CD163+ tissue macrophages having a round morphology increased (p<0.001 by one-way ANOVA) concurrently with a decrease in the proportion of dendritiform CD163+ cells.The results of the present study indicate that TLR4 expression increased in the iris and iris tissue macrophages expressed TLR4 during EIU.This has significant implications for the understanding of ocular inflammation and for interpreting the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.

ABSTRACT

Purpose: To investigate the dynamics and distribution of toll-like receptor 4 (TLR4)-positive cells and resident tissue macrophages in the uvea during endotoxin-induced uveitis (EIU) in Wistar rats.

Methods: Wistar rats (n=40) received a footpad injection of 200 microg of Vibrio cholera lipopolysaccharide (LPS), and the intensity of anterior segment inflammation was evaluated after the LPS injection. Ten rats each were killed 6, 12, 24 and 48 h after injection. Ten normal Wistar rats were killed as controls (0 h). The iris-ciliary body complex and choroids from each eye were removed and subdivided into segments. Immunohistochemical localization of TLR4 and a resident tissue macrophage marker, cluster of differentiation 163 (CD163), was performed on whole mount isolated iris-ciliary body complexes and choroids. TLR4+ and CD163+ cells in the iris were manually counted, and the cell density (cells/mm(2)) was calculated. The distribution patterns and phenotypes of cells expressing these two proteins were further characterized by double-labeled immunofluorescence studies.

Results: The iris-ciliary body complex did not express TLR4 in normal rats. TLR4+ cells were detectable in the iris stroma 6 h after injection, and the number significantly increased (p<0.001 by one-way ANOVA) 12, 24, and 48 h after injection. The morphology of TLR4+ cells hardly changed 12-48 h after injection. CD163 was expressed in the uvea in all rats. During the inflammatory response phase (0-48 h after injection), the proportion of CD163+ tissue macrophages having a round morphology increased (p<0.001 by one-way ANOVA) concurrently with a decrease in the proportion of dendritiform CD163+ cells. These changes occurred mainly in the macrophages located in the stroma bordering the iris endothelial layer. Double-labeling immunofluorescence demonstrated the co-expression of TLR4 and CD163 in round stroma cells with TLR4 located at the cell membrane and CD163 in the cytoplasm. TLR4+ cells could not be detected in choroids in any of the rats.

Conclusions: The results of the present study indicate that TLR4 expression increased in the iris and iris tissue macrophages expressed TLR4 during EIU. This has significant implications for the understanding of ocular inflammation and for interpreting the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis.

Show MeSH
Related in: MedlinePlus