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Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

Rutar M, Natoli R, Chia RX, Valter K, Provis JM - J Neuroinflammation (2015)

Bottom Line: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10.In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE).This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Canberra, ACT 2601, Australia. matt.rutar@anu.edu.au.

ABSTRACT

Background: Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration.

Methods: Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR.

Results: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

Conclusions: Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

No MeSH data available.


Related in: MedlinePlus

In situhybridization for Ccl3 and Ccl4 mRNA in the retina following exposure to 24 hours of light damage.In situ hybridization for Ccl3 is documented in A-I, while Ccl4 is shown in J-R. A: In retinas from dim-reared animals, staining for Ccl3 was not observed in the retina. B-D: After 24 hrs light damage (LD), there was staining for Ccl3 in irregularly shaped nuclei - ranging from elongated to globular - traversing the outer nuclear layer (ONL) of the superior retina (arrowheads). Conversely, few Cc3-expressing cells were found in the inferior retina (data not shown). E-I: Nuclei stained for Ccl3 mRNA (red) showed strong correlation (arrowheads) to activated microglia, which were counterimmunolabeled with an antibody to IBA1 (green). J: Ccl4 mRNA staining was not apparent in sections of dim-reared retinas. J-M: At 24 hrs, LD staining for Ccl4 was detected among clusters of irregular nuclei in the ONL from the superior retina (arrowheads); little-to-none were observed in the inferior retina (data not shown). N-R: Ccl4-expressing nuclei (red) in retinal sections show strong immunofluorescence (arrowheads) for IBA1+ microglia (green), much like Ccl3 (E-I). INL, inner nuclear layer; IHC, immunohistochemistry; ISH, in situ hybridization; ONL, outer nuclear layer; OS, outer segments.
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Fig3: In situhybridization for Ccl3 and Ccl4 mRNA in the retina following exposure to 24 hours of light damage.In situ hybridization for Ccl3 is documented in A-I, while Ccl4 is shown in J-R. A: In retinas from dim-reared animals, staining for Ccl3 was not observed in the retina. B-D: After 24 hrs light damage (LD), there was staining for Ccl3 in irregularly shaped nuclei - ranging from elongated to globular - traversing the outer nuclear layer (ONL) of the superior retina (arrowheads). Conversely, few Cc3-expressing cells were found in the inferior retina (data not shown). E-I: Nuclei stained for Ccl3 mRNA (red) showed strong correlation (arrowheads) to activated microglia, which were counterimmunolabeled with an antibody to IBA1 (green). J: Ccl4 mRNA staining was not apparent in sections of dim-reared retinas. J-M: At 24 hrs, LD staining for Ccl4 was detected among clusters of irregular nuclei in the ONL from the superior retina (arrowheads); little-to-none were observed in the inferior retina (data not shown). N-R: Ccl4-expressing nuclei (red) in retinal sections show strong immunofluorescence (arrowheads) for IBA1+ microglia (green), much like Ccl3 (E-I). INL, inner nuclear layer; IHC, immunohistochemistry; ISH, in situ hybridization; ONL, outer nuclear layer; OS, outer segments.

Mentions: Chemokine ligands Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10 were selected for further characterization in 24-hr LD retinas, by in situ hybridization (Figures 3, 4, 5, 6). No expression of chemokines was detected in retinas of dim-reared animals (Figures 3, 4, 5, 6). For Ccl3 and Ccl4, mRNA staining was evident by 24 hrs of LD in irregular-shaped nuclei/processes (Figure 3A-D, J-M), scattered throughout the ONL. These cells were more numerous in the superior part of the retina, which is the focal region for LD-mediated degeneration (data not shown). Counter immunolabeling for IBA1 revealed a strong co-localization of both Ccl3 (Figure 3E-I) and Ccl4 (Figure 3N-R) with activated IBA1+ microglia in the ONL at 24 hrs post exposure/LD.Figure 3


Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

Rutar M, Natoli R, Chia RX, Valter K, Provis JM - J Neuroinflammation (2015)

In situhybridization for Ccl3 and Ccl4 mRNA in the retina following exposure to 24 hours of light damage.In situ hybridization for Ccl3 is documented in A-I, while Ccl4 is shown in J-R. A: In retinas from dim-reared animals, staining for Ccl3 was not observed in the retina. B-D: After 24 hrs light damage (LD), there was staining for Ccl3 in irregularly shaped nuclei - ranging from elongated to globular - traversing the outer nuclear layer (ONL) of the superior retina (arrowheads). Conversely, few Cc3-expressing cells were found in the inferior retina (data not shown). E-I: Nuclei stained for Ccl3 mRNA (red) showed strong correlation (arrowheads) to activated microglia, which were counterimmunolabeled with an antibody to IBA1 (green). J: Ccl4 mRNA staining was not apparent in sections of dim-reared retinas. J-M: At 24 hrs, LD staining for Ccl4 was detected among clusters of irregular nuclei in the ONL from the superior retina (arrowheads); little-to-none were observed in the inferior retina (data not shown). N-R: Ccl4-expressing nuclei (red) in retinal sections show strong immunofluorescence (arrowheads) for IBA1+ microglia (green), much like Ccl3 (E-I). INL, inner nuclear layer; IHC, immunohistochemistry; ISH, in situ hybridization; ONL, outer nuclear layer; OS, outer segments.
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Fig3: In situhybridization for Ccl3 and Ccl4 mRNA in the retina following exposure to 24 hours of light damage.In situ hybridization for Ccl3 is documented in A-I, while Ccl4 is shown in J-R. A: In retinas from dim-reared animals, staining for Ccl3 was not observed in the retina. B-D: After 24 hrs light damage (LD), there was staining for Ccl3 in irregularly shaped nuclei - ranging from elongated to globular - traversing the outer nuclear layer (ONL) of the superior retina (arrowheads). Conversely, few Cc3-expressing cells were found in the inferior retina (data not shown). E-I: Nuclei stained for Ccl3 mRNA (red) showed strong correlation (arrowheads) to activated microglia, which were counterimmunolabeled with an antibody to IBA1 (green). J: Ccl4 mRNA staining was not apparent in sections of dim-reared retinas. J-M: At 24 hrs, LD staining for Ccl4 was detected among clusters of irregular nuclei in the ONL from the superior retina (arrowheads); little-to-none were observed in the inferior retina (data not shown). N-R: Ccl4-expressing nuclei (red) in retinal sections show strong immunofluorescence (arrowheads) for IBA1+ microglia (green), much like Ccl3 (E-I). INL, inner nuclear layer; IHC, immunohistochemistry; ISH, in situ hybridization; ONL, outer nuclear layer; OS, outer segments.
Mentions: Chemokine ligands Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10 were selected for further characterization in 24-hr LD retinas, by in situ hybridization (Figures 3, 4, 5, 6). No expression of chemokines was detected in retinas of dim-reared animals (Figures 3, 4, 5, 6). For Ccl3 and Ccl4, mRNA staining was evident by 24 hrs of LD in irregular-shaped nuclei/processes (Figure 3A-D, J-M), scattered throughout the ONL. These cells were more numerous in the superior part of the retina, which is the focal region for LD-mediated degeneration (data not shown). Counter immunolabeling for IBA1 revealed a strong co-localization of both Ccl3 (Figure 3E-I) and Ccl4 (Figure 3N-R) with activated IBA1+ microglia in the ONL at 24 hrs post exposure/LD.Figure 3

Bottom Line: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10.In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE).This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Canberra, ACT 2601, Australia. matt.rutar@anu.edu.au.

ABSTRACT

Background: Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration.

Methods: Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR.

Results: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

Conclusions: Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

No MeSH data available.


Related in: MedlinePlus