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. 2007 Oct;48(10):4616-25.
doi: 10.1167/iovs.07-0233.

Altered chemokine profile associated with exacerbated autoimmune pathology under conditions of genetic interferon-gamma deficiency

Shao Bo Su  1 , Rafael S Grajewski, Dror Luger, Rajeev K Agarwal, Phyllis B Silver, Jun Tang, Jingsheng Tuo, Chi-Chao Chan, Rachel R Caspi
Affiliations

Altered chemokine profile associated with exacerbated autoimmune pathology under conditions of genetic interferon-gamma deficiency

Shao Bo Su et al. Invest Ophthalmol Vis Sci. 2007 Oct.

Abstract

Purpose: A prior study showed that mice deficient in IFN-gamma (GKO) are more susceptible to experimental autoimmune uveitis (EAU) than are wild-type (WT) mice. Histopathology of uveitic eyes revealed that the ocular infiltrate in GKO mice was dominated by neutrophils and eosinophils rather than by mononuclear cells, as in WT mice. The present study was conducted to explore the differential expression of chemokine(s) likely to account for the distinct inflammatory cell composition in uveitic eyes of WT and GKO mice.

Methods: Mice were immunized to induce EAU. Lymph nodes draining the site of the immunization and the eyes were collected at different time points for chemokine analysis. Microarray, real-time PCR and protein analyses were performed to examine the expression of chemokines in WT and GKO mice.

Results: Many chemokines were differentially upregulated in GKO versus WT mice. Expression of the Th1-associated chemokines CXCL10, CXCL9, CCL5, and CXCL11 was elevated in WT mice, whereas the Th2-associated chemokines CCL11, CCL17, and CCL1 and the Th17-associated chemokines CCL22 and CXCL2 were elevated in the GKO mice. Depletion of granulocytes abrogated EAU in both WT and GKO mice.

Conclusions: These results suggest that Th1-associated chemokines play a critical role in the attraction of mononuclear cells to the eyes in the presence of IFN-gamma, while in the absence of this cytokine, Th2- and Th17-related chemokines may be the key elements for influx of granulocytes.

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Figures

Figure 1
Figure 1
EAU and related immune responses in WT and GKO mice. GKO and control WT mice were immunized with 150 μg IRBP emulsified in CFA and received 0.5 μg of PT IP. (A) The EAU score was evaluated by histopathology at 21 days after immunization. The EAU score is the average of all the mice in the group. EAU incidence (positive/total) is shown in each bar. Shown is a representative experiment of three. (B) DTH responses were elicited on day 19 and were evaluated on day 21 after immunization. (C) Ag-specific proliferation of lymph nodes from immunized mice was determined by incorporation of [3H]thymidine. Shown are counts per minute from one representative experiment of three (average of triplicates). *Statistically significant difference in scores versus WT (P < 0.05).
Figure 2
Figure 2
Ocular histopathology of WT and GKO mice. (A) EAU (score, 0.5) in WT mice with mononuclear cell infiltration. (B) EAU (score, 2) in GKO mice with massive granulocyte infiltration. Hematoxylin and eosin. Magnification, ×200.
Figure 3
Figure 3
Chemokine mRNA expression by real-time RT-PCR in eyes, lymph nodes, or lymph node cells stimulated with IRBP from WT and GKO mice. EAU was induced by immunization with IRBP/CFA and PT, and RNA extraction, real-time RT-PCR were performed. The relative changes in gene expression were calculated using the 2−ΔΔCT method. A twofold change was considered significant. (A) Chemokine expression in normal eyes versus (B) in eyes at EAU onset, 12 days after immunization. (C) Chemokine expression in normal lymph nodes versus (D) in lymph nodes from mice 3 days after IRBP immunization. (E) Background chemokine expression in draining lymph node cells 21 days after immunization cultured with medium alone versus (F) in lymph nodes stimulated with IRBP overnight. The experiment was repeated twice with similar results. LN, lymph node.
Figure 4
Figure 4
Chemokine protein in eyes and in culture supernatant of draining lymph node cells from the mice immunized with IRBP. EAU was induced by immunization with IRBP/CFA and PT. (A) The eyes from day 0 (normal eyes), day 13 (1 day after disease onset), and day 21 (mature of the disease) after immunization were collected, and eye extract was prepared. (B) Lymph node cells harvested 21 days after immunization from IRBP-immunized mice were pooled within groups, and cultures of 5 ′ 106 cells/mL were stimulated with IRBP. The supernatants were collected after 48 hours. Shown is chemokine content as assayed by ELISA in eye extracts and in culture (a representative experiment of three). Samples were pooled for analysis, therefore no error bars could be generated. LN, lymph node.
Figure 5
Figure 5
Kinetics of disease progression in GKO and WT mice. GKO mice (n = 11) and control WT mice (n = 10) were immunized with 150 μg IRBP emulsified in CFA and received 0.5 μg of PT IP. Clinical development of disease was followed by fundus examination at the specified time points after immunization. The EAU score is the average of all the mice in the group ± SE.
Figure 6
Figure 6
Effect of eosinophil or granulocyte depletion on EAU development. EAU was induced by immunization with IRBP in CFA plus PT. (A) Mice were injected IP with 0.5 mg anti-CCR3 Ab daily from day −1 to day 13. Shown is a representative experiment of three. (B) Mice were injected IP with 0.1 mg of anti-Gr-1 Ab on days −1, 3, 6, 9, 12, and 15, Shown is a representative experiment of two. EAU scores were evaluated by histopathology 21 days after immunization. The score is the average of all the mice in the group. EAU incidence (positive/total) is shown within the bars. *Statistically significant difference in scores versus the untreated group (P < 0.05).
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