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. 1999 Apr;83(4):478–485. doi: 10.1136/bjo.83.4.478

Modulating phenotype and cytokine production of leucocytic retinal infiltrate in experimental autoimmune uveoretinitis following intranasal tolerance induction with retinal antigens

B Laliotou 1, A Dick 1
PMCID: PMC1723017  PMID: 10434874

Abstract

BACKGROUND/AIM—Nasal administration of retinal antigens induces systemic tolerance which results in suppression of experimental autoimmune uveoretinitis (EAU) when subsequently exposed to antigen. The aim was to establish if tolerance induction alters retinal infiltrating leucocyte phenotype and cytokine profile in tolerised animals when there is significantly reduced tissue destruction despite immunisation with retinal antigen.
METHODS—Female Lewis rats were tolerised by intranasal administration with retinal extract (RE) before immunisation with RE to induce EAU. Control animals were administered phosphate buffered saline (PBS) intranasally. Post immunisation, daily clinical responses were recorded and at the height of disease, retinas were removed and either infiltrating leucocytes isolated for flow cytometric phenotype assessment and intracellular cytokine production, or chorioretina processed for immunohistochemistry. Fellow eyes were assessed for cytokine mRNA by semiquantitative RT-PCR.
RESULTS—Flow cytometric analysis showed that before clinical onset of EAU there is no evidence of macrophage infiltration and no significant difference in circulating T cell populations within the retina. By day 14 a reduced retinal infiltrate in tolerised animals was observed and in particular a reduction in numbers of "activated" (with respect to CD4 and MHC class II expression) macrophages. Immunohistochemistry confirmed these findings and additionally minimal rod outer segment destruction was observed histologically. Cytokine analysis revealed that both IL-10 mRNA and intracellular IL-10 production was increased in tolerised eyes 7 days post immunisation. Although by day 14 post immunisation, IL-10 production was equivalent in both groups, a reduced percentage of IFN-γ+ macrophages and IFN-γ+ CD4+ T cells with increased percentage of IL-4+ CD4+ T cells were observed in tolerised animals.
CONCLUSIONS—Leucocytic infiltrate is not only reduced in number but its distinct phenotype compared with controls implies a reduced activation status of infiltrating monocytes to accompany increased IL-10 and reduced IFN-γ production in tolerised animals. This modulation may in turn contribute towards protection against target organ destruction in EAU.

 Keywords: intranasal tolerance; experimental autoimmune uveoretinitis; retina; cytokines; flow cytometry

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Figure 1  .

Figure 1  

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Suppression of clinicopathological features of experimental autoimmune uveoretinitis (EAU) by intranasal tolerance induction. (A) Histological grading12 of EAU shows extent of histopathological damage is reduced in tolerised animals. (B) Clinical inflammatory scores12 are significantly suppressed in tolerised animals.

Figure 2  .

Figure 2  

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Composite of immunohistochemical analysis of chorioretina from tolerised and control animals. By day 9 post immunisation ED1+ macrophages are found infiltrating both the choroid and retina (arrows) (a), concomitant with an increase in MHC class II staining (b) particularly at the choroid/RPE, inner retinal vessels (arrow), and retinal parenchyma (arrowhead representing parenchymal microglia), both panels are from control eyes. During active inflammation (day 11 post immunisation) ED1+ cell infiltrate increases in number in both control (c) and tolerised animals (d), although staining is more intense in control animals (c). By day 14 post immunisation with persistent ED1+ infiltrate, retinal architecture (particularly ROS (R)) is markedly damaged in control animals (e) whereas tolerised animals display preserved ROS (R) despite persistent ED1+ infiltrate (f). This is further confirmed by day 21 post immunisation. Although leucocytic infiltrate has largely resolved total ROS loss (R) can be observed in retinas of control (g) and not tolerised animals (h). Original magnifications: a-f ×350 and g, h ×300.

Figure 3  .

Figure 3  

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Flow cytometric analysis of retinal leucocytes in tolerised and control animals during EAU. Retinal leucocytes were isolated day 14 post immunisation The cells were derived from a pool of four eyes per experimental group, but comparable data were obtained in another identical experiment. Populations identified were based on two colour flow cytometric analysis including CD4 v αβTCR (CA and DC) and ED7 vMHC class II (BE and FD) and ED7 v CD4 (G and H)); see text. Plots A and B show scatter profile of retinal cell isolates demonstrating an increased population of granulocytes (arrow) in control animals (see text). Population 1 (plots C and D) identifies a CD4highαβ TCR- population in control animals (CD4 MFI of 110), distinct from CD4lowαβTCR- (arrow) population (CD4 MFI of 43), representing microglia (MG) and non-activated infiltrating macrophages.22 25 26 MG, further shown (population 2) on plots E-H and characterised by ED7low expression (see text), are equal in number (data not shown) and express similar MHC class II and CD4 between the two groups. Population 3 represent ED7high cells (granulocytes are excluded for calculation of macrophage numbers by appropriate backgating to scatter plot). Increased numbers of macrophages express MHC class II (plots E and F) and high levels of CD4 (plots G and H) in control animals (see text). E and F do not show granulocyte population whereas plots G and H include granulocytes (population 4) to show that they do not express CD4 and are markedly reduced in number in tolerised animals.

Figure 4  .

Figure 4  

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(A) RT-PCR cytokine analysis from eyes of control and tolerised animals during EAU. Values are from a representative experiment calculated from means of two animals/group at day 7 post immunisation each time point. (B) PCR IL-10 blots at day 7 post immunisation. Lanes 1 and 3, tolerised IL-10; lanes 2 and 4, tolerised β actin; lanes 5 and 7, control IL-10; lanes 6 and 8 control β actin. (C) Flow cytometric intracellular cytokine analysis day 7 post immunisation in control and tolerised animals. IL-10 expression represented as percentage of OX1+ cells. Values are mean of two animals/group.

Figure 5  .

Figure 5  

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Flow cytometric intracellular cytokine analysis day 14 post immunisation in control and tolerised animals. Cytokine expression represented as percentage of either monocyte/macrophage gate or CD4+ T cells. Values are mean of two animals/group.

Selected References

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