A penetrating ocular injury can affect the induction of anterior chamber-associated immune deviation

Abstract

Purpose: To determine the effect of penetrating ocular injury on the induction of anterior chamber-associated immune deviation (ACAID).

Methods: An injection of 5 mul ovalbumin (OVA, 20 mg/ml) into the anterior chamber (AC) of female BALB/c mice was performed to induce ACAID. A penetrating ocular injury was induced via the limbus on OVA-inoculated eyes at 24 h, 48 h, 72 h, and 120 h following AC injection. The mice receiving an OVA inoculation without the ocular injury served as the AC-injection group. Delayed type hypersensitivity (DTH) was examined to evaluate the induction of ACAID. The levels of transforming growth factor (TGF)-beta1, interleukin (IL)-10, and interferon (IFN)-gamma produced by splenocytes were detected by enzyme-linked immunosorbent assays (ELISA). The frequency of CD4(+)CD25(+)Foxp3(+)T cells in the splenocytes was detected by flow cytometry.

Results: A significantly decreased DTH response was observed in the AC-injection group as well as in mice that received a penetrating injury at 72 h and 120 h following AC-injection of OVA. The levels of TGF-beta1 and IL-10 produced by splenocytes of mice in the AC-injection group and in the 72-h and 120-h group were significant higher than those in the 24-h and 48-h group. However, the levels of IFN-gamma produced by splenocytes of the AC-injection group and the 72-h and 120-h group were significantly lower than those in the 24-h and 48-h group. An increased frequency of CD4(+)CD25(+)Foxp3(+)T cells was found in the AC-injection group and the 72-h and 120-h group.

Conclusions: Penetrating ocular injury preformed shortly (24 h-48 h) after an AC injection of an antigen was able to abrogate ACAID and was associated with a decreased production of TGF-beta1 and IL-10, an increased production of IFN-gamma, and a decreased expression of CD4(+)CD25(+)Foxp3(+)T cells.

Figure 1

The method for preparation of a 1 mm-depth penetrating ocular injury at six o’clock of the murine limbus. A modified Standard Angle Blade is shown in the picture (A). A copper sheet was stuck 1 mm posterior to the blade tip. The diagram (B) showed that a 1 mm-depth penetrating ocular injury was made perpendicularly to the limbus using the modified standard Angle Blade.

Figure 2

Delayed type hypersensitivity measurements were performed after an anterior chamber injection of ovalbumin. Mice that were immunized via an s.c. injection of OVA in CFA seven days after AC injection of OVA acted as the AC-injection group. Mice received a penetrating ocular injury 24 h (24-h group), 48 h (48-h group), 72 h (72-h group), or 120 h (120-h group) after an AC injection of OVA on the OVA-inoculated eyes. This was followed by an s.c. injection of OVA in CFA seven days after the AC injection of OVA. Mice without any treatment acted as normal controls. Mice immunized only with OVA in CFA acted as the positive controls. Mice receiving an AC injection of sterile PBS and an s.c. injection of OVA in CFA seven days after the AC injection acted as PBS controls. All mice received an ear challenge seven days after s.c. immunization. Both ear pinnae were measured before (0 h) and 24 h after the ear challenge. Mean ± SEM ear-swelling responses are presented (n=6 per group). The asterisk indicates that p<0.05 when compared with the positive control.

Figure 3

Detection of TGF-β1 produced by splenocyte. Mice were sacrificed seven days after s.c. immunization. Spleens were removed and grinded to prepare a single-cell suspension. These cells were incubated in a complete RPMI 1640 medium with OVA (100 μg/ml) for 48 h in 24-well culture plates (2×10⁶ cells/well). Supernatants were collected for determining the quantity of TGF-β1 by ELISA. Data are the mean ± SEM (n=6 per group). The asterisk indicates that p<0.05 as compared with the positive control.

Figure 4

Detection of IL-10 produced by splenocytes. Spleens from each group of mice were harvested seven days after s.c. immunization and were used for preparing a single-cell suspension. The cells were incubated in a complete RPMI 1640 medium with OVA (200 μg/ml) for 48 h in 24-well culture plates (4×10⁶ cells/well). IL-10 in supernatants was quantified by ELISA. Data are the mean ± SEM (n=6 per group). The asterisk means that p<0.05 as compared with the positive control.

Figure 5

Detection of IFN-γ produced by splenocytes. Splenocytes were obtained from each group of mice seven days after s.c. immunization. The cells were incubated in a complete RPMI 1640 medium with OVA (200 μg/ml) for 48 h in 24-well culture plates (4×10⁶ cells/well). The levels of IFN-γ in supernatants were determined by ELISA. Data are the mean ± SEM (n=6 per group). The asterisk indicates that p<0.05 when compared with the positive control. The hash mark means that the production of IFN-γ was lower than the detection limit of the assay.

Figure 6

Flow cytometry analysis of CD4⁺CD25⁺Foxp3⁺T cells. Flow cytometry analysis of CD4+CD25+Foxp3+T cells per 1×106 splenocytes in mice from different groups. The mice in the groups listed as AC-injection 24h, 48h, 72h, 120h, normal controls, and PBS controls were as same as described in Figure 2. Spleens from different groups were removed 7 days after s.c. immunization and used for preparing a single-cell suspension. The cells were stained with anti-CD3, anti-CD4, anti-CD25, and anti-Foxp3 mAb. Data in A indicate the percentage of CD25+Foxp3+ cells which derive from gating on CD3+ CD4+ cells. The results shown in the histogram (B) are the mean SEM (n=6 per group). The asterisk indicates that p<0.05 when compared with the positive control.