The expression of cytokines in the aqueous humor and serum during endotoxin-induced uveitis in C3H/HeN mice

Abstract

Purpose: The cytokines present in the aqueous humor and serum of C3H/HeN mice with endotoxin-induced acute anterior uveitis were analyzed, and the potential role of the cytokines in the pathogenesis of the disease was investigated.

Methods: One hundred and eighty C3H/HeN mice were divided into an experimental group (n=150) and a control group (n=30). The mice in the experimental group were footpad-injected with 200 microg Vibro cholerae endotoxin (classical biotype, serotype Ogawa). The mice were then executed 4 h, 8 h, 16 h, 24 h, and 48 h after the injection of endotoxin. Aqueous humor and peripheral blood samples were collected using a microinjector. Ten samples were pooled together for analysis and centrifuged at 705x g, at 4 degrees C, for 3 min. The supernatant was collected and stored at -80 degrees C. The concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-gamma (IFN-gamma) in the samples were measured using a Cytometric Bead Array (CBA).

Results: Acute anterior uveitis was successfully induced in C3H/HeN mice. At roughly 16 h post-injection, the concentrations of both IL-1 and IL-6 reached peak levels, and were significantly different from the control group (p=0.001 and p=0.001, respectively). At 24 h post-injection, the concentrations of IFN-gamma and IL-10 in the aqueous humor reached peak levels, and were significantly different from the control group (p=0.022 and p=0.003). The concentrations of IFN-gamma in serum at 4 and 24 hours were significantly different from the control group (p=0.033 and p=0.032). The concentration of IL-10 in serum, at 24 h post-injection, was also found to be significantly different from the control group (p=0.003). The cytokine expression levels in the aqueous humor were consistent with what would be expected during the process of inflammation.

Conclusions: Both IL-1 and IL-6 appear to play an important role in acute anterior uveitis; furthermore, the severity of inflammation may be associated with the dynamic balance of IFN-gamma and IL-10. Our results suggest that the cytokine network might be a useful therapeutic target in the treatment of acute anterior uveitis.

Figure 1

Clinical manifestations of endotoxin-induced uveitis in C3H/HeN mice. The image shows the eye of an animal 24 h after injection with lipopolysaccharides. The arrow points out the pupil adhesion.

Figure 2

Hematoxylin-eosin stain study for neutrophils in the aqueous humor. The image shows the eye 24 h after injection of lipopolysaccharides. Arrows indicate neutrophils in the anterior chamber.

Figure 3

The expression levels tumor necrosis factor-α in the aqueous humor and serum at different time points (expressed as the mean μg/l±SD). The concentration of tumor necrosis factor-α in the aqueous humor reached a peak at 4 h. In the serum, tumor necrosis factor-α reached a peak concentration 16 h after injection. After 48 h, tumor necrosis factor-α concentrations in both the aqueous humor and the serum returned to their original levels.

Figure 4

The expression levels of interleukin-1 in the aqueous humor and serum at different time points (expressed as the mean μg/l±SD). The concentration of interleukin-1 in the aqueous humor increased significantly at 4 h, reached a peak at 16 h, and finally returning to its original concentration at 48 h. But it showed little difference in the serum.

Figure 5

The expression of interleukin-6 in the aqueous humor and serum at different time points (expressed as the mean μg/l±SD). The concentration of interleukin-6 in the aqueous humor was consistently much higher than in the serum. The interleukin-6 concentration in the aqueous humor rapidly increased in the first 4 h, reached a peak at 16 h, and returned to the original concentration after 48 h.

Figure 6

The expression of interleukin-10 in the aqueous humor and serum at different time points (expressed as the mean μg/l±SD). Interleukin-10 reached a peak concentration at 24 h, and returned to its original concentration at 48 h in aqueous humor. The concentration of interleukin-10 observed in the serum was similar to that in the aqueous humor, increasing at 8 h and reaching a maximum concentration at 16 h.

Figure 7

The expression of interferon-γ in the aqueous humor and serum at different time points (expressed as the mean μg/l±SD). Interferon-γ concentrations in the aqueous humor and serum were not significantly different at 8 h or 16 h in comparison to the control group.