Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9

Abstract

Background: IL-2 has classically been considered a cytokine that regulates T cell proliferation and differentiation, signaling through its heterotrimeric receptor (IL-2R) consisting of alpha (CD25), beta (CD122), gamma chains (CD132). Expression of IL-2R has also been detected in mucosal epithelial cells. Soluble IL-2Ralpha (CD25) has been reported as an inflammatory marker. We evaluated the expression of CD25 and CD122 in the ocular surface epithelium and investigated the mechanism of proteolytic cleavage of CD25 from these cells.

Methods: Desiccating stress (DS) was used as an inducer of matrix metalloproteinase 9 (MMP-9). DS was created by subjecting C57BL/6 and MMP-9 knockout (BKO) mice and their wild-type littermates (WT) mice to a low humidity and drafty environment for 5 days (DS5). A separate group of C57BL/6 mice was subjected to DS5 and treatment with topical 0.025% doxycycline, a MMP inhibitor, administered QID. The expression of CD25 and CD122 was evaluated in cryosections by dual-label laser scanning confocal microscopy. Western blot was used to measure relative levels of CD25 in epithelial lysates. Gelatinase activity was evaluated by in situ zymography. Soluble CD25 in tear fluid was measured by an immunobead assay.

Results: CD25 and CD122 were abundantly expressed in cornea (all layers) and conjunctiva epithelia (apical and subapical layers) in nonstressed control mice. After desiccating stress, we found that immunoreactivity to CD25, but not CD122, decreased by the ocular surface epithelia and concentration of soluble CD25 in tears increased as MMP-9 staining increased. CD25 was preserved in C57BL/6 mice topically treated with an MMP-9 inhibitor and in MMP-9 knock-out mice. MMP-9 treatment of human cultured corneal epithelial cells decreased levels of CD25 protein in a concentration dependent fashion.

Conclusion: Our results indicate that functional IL-2R is produced by the ocular surface epithelia and that CD25 is proteolytic cleaved to its soluble form by MMP-9, which increases in desiccating stress. These findings provide new insight into IL-2 signaling in mucosal epithelia.

Figure 1

A: Dual label immunofluorescent laser scanning confocal microscopy of ocular surface tissue sections from C57BL/6 mice for interleukin 2 receptor alpha (CD25, green) and beta chains (CD122, blue) with propidium iodide (red) nuclear counterstaining in nonstressed controls (NS), 5 days (D) of desiccating stress (DS5) and DS5 treated with topical doxycycline (DS5+Doxy) in C57BL/6 mice. A turquoise color indicates co-localization of both markers. Note partial disappearance of CD25 with preservation of CD122 after DS5 (arrows) in the conjunctival epithelia. Scale bar = 50 μm 1. B. Tissue sections prepared for in situ zymography (in situ Z) in nonstressed controls (NS), 5 days (D) of desiccating stress (DS5) and DS5 treated with topical doxycycline (DS5+Doxy) in C57BL/6 mice. Scale bar = 100 μm. 1. C. Merged images of laser scanning confocal fluorescent microscopy of ocular surface tissue sections stained for matrix metalloproteinase 9 (MMP-9, in green) with propidium iodide (PI, red) nuclear counterstaining in NS controls, DS5 and DS5+Doxy groups in C57BL/6 mice. Scale bar = 50 μm 1. D. Representative Western blot showing effect of DS on CD25 expression in corneal (CO) and conjunctival epithelial (CJ) lysates. 1. E. Bar graphs are mean + standard error mean of CD25 band intensities in 3 independent Western blots (arbitrary units).

Figure 2

Mean ± standard deviation of fluorescent intensity measurements in corneal and Conjunctival epithelia stained for CD25, CD122, MMP-9 in nonstressed (NS) C57BL/6 (A) or BKO and WT (B) and mice subjected for desiccating stress for 5 days (DS5). A separate group of DS5 mice were topically treated with doxycycline (DS5+Doxy) (C) Mean ± standard deviation of fluorescent intensity measurements in corneal and conjunctival epithelia stained for Fas-L in nonstressed (NS) C57BL/6 mice (B6-NS), desiccating stressed C57BL/6 mice for 5 days (B6-DS5), NS C57BL/6 mice treated with bovine serum albumin (injection control, B6-NS+BSA) or IL-2 subconjunctival injections (B6-NS+IL-2), CD25 knock-out mice (CD25KO-NS). *P < 0.05; ** = P < 0.01; *** = P < 0.001.

Figure 3

Merged images of laser scanning confocal fluorescent microscopy ocular surface tissue sections stained for interleukin 2 receptor alpha chain (CD25, in green) and interleukin 2 receptor beta chain (CD12, in green) with propidium iodide (PI, red) nuclear counterstaining in nonstressed controls (NS), 5 days (D) of desiccating stress (DS5) in MMP-9 knock-out (BKO) and wild-type (WT) mice. All images shown are the merged image of CD25 and CD122 (in green) with PI counterstaining. Scale bar = 50 μm.

Figure 4

Merged images of laser scanning confocal fluorescent microscopy of cornea (CO) and conjunctiva (CJ) sections stained for Fas-ligand (green) with propidium iodide (PI, red) nuclear counterstaining in nonstressed (NS) C57BL/6 mice (B6-NS), desiccating stressed C57BL/6 mice for 5 days (B6-DS5), NS C57BL/6 mice treated with bovine serum albumin (injection control, B6-NS+BSA) or IL-2 subconjunctival injections (B6-NS+IL-2), CD25 knock-out mice (CD25KO-NS). Scale bar = 100 μm 3. B. Laser scanning confocal fluorescent microscopy of human tissue sections (cornea, limbus and conjunctiva) stained for interleukin 2 receptor alpha chain (CD25, green) with PI (red) nuclear counterstaining. For the limbus, the CD25 and CD122 image (in green) is shown besides the merged image with PI nuclear staining. Note absence of staining on the basal epithelial layer of the limbus. Scale bar = 50 μm 3. C. Laser scanning confocal microscopy of human corneal epithelial cells grown on an eight chamber slide and stained for CD25 after treatment with increasing concentrations of MMP-9 for 48 hours. Scale bar = 50 μm 3. D. Representative Western blot showing effect of MMP-9 treatment on CD25 expression by cultured human corneal epithelial cells lysates. Increasing MMP-9 concentration decreased CD25 levels.