J Bacteriol Virol.  2007 Jun;37(2):119-128. 10.4167/jbv.2007.37.2.119.

The Expression Pattern of Toll-like Receptor (TLR) and Cytokine Production to TLR Agonists in Human Retinal Pigment Epithelial Cells

• Choi SJ
• Lee KH
• Park SJ
• Park HS
• Kim J
• Kim SK
• Park JY

• KMID: 2055046
• DOI: http://doi.org/10.4167/jbv.2007.37.2.119

Abstract

Retinal pigment epithelium (RPE) constituting the outer blood-retina barrier plays an important role in ocular defense mechanism. Many studies reported that RPE participates in ongoing immune responses in the retina. However, the exact mechanism is still uncertain. Toll-like receptors (TLRs) participate in the recognition of pathogen-associated molecular patterns (PAMP), such as LPS, zymosan, lipoprotein, and dsRNA. The expression and function of TLRs in human RPE have not been established. In this study, we investigated TLRs expression in human fetal RPE and their recognition of PAMP to determine how human RPE participates in ocular defense mechanism against microbial component. RT-PCR and real time PCR revealed that TLR1 through 5 were constitutively expressed in human fetal RPE, and their expressions were slightly increased by LPS. We determined the TNF-alpha, IL-6, and IL-8 expression in human fetal RPE after treatment with LPS, zymosan, petidoglycan, or poly I:C. RT-PCR demonstrated that LPS and poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human fetal RPE. LPS showed more potent effects on TNF-alpha and IL-8 production. Peptidoglycan and zymosan did not induce the production of TNF-alpha. CD14, the co-receptor of LPS was weakly expressed and functioned in recognizing LPS in human fetal RPE. These results suggest that human RPE may participate in ocular defense mechanism against microbial component through toll-like receptors.

Keyword

Toll-like receptor; Pathogen-associated molecular pattern; Retinal pigment epithelium; Cytokine

MeSH Terms

Epithelial Cells*
Humans*
Interleukin-6
Interleukin-8
Lipoproteins
Peptidoglycan
Real-Time Polymerase Chain Reaction
Retina
Retinal Pigment Epithelium
Retinaldehyde*
Toll-Like Receptors*
Tumor Necrosis Factor-alpha
Zymosan
Interleukin-6
Interleukin-8
Lipoproteins
Peptidoglycan
Retinaldehyde
Toll-Like Receptors
Tumor Necrosis Factor-alpha
Zymosan

Figure

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  Figure 1. Culture of human fetal RPE. (A) Phase contrast microscope ×100 (B) Indirect immunostaining of pan-cytokeratin in primarily cultured human fetal RPE.

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  Figure 2. The expression of TLR1, TLR2, TLR3, TLR4, TLR5, and CD14 in human fetal RPE and U937. (A) The human fetal RPE were cultured in DMEM containing 10% serum. Total RNA was isolated and RT-PCR was performed. (B) The U937 cells were cultured in RPMI 1640 containing 10% serum. Total RNA was isolated and RT-PCR was performed.

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  Figure 3. The effect of LPS on TLR2, TLR3, and TLR4 expression in human fetal RPE. (A) After LPS treatment for 7h at variable dosages, total RNA was isolated and RT-PCR was performed. (B) After treatment with 10 ng/ml of LPS, total RNA was isolated and RT-PCR was performed. (C) After LPS treatment for 7h at variable dosages, real time PCR was performed using primers of GAPDH, TLR2, TLR3, and TLR4. Comparative expression levels were calculated.

• 

  Figure 4. The effect of microbial components on cytokine expression in human fetal RPE. (A) The human fetal RPE was cultured in the presence or absence of LPS (10 ng/ml), poly I:C (50 μg/ml), peptidoglycan (100 ng/ml), or zymosan (100 μg/ml) for 3h. Total RNA was isolated and mRNA levels of TNF-α, IL-6, and IL-8 were analyzed by RT-PCR. (B) The human fetal RPE was treated with variable dosages of LPS, poly I:C, peptidoglycan, or zymosan for 3h. The levels of TNF-α mRNA were analyzed by RT-PCR. PTG; peptidoglycan, Zymo; zymosan.

• 

  Figure 5. The effect of LPS and poly I:C on TNF-α, IL-6, IL-8 expression. The human fetal RPE was incubated in the presence or absence of LPS (10 ng/ml) or poly I:C (50 ng/ml). At different time interval, total RNAs were isolated. Then, mRNA levels of TNF-α (A), IL-6 and IL-8 (B) were analyzed using RT-PCR. (C) The cultures were incubated with LPS for 24h and culture supernatants were then harvested. The levels of IL-8 in the culture supernatants were measured using ELISA. PIC; poly I:C

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  Figure 6. The effect of serum and soluble CD14 on LPS induced TNF-α expression in human fetal RPE. (A) After LPS treatment for 7h at variable dosages, total RNA was isolated and RT-PCR was performed. (B) The human fetal RPE was cultured in serum-free DMEM. After LPS treatment alone or with soluble CD14 (0.1 ng/ml) for 3h, total RNA was isolated. Then, mRNA levels of TNF-α were analyzed by RT-PCR.

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