Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid

Abstract

Purpose: To understand the expression of genes involved in different complement pathways in the retina and retinal pigment epithelium (RPE)/choroid under physiologic conditions and how their expression is regulated by inflammatory cytokines.

Methods: The expression of complement components of the classical pathway (CP), mannose-binding lectin (MBL) pathway, alternative pathway (AP), and terminal pathway in the retina and RPE/choroid was determined by conventional reverse transcription polymerase chain reaction (RT-PCR). The effect of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α, 20 ng/ml), interleukin (IL)-6 (10 ng/ml), interferon-gamma (IFN-γ, 100 ng/ml) or lipopolysaccharides (LPS, 1 μg/ml) on the expression of these complement component genes was tested in vitro in primary cultured RPE cells and a microglial cell line (BV2 cells) and quantified by real-time RT-PCR.

Results: In the CP, complements C1qb, C1r, C1s, C2, and C4 were constitutively expressed by retina and RPE/choroid. Complement factor H and factor B of the AP as well as C3 were also detected in the retinal and RPE/choroidal tissues. In the MBL pathway, low levels of mannose-binding lectin (MBL)-associated serine protease (MASP)-1 in the retina and RPE/choroid and MASP2L in the retina were detected. Other components, including mannose-binding lectin 1 (MBL1), mannose-binding lectin 2 (MBL2), complement factor I (CFI), complement component 5 (C5) and complement factor H-related protein 1 (CFHR1), were not detected in either the retina or the RPE/choroid. The expression of CP- and AP-complement component genes in RPE and microglial cells was upregulated by interferon (IFN)-γ treatment. Treatment with TNF-α selectively upregulated the expression of C1s and C3 genes but downregulated complement factor H gene expression in RPE and microglial cells. The expression of genes involved in the MBL pathway was not affected by the inflammatory cytokines tested in this study.

Conclusions: Retina and RPE/choroid express a variety of complement components that are involved mainly in the CP and AP. RPE and microglial cells are the main sources of retinal complement gene expression. Retinal complement gene expression is regulated by inflammatory cytokines, such as IFN-γ and TNF-α.

Figure 1

The expression of selected classical pathway-related complement genes. The expression of C1qb, C1r, C1s, C2, and C4 in retina and retinal pigment epithelium (RPE)/choroid (A) and primary cultured RPE cells, B6-RPE07 cell line, primary cultured retinal microglia (Ret_microglia), brain microglia (Bri_microglia), and BV-2 cells (B) were analyzed by reverse transcriptase-PCR. Total RNA from mouse liver tissue was used as a positive control.

Figure 2

The expression of selected mannose binding lectin (MBL) pathway-related complement genes. The expression of MBL1, MBL2, MASP1, MSAP2L, and MASP2S in retina and RPE/choroid (A) and primary cultured retinal pigment epithelium (RPE) cells, B6-RPE07 cell line, primary cultured retinal microglia (Ret_microglia), brain microglia (Bri_microglia), and BV-2 cells (B) were analyzed by reverse transcriptase-PCR. Total RNA from mouse liver tissue was used as a positive control.

Figure 3

The expression of selected alternative pathway-related complement genes. The expression of CFI, CFH, CFB, and CFHR1 in retina and retinal pigment epithelium (RPE)/choroid (A) and primary cultured RPE cells, B6-RPE07 cell line, primary cultured retinal microglia (Ret_microglia), brain microglia (Bri_microglia), and BV-2 cells (B) were analyzed by reverse transcriptase-PCR. Total RNA from mouse liver tissue was used as a positive control. CFH, complement factor H; CFB, complement factor B; CFI, complement factor I CFHR1, complement factor H-related protein 1.

Figure 4

The expression of complement C3 and C5 genes. The expression of C3 and C5 in retina and retinal pigment epithelium (RPE)/choroid (A) and primary cultured RPE cells, B6-RPE07 cell line, primary cultured retinal microglia (Ret_microglia), brain microglia (Bri_microglia), and BV-2 cells (B) were analyzed by reverse transcriptase-PCR. Total RNA from mouse liver tissue was used as a positive control.

Figure 5

The effect of inflammatory cytokine on classical pathway-related complement gene expression in BV-2 and retinal pigment epithelium cells. BV-2 cells (A) and primary cultured mouse RPE cells (B, C) were treated with tumor necrosis factor (TNF)-α (20 ng/ml), interleukin (IL)-6 (10 ng/ml), interferon (IFN)-γ (100 ng/ml), or lipopolysaccharides (LPS; 1µg/ml) for 20 h. Cells were then collected for real-time RT–PCR (A, B) or conventional RT–PCR (C). Mouse liver RNA was used as a positive control in C. n=3 in each group. *p<0.05; **p<0.01; ***p<0.001 as compared to untreated control group using unpaired Student's t-test. Experiments were repeated twice.

Figure 6

The effect of inflammatory cytokine on mannose binding lectin pathway-related complement gene expression in BV-2 and retinal pigment epithelium cells. Primary RPE cells and BV-2 cells were treated with pro-inflammatory cytokines tumor necrosis factor (TNF)-α (20 ng/ml), interferon (IFN)-γ (100 ng/ml), interleukin (IL)-6 (10 ng/ml), and lipopolysaccharides (LPS; 1 µg/ml) for 20 h. Cells were then collected for real-time RT–PCR (A) or conventional RT–PCR (B, C). Mouse liver RNA was used as a positive control in B and C. n=3 in each group. Experiments were repeated twice.

Figure 7

The effect of inflammatory cytokine on alternative pathway-related complement gene expression in BV-2 and retinal pigment epithelium cells. Primary RPE cells and BV-2 cells were treated with pro-inflammatory cytokines TNF-α (20 ng/ml), interferon (IFN)-γ (100 ng/ml), interleukin (IL)-6 (10 ng/ml), and lipopolysaccharides (LPS; 1 µg/ml) for 20 h. Cells were then collected for real-time RT–PCR (A) or conventional RT–PCR (B). Mouse liver RNA was used as a positive control in B. A: The effect of inflammatory cytokines on CFH and CFB genes expression in BV-2 cells. B: The effect of inflammatory cytokines on CFI and CFHR1 gene expression in BV-2 and RPE cells. n=3 in each group. *p<0.05 when compared to untreated control group using unpaired Student's t test. Experiments were repeated twice.

Figure 8

The effect of inflammatory cytokine on complement C3 and C5 gene expression in BV-2 and retinal pigment epithelium cells. Primary RPE cells and BV-2 cells were treated with pro-inflammatory cytokines tumor necrosis factor (TNF)-α (20 ng/ml), interferon (IFN)-γ (100 ng/ml), interleukin (IL)-6 (10 ng/ml), and lipopolysaccharides (LPS;1 µg/ml) for 20 h. Cells were then collected for real-time RT–PCR analysis of C3 gene expression (A) or conventional RT–PCR analysis of C5 gene expression (B). Mouse liver RNA was used as a positive control in B. n=3 in each group. *p<0.05; **p<0.01 when compared to untreated controls using unpaired Student's t test. Experiments were repeated twice.