Thrombin regulates chemokine induction during human retinal pigment epithelial cell/monocyte interaction

Abstract

Thrombin, an important clotting factor, extravasates at sites of blood-retina barrier breakdown that is often associated with many retinal diseases. Here we investigated the effects of thrombin on human retinal pigment epithelial (HRPE) cells, monocytes, and HRPE cell/monocyte co-cultures. Thrombin induced secretion and mRNA expression of HRPE interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Thrombin also enhanced IL-8 and MCP-1 by HRPE cell/monocyte co-cultures, by apparently enhancing cell-cell contact mechanisms. The thrombin effects on IL-6 secretion were similar to those on chemokine secretion. Thrombin-induced chemokines by co-cultures were inhibited by anti-tumor necrosis factor-alpha (TNF-alpha) antibody, but not by anti-IL-1beta antibody. TNF-alpha was detected in cell lysates of monocytes detached from HRPE cells after co-culture stimulation with thrombin. HRPE cells mainly produced these chemokines. However, thrombin generally potentiated exogenous IL-1beta- and TNF-alpha-induced chemokine production by HRPE cells, monocytes, and co-cultures. Interferon-gamma potentiated chemokine secretion by co-cultures with or without thrombin. Our results indicate that thrombin may cause leukocyte recruitment by inducing HRPE cell and monocyte chemokine and by enhancing HRPE cell/monocyte interactions, in part because of monocyte TNF-alpha induction, suggesting important mechanisms for ocular inflammation during blood-retina barrier breakdown and intra-ocular hemorrhage.

Figure 1.

Induction of HRPE IL-8 and MCP-1 secretion by thrombin. A and B: Thrombin induced HRPE IL-8 (A) and MCP-1 (B) in a dose-dependent manner. HRPE cells were incubated in medium containing various concentrations of thrombin (0 to 10 U/ml) or thrombin (10 U/ml) and hirudin (20 U/ml). After 24 hours, supernatants were collected and IL-8 and MCP-1 protein levels in supernatants were measured by ELISA. *, P < 0.05; **, P < 0.01, compared with control (n = 3). C and D: Thrombin-induced IL-8 (C) and MCP-1 (D) secretion was time-dependent. HRPE cells were incubated with (black bars) or without (gray bars) thrombin (10 U/ml) for different times and IL-8 and MCP-1 protein levels in supernatants were measured (n = 3).

Figure 2.

Thrombin induction of HRPE IL-8 and MCP-1 mRNA. A: HRPE cells were incubated with thrombin (10 U/ml) for different times, total RNA was extracted, and semiquantitative RT-PCR was performed. These representative data are from one of three independent experiments. B: Results are expressed as a ratio of each PCR product/β-actin band density. Values represent means ± SEM (n = 3).

Figure 3.

Effect of thrombin on IL-8 and MCP-1 secretion by HRPE cell/monocyte co-cultures. A and B: Thrombin enhanced IL-8 (A) and MCP-1 (B) secretion by HRPE cell/monocyte co-cultures. Various concentrations of thrombin (0 to 10 U/ml) or thrombin (10 U/ml) and hirudin (20 U/ml) were added to HRPE cell/monocyte co-cultures (HRPE + Mo). After 24 hours, supernatants were collected and IL-8 and MCP-1 protein levels in supernatants were measured by ELISA. *, P < 0.05; **, P < 0.01, compared with chemokine secretion from HRPE cell/monocyte co-cultures without thrombin (n = 3). HRPE, HRPE cells; Mo, monocytes. C and D: Thrombin-induced IL-8 (C) and MCP-1 (D) secretion was time-dependent. HRPE cells were incubated with (black bars) or without (gray bars) thrombin (10 U/ml) for different times and IL-8 and MCP-1 protein levels in supernatants were measured (n = 3).

Figure 4.

Effect of thrombin on IL-6 secretion by HRPE cell, monocytes, HRPE cell/monocyte co-cultures. Thrombin induced IL-6 secretion by HRPE cells and HRPE cell/monocyte co-cultures. HRPE cell, monocytes, and HRPE cell/monocyte co-cultures were incubated with thrombin (10 U/ml). After 24 hours, supernatants were collected, and IL-6 protein levels in supernatants were measured by ELISA (n = 3).

Figure 5.

Thrombin modulated cell-associated IL-8 (A) and MCP-1 (B). HRPE cells (HRPE), monocytes (Mo), and HRPE cell/monocyte co-cultures were incubated with thrombin (10 U/ml) for 4 hours. HRPE cells and monocytes in the co-cultures were separated, and cells were lysed, as described in Materials and Methods. IL-8 and MCP-1 levels were measured by ELISA. c versus d, P < 0.05; a versus b, a versus e, e versus g, e versus f, f versus h, g versus h, i versus j, i versus m, m versus o, m versus n, and n versus p; P < 0.01 (n = 3).

Figure 6.

Cell-cell contact enhanced thrombin induction of IL-8 (A), MCP-1 (B), and IL-6 (C) secretion by co-cultures. HRPE cells co-incubated with monocytes in the same cultures, but separated by porous polycarbonate filters (HRPE/Mo), and HRPE cells overlayed with monocytes directly (HRPE + Mo) were incubated with thrombin (10 U/ml). After 24 hours, supernatants were collected and IL-8, MCP-1, and IL-6 protein levels in supernatants were measured by ELISA. a versus c, b versus d, e versus g, f versus h, i versus k, and j versus l; P < 0.01 (n = 3).

Figure 7.

Anti-TNF-α Ab, but not anti-IL-1β Ab, inhibited thrombin-induced IL-8 (A) and MCP-1 (B) secretion by HRPE cell/monocyte co-cultures. HRPE cell/monocyte co-cultures (HRPE + Mo) were incubated with anti-TNF-α Ab or anti-IL-1β Ab in the presence or absence of thrombin (10 U/ml) for 24 hours. Supernatants were collected and IL-8 and MCP-1 protein levels in supernatants were measured by ELISA (n = 3).

Figure 8.

Effect of thrombin on rhIL-1β-induced (A and B) and rhTNF-α-induced (C and D) IL-8 (A and C), MCP-1 (B and D), and IL-6 (E) secretion. Thrombin had synergistic effects on rhIL-1β- and rhTNF-α-induced IL-8, MCP-1, and IL-6 secretion. HRPE cells (HRPE), monocytes (Mo), and HRPE cell/monocyte co-cultures (HRPE + Mo) were incubated in the medium containing rhIL-1β (0.2 ng/ml) or rhTNF-α (2 ng/ml), either alone or in combination with thrombin for 24 hours. Supernatants were collected and IL-8, MCP-1, and IL-6 protein levels in supernatants were measured by ELISA. *, P < 0.05; **, P < 0.01, compared with chemokine secretion from cells with rhIL-1β or rhTNF-α alone (n = 3).

Figure 9.

Modulation of IL-8 (A), MCP-1 (B), and IL-6 (C) secretion by IFN-γ. HRPE cells (HRPE), monocytes (Mo), and HRPE cell/monocyte co-cultures (HRPE + Mo) were incubated in the medium containing IFN-γ (1000 U/ml), either alone or in combination with thrombin for 24 hours. Supernatants were collected and IL-8, MCP-1, and IL-6 protein levels in supernatants were measured by ELISA (n = 3).