RvE1 uses the LTB4 receptor BLT1 to increase [Ca2+]i and stimulate mucin secretion in cultured rat and human conjunctival goblet cells

Abstract

Purpose: Specialized pro-resolving lipid mediator resolvin (Rv) E1 stimulates secretion including mucins from conjunctival goblet cells. RvE1 can use both its ChemR23 receptor and the LTB₄ receptor BLT1 to increase [Ca²⁺]_i. The purpose of this study was to determine the expression of ChemR23 and BLT1 and receptors on conjunctival goblet cells and the respective roles these two receptors play in goblet cell responses to RvE1.

Methods: Goblet cells were cultured from male rat or human conjunctiva from both sexes. Western blotting analysis, reverse transcription PCR and immunofluorescence microscopy were used to demonstrate the expression of ChemR23 and BLT1 in conjunctival goblet cells. High molecular weight glycoprotein secretion was determined using an enzyme-linked lectin assay. Signaling pathways were studied by measuring the increase in [Ca²⁺]_i using fura 2/AM.

Results: ChemR23 and BLT1 and receptors were present on both rat and human conjunctival goblet cells. The BLT1 inhibitors LY293111 and U75302 significantly blocked RvE1-and LTB₄-stimulated [Ca²⁺]_i increase. RvE1-and LTB₄-stimulated [Ca²⁺]_i and secretion increases were blocked by BLT1-targeted siRNA. RvE1-stimulated [Ca²⁺]_i and secretion increases were also blocked by ChemR23-targeted siRNA. Addition of RvE1 2 min before or simultaneously with LTB₄ desensitized the LTB₄ [Ca²⁺]_i response. Addition of RvE1 and LTB₄ simultaneously caused secretion that was decreased compared to either response alone.

Conclusion: RvE1, in addition to the ChemR23 receptor, uses the BLT1 receptor to increase [Ca²⁺]_i and stimulate secretion in both rat and human cultured conjunctival goblet cells.

Keywords: Conjunctiva; Dry eye syndromes; Goblet cells; Inflammation; Resolution; Secretion.

Fig 1.. Identification of BLT1 and ChemR23 receptors in cultured rat conjunctival goblet cells and rat conjunctival epithelium.

RNA was isolated from cultured rat goblet cells and RT-PCR performed using primers for rat BLT1 or ChemR23 receptors and the standard control β-actin (A). L=molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using anti-bodies to BLT1 (B upper panel) and ChemR23 (B lower panel). Each lane indicates a separate animal. Immunofluorescence microscopy was performed on cultured goblet cells (C and D) using antibodies to BLT 1 (C) and ChemR23 (D). In C the left panel indicates immunofluorescence to BLT1 (red); the center panel to the directly conjugated lectin UEA-1 (goblet cell secretory product; green); and the right panel indicates an overlay of anti-BLT1, UEA-1, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to UEA-1 (green); the right panel indicates an overlay of anti-ChemR23, UEA-1, and DAPI (blue). Micrographs are representative of results from 3 rats. Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide for overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with the BLT1 blocking peptide (red) and with DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (Red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with the ChemR23 blocking peptide (red) and DAPI (blue). In G the top left panel indicates anti-BLT1 immunoreactivity in epithelial sections, top middle panel indicates overlay of BLT1 immunoreactivity (red), UEA-1 (green, indicates secretory granules), and DAPI. The inset and top right panel indicates a higher magnification image (1000×). In the bottom left panel in E indicates the isotype control for BLT1. The bottom right panel in E indicates an overlay of UEA-1 fluorescence (green) and DAPI (blue). Magnification 400×.

Fig 1.. Identification of BLT1 and ChemR23 receptors in cultured rat conjunctival goblet cells and rat conjunctival epithelium.

RNA was isolated from cultured rat goblet cells and RT-PCR performed using primers for rat BLT1 or ChemR23 receptors and the standard control β-actin (A). L=molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using anti-bodies to BLT1 (B upper panel) and ChemR23 (B lower panel). Each lane indicates a separate animal. Immunofluorescence microscopy was performed on cultured goblet cells (C and D) using antibodies to BLT 1 (C) and ChemR23 (D). In C the left panel indicates immunofluorescence to BLT1 (red); the center panel to the directly conjugated lectin UEA-1 (goblet cell secretory product; green); and the right panel indicates an overlay of anti-BLT1, UEA-1, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to UEA-1 (green); the right panel indicates an overlay of anti-ChemR23, UEA-1, and DAPI (blue). Micrographs are representative of results from 3 rats. Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide for overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with the BLT1 blocking peptide (red) and with DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (Red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with the ChemR23 blocking peptide (red) and DAPI (blue). In G the top left panel indicates anti-BLT1 immunoreactivity in epithelial sections, top middle panel indicates overlay of BLT1 immunoreactivity (red), UEA-1 (green, indicates secretory granules), and DAPI. The inset and top right panel indicates a higher magnification image (1000×). In the bottom left panel in E indicates the isotype control for BLT1. The bottom right panel in E indicates an overlay of UEA-1 fluorescence (green) and DAPI (blue). Magnification 400×.

Fig 1.. Identification of BLT1 and ChemR23 receptors in cultured rat conjunctival goblet cells and rat conjunctival epithelium.

RNA was isolated from cultured rat goblet cells and RT-PCR performed using primers for rat BLT1 or ChemR23 receptors and the standard control β-actin (A). L=molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using anti-bodies to BLT1 (B upper panel) and ChemR23 (B lower panel). Each lane indicates a separate animal. Immunofluorescence microscopy was performed on cultured goblet cells (C and D) using antibodies to BLT 1 (C) and ChemR23 (D). In C the left panel indicates immunofluorescence to BLT1 (red); the center panel to the directly conjugated lectin UEA-1 (goblet cell secretory product; green); and the right panel indicates an overlay of anti-BLT1, UEA-1, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to UEA-1 (green); the right panel indicates an overlay of anti-ChemR23, UEA-1, and DAPI (blue). Micrographs are representative of results from 3 rats. Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide for overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with the BLT1 blocking peptide (red) and with DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (Red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with the ChemR23 blocking peptide (red) and DAPI (blue). In G the top left panel indicates anti-BLT1 immunoreactivity in epithelial sections, top middle panel indicates overlay of BLT1 immunoreactivity (red), UEA-1 (green, indicates secretory granules), and DAPI. The inset and top right panel indicates a higher magnification image (1000×). In the bottom left panel in E indicates the isotype control for BLT1. The bottom right panel in E indicates an overlay of UEA-1 fluorescence (green) and DAPI (blue). Magnification 400×.

Fig 1.. Identification of BLT1 and ChemR23 receptors in cultured rat conjunctival goblet cells and rat conjunctival epithelium.

RNA was isolated from cultured rat goblet cells and RT-PCR performed using primers for rat BLT1 or ChemR23 receptors and the standard control β-actin (A). L=molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using anti-bodies to BLT1 (B upper panel) and ChemR23 (B lower panel). Each lane indicates a separate animal. Immunofluorescence microscopy was performed on cultured goblet cells (C and D) using antibodies to BLT 1 (C) and ChemR23 (D). In C the left panel indicates immunofluorescence to BLT1 (red); the center panel to the directly conjugated lectin UEA-1 (goblet cell secretory product; green); and the right panel indicates an overlay of anti-BLT1, UEA-1, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to UEA-1 (green); the right panel indicates an overlay of anti-ChemR23, UEA-1, and DAPI (blue). Micrographs are representative of results from 3 rats. Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide for overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with the BLT1 blocking peptide (red) and with DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (Red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with the ChemR23 blocking peptide (red) and DAPI (blue). In G the top left panel indicates anti-BLT1 immunoreactivity in epithelial sections, top middle panel indicates overlay of BLT1 immunoreactivity (red), UEA-1 (green, indicates secretory granules), and DAPI. The inset and top right panel indicates a higher magnification image (1000×). In the bottom left panel in E indicates the isotype control for BLT1. The bottom right panel in E indicates an overlay of UEA-1 fluorescence (green) and DAPI (blue). Magnification 400×.

Fig 2.. Identification of BLT1 and ChemR23 receptors in cultured human goblet cells.

RNA was isolated from cultured human goblet cells and RT-PCR performed using primers for human BLT1 and ChemR23 receptors and the standard control β-actin (A). L= molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using antibodies to BLT1 (B left upper panel) and ChemR23 (B right upper panel). Homogenized THP1 cells were the positive control and HEK cells the negative control for BLT1 (B left lower panel) and ChemR23 (right lower panel). In B each lane indicates a separate individual. In C the left panel indicates immunofluorescence to BLT1 (red); the center panel indicates the directly conjugated lectin HPA that indicates goblet cell secretory product (green); the right panel indicates an overlay of anti-BLT1, HPA, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to HPA (green); and the right panel indicates an overlay of anti-ChemR23, HPA, and DAPI (blue). Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with BLT1 blocking peptide (red) and DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with ChemR23 blocking peptide and DAPI (blue). Micrographs are representative of results from 3 individuals. Magnification 400×.

Fig 2.. Identification of BLT1 and ChemR23 receptors in cultured human goblet cells.

RNA was isolated from cultured human goblet cells and RT-PCR performed using primers for human BLT1 and ChemR23 receptors and the standard control β-actin (A). L= molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using antibodies to BLT1 (B left upper panel) and ChemR23 (B right upper panel). Homogenized THP1 cells were the positive control and HEK cells the negative control for BLT1 (B left lower panel) and ChemR23 (right lower panel). In B each lane indicates a separate individual. In C the left panel indicates immunofluorescence to BLT1 (red); the center panel indicates the directly conjugated lectin HPA that indicates goblet cell secretory product (green); the right panel indicates an overlay of anti-BLT1, HPA, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to HPA (green); and the right panel indicates an overlay of anti-ChemR23, HPA, and DAPI (blue). Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with BLT1 blocking peptide (red) and DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with ChemR23 blocking peptide and DAPI (blue). Micrographs are representative of results from 3 individuals. Magnification 400×.

Fig 2.. Identification of BLT1 and ChemR23 receptors in cultured human goblet cells.

RNA was isolated from cultured human goblet cells and RT-PCR performed using primers for human BLT1 and ChemR23 receptors and the standard control β-actin (A). L= molecular weight ladder. Protein was homogenized from cultured goblet cells and western blot performed using antibodies to BLT1 (B left upper panel) and ChemR23 (B right upper panel). Homogenized THP1 cells were the positive control and HEK cells the negative control for BLT1 (B left lower panel) and ChemR23 (right lower panel). In B each lane indicates a separate individual. In C the left panel indicates immunofluorescence to BLT1 (red); the center panel indicates the directly conjugated lectin HPA that indicates goblet cell secretory product (green); the right panel indicates an overlay of anti-BLT1, HPA, and DAPI (blue, indicates cell nuclei). In D the left panel indicates immunofluorescence to ChemR23 (red); the center panel to HPA (green); and the right panel indicates an overlay of anti-ChemR23, HPA, and DAPI (blue). Anti-BLT1 antibody and anti-ChemR23 antibody were incubated with associated blocking peptide overnight (E and F). In E the left panel indicates BLT1 immunofluorescence (Red) with DAPI (blue) and in F the left panel indicates the anti-BLT1 antibody pre-incubated with BLT1 blocking peptide (red) and DAPI (blue). In E the right panel indicates ChemR23 immunofluorescence (red) and DAPI (blue) and in F the right panel indicates the anti-ChemR23 antibody pre-incubated with ChemR23 blocking peptide and DAPI (blue). Micrographs are representative of results from 3 individuals. Magnification 400×.

Fig 3.. RvE1 triggered [Ca²⁺]_i increase can be blocked by BLT receptor inhibitor LY293111 in both rat and human conjunctival goblet cells.

Cultured rat or human goblet cells were incubated with vehicle or LY293111 (10⁻⁷ M and 10⁻⁶ M) for 30 min prior to addition of RvE1 at 10⁻⁸ M (A and C) or LTB₄ at 10⁻⁸ M (B and C) in rat cells and RvE1 at 10⁻⁸ M (D and F) or LTB₄ at 10⁻⁸ M (E and F) in human cells and [Ca²⁺]_i was measured by fura2. The average [Ca²⁺]_i level over time was shown in (A and B for rat; and D and E for human cells); Change in peak [Ca²⁺]_i was calculated and shown in (C and F). Data are mean ± SEM from 6 rats and 5 humans. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from RvE1 or LTB₄ alone.

Fig 3.. RvE1 triggered [Ca²⁺]_i increase can be blocked by BLT receptor inhibitor LY293111 in both rat and human conjunctival goblet cells.

Cultured rat or human goblet cells were incubated with vehicle or LY293111 (10⁻⁷ M and 10⁻⁶ M) for 30 min prior to addition of RvE1 at 10⁻⁸ M (A and C) or LTB₄ at 10⁻⁸ M (B and C) in rat cells and RvE1 at 10⁻⁸ M (D and F) or LTB₄ at 10⁻⁸ M (E and F) in human cells and [Ca²⁺]_i was measured by fura2. The average [Ca²⁺]_i level over time was shown in (A and B for rat; and D and E for human cells); Change in peak [Ca²⁺]_i was calculated and shown in (C and F). Data are mean ± SEM from 6 rats and 5 humans. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from RvE1 or LTB₄ alone.

Fig 4.. RvE1 triggered [Ca²⁺]_i increase can be blocked by BLT1 receptor inhibitor U75302 in both rat and human conjunctival goblet cells.

Cultured rat or human goblet cells were incubated with vehicle or U75302 (10⁻⁶ M) for 30 min prior to addition of RvE1 at 10⁻⁸ M or LTB₄ at 10⁻⁸ M (A and B rat cells and C and D human cells) and [Ca²⁺]_i was measured by fura2. The average [Ca²⁺]_i level over time was shown in (A and C). Change in peak [Ca²⁺]_i was calculated and shown in (B and D). Data are mean ± SEM from 6 rats or 5 humans. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from RvE1 or LTB₄ alone.

Fig 4.. RvE1 triggered [Ca²⁺]_i increase can be blocked by BLT1 receptor inhibitor U75302 in both rat and human conjunctival goblet cells.

Cultured rat or human goblet cells were incubated with vehicle or U75302 (10⁻⁶ M) for 30 min prior to addition of RvE1 at 10⁻⁸ M or LTB₄ at 10⁻⁸ M (A and B rat cells and C and D human cells) and [Ca²⁺]_i was measured by fura2. The average [Ca²⁺]_i level over time was shown in (A and C). Change in peak [Ca²⁺]_i was calculated and shown in (B and D). Data are mean ± SEM from 6 rats or 5 humans. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from RvE1 or LTB₄ alone.

Fig 5.. Depletion of BLT1 or ChemR23 blocked the RvE1 triggered [Ca²⁺]_i increase in rat conjunctival goblet cells.

Cultured rat goblet cells were transfected with BLT1 and ChemR23 siRNA at 50 or 100 nM, scrambled siRNA (scsiRNA), or not transfected (NT). Western blot analysis of cells treated with scsiRNA or siRNA knockdown of BLT1 (upper panel) or ChemR23 (lower panel) receptor was shown in (A). RvE1 at 10⁻⁸ M was added and average [Ca²⁺]_i over time was shown in (B). The change in peak [Ca²⁺]_i was calculated and shown in (C). RvE1 was added and the glycoprotein secretion was measured after a 2-hr incubation (D). Data are mean ± SEM from 4 rats each for [Ca²⁺]_i measurement and secretion measurement. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from non-transfected group.

Fig 5.. Depletion of BLT1 or ChemR23 blocked the RvE1 triggered [Ca²⁺]_i increase in rat conjunctival goblet cells.

Cultured rat goblet cells were transfected with BLT1 and ChemR23 siRNA at 50 or 100 nM, scrambled siRNA (scsiRNA), or not transfected (NT). Western blot analysis of cells treated with scsiRNA or siRNA knockdown of BLT1 (upper panel) or ChemR23 (lower panel) receptor was shown in (A). RvE1 at 10⁻⁸ M was added and average [Ca²⁺]_i over time was shown in (B). The change in peak [Ca²⁺]_i was calculated and shown in (C). RvE1 was added and the glycoprotein secretion was measured after a 2-hr incubation (D). Data are mean ± SEM from 4 rats each for [Ca²⁺]_i measurement and secretion measurement. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from non-transfected group.

Fig 6.. RvE1 and LTB₄ activate a common receptor in rat and human conjunctival goblet cells.

Rat (A and B) or human (C and D) cells were stimulated with RvE1 at 10⁻⁸ M or LTB₄ at 10⁻⁸ M alone followed by RvE1 or LTB₄ after 2 min. Average [Ca²⁺]_i over time was shown in time was shown in (A and C). The change in peak [Ca²⁺]_i was calculated and shown in (B and D). Data are mean ± SEM from 4 rats and 5 humans. Arrows indicate the addition of the first and second stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from adding stimuli alone.

Fig 6.. RvE1 and LTB₄ activate a common receptor in rat and human conjunctival goblet cells.

Rat (A and B) or human (C and D) cells were stimulated with RvE1 at 10⁻⁸ M or LTB₄ at 10⁻⁸ M alone followed by RvE1 or LTB₄ after 2 min. Average [Ca²⁺]_i over time was shown in time was shown in (A and C). The change in peak [Ca²⁺]_i was calculated and shown in (B and D). Data are mean ± SEM from 4 rats and 5 humans. Arrows indicate the addition of the first and second stimuli. ^*Indicates significance difference from basal. ^#Indicates significance difference from adding stimuli alone.

Fig 7.. Activation of a common receptor rather than depletion of Ca²⁺_i accounted for the effect of RvE1 and LTB₄ added simultaneously to rat conjunctival goblet cells.

Rat cells were stimulated with RvE1 (10⁻⁸ M) or LTB₄ (10⁻⁸ M) alone, or RvE1 and LTB₄ added together. The average [Ca²⁺]_i level over time was shown in (A); change in peak [Ca²⁺]_i was calculated and shown in (B). RvE1 (10⁻⁸ M) or LTB₄ (10⁻⁸ M) alone or a mixture of RvE1 and LTB₄ together was added and the glycoprotein secretion was measured after a 2-hr incubation (C). Data are mean ± SEM from 6 rats for calcium experiments and 4 rats for secretion experiments. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#indicates significance difference from adding stimuli alone.

Fig 7.. Activation of a common receptor rather than depletion of Ca²⁺_i accounted for the effect of RvE1 and LTB₄ added simultaneously to rat conjunctival goblet cells.

Rat cells were stimulated with RvE1 (10⁻⁸ M) or LTB₄ (10⁻⁸ M) alone, or RvE1 and LTB₄ added together. The average [Ca²⁺]_i level over time was shown in (A); change in peak [Ca²⁺]_i was calculated and shown in (B). RvE1 (10⁻⁸ M) or LTB₄ (10⁻⁸ M) alone or a mixture of RvE1 and LTB₄ together was added and the glycoprotein secretion was measured after a 2-hr incubation (C). Data are mean ± SEM from 6 rats for calcium experiments and 4 rats for secretion experiments. Arrow indicates the addition of stimuli. ^*Indicates significance difference from basal. ^#indicates significance difference from adding stimuli alone.

Fig 8.. Schematic of the activation of BLT1 and ChemR23 receptors by LTB₄ and RvE1.

LTB₄ activates BLT1 receptor in the basal membrane leading to the increase of [Ca²⁺]_i and secretion of high molecular weight glycoprotein across the apical membrane into tears. The activation of BLT1 receptor by LTB₄ is blocked by the BLT inhibitor LY293111, the BLT1 receptor U75302, and BLT1 siRNA. RvE1 activates ChemR23 receptor in the basal membrane as well as BLT 1 receptor leading to the increase of [Ca²⁺]_i and secretion of high molecular weight glycoprotein across the apical membrane into the tears. The RvE1-ChemR23 signal is blocked by ChemR23 siRNA.