Neuraminidase activity mediates IL-6 production by activated lupus-prone mesangial cells

Abstract

The development of nephritis is a leading cause of morbidity and mortality in lupus patients. Although the general pathophysiological progression of lupus nephritis is known, the molecular mediators and mechanisms are incompletely understood. Previously, we demonstrated that the glycosphingolipid (GSL) catabolic pathway is elevated in the kidneys of MRL/lpr lupus mice and human lupus patients with nephritis. Specifically, the activity of neuraminidase (NEU) and expression of Neu1, an enzyme in the GSL catabolic pathway is significantly increased. To better understand the role and mechanisms by which this pathway contributes to the progression of LN, we analyzed the expression and effects of NEU activity on the function of MRL/lpr lupus-prone mesangial cells (MCs). We demonstrate that NEU1 and NEU3 promote IL-6 production in MES13 MCs. Neu1 expression, NEU activity, and IL-6 production are significantly increased in stimulated primary MRL/lpr lupus-prone MCs, and blocking NEU activity inhibits IL-6 production. NEU1 and NEU3 expression overlaps IgG deposits in MCs in vitro and in renal sections from nephritic MRL/lpr mice. Together, our results suggest that NEU activity mediates IL-6 production in lupus-prone MCs possibly through an IgG-receptor complex signaling pathway.

Keywords: IL-6; lupus nephritis; mesangial cells; neuraminidase.

Fig. 1.

Renal NEU activity is elevated and NEU1 and NEU3 are highly expressed in MCs of nephritic lupus mice. A: NEU activity was measured in kidney homogenates from 8- to 10-wk-old prenephritic and 23- to 28-wk-old nephritic NZM2410 mice. P value was calculated as described in materials and methods. Immunohistochemistry analyses and semiquantitative measures of staining for NEU1 and NEU3 on renal sections of early and late disease stage MRL/lpr (B) and NZM2410 (C) mice. Images are representative of three mice from each group, which were evaluated by a pathologist. Percentage of high positive and positive NEU1- and NEU3-stained glomeruli are shown to the right of images. Staining in 8–12 glomeruli per animal were evaluated as described in materials and methods with values of individual glomeruli presented.

Fig. 2.

GSL catabolism upregulates Il-6 gene expression and IL-6 production by MES13 MCs. A: MES13 MCs were cultured with LacCer and GlcCer, mRNA isolated, and cytokine gene expression was measured by real-time PCR in reverse-transcribed mRNA. Data are an average of two or three independent experiments with gene expression presented as fold increase in LacCer/GlcCer-treated cells over the gene expression in vehicle-treated cells. B and C: MES13 mouse MCs were transfected with a NEU1, NEU3, or empty expression plasmid in the presence or absence of HA-IgG at increasing concentrations for 24 h. IL-6 (B) and MCP-1 (C) in media were measured by ELISA and normalized to total protein. The data are representative of three independent experiments with similar results. P values were calculated as described in materials and methods.

Fig. 3.

MES13 MCs produce IL-6 in a dose-dependent manner in response to NEU1 or NEU3 overexpression in the absence of stimulation. MES13 mouse MCs were transfected with increasing concentrations of a NEU1 (A and B) or NEU3 (C and D) expression plasmid and media collected 24 h (not shown) and 48 h (presented) posttransfection. IL-6 (A and C) and MCP-1 (B and D) levels in media were measured by ELISA and normalized to total protein. Data are representative of at least three transfections with similar results. P values were calculated as described in materials and methods. Type 3 main effect P value for A and C, P < 0.0001. Post hoc P values are provided in the graphs.

Fig. 4.

HA-IgG stimulation of primary MRL/lpr lupus-prone MCs increases Neu1 message levels, NEU activity, IL-6, and MCP-1 production. Primary MRL/lpr MCs were grown out of glomeruli isolated from prenephritic mice and stimulated with HA-IgG. A: cells were treated with increasing concentrations of HA-IgG (0.5, 2.5, 7.5, 22.5, and 67.5 μg/ml) for 24 h. Neu1 and Neu3 mRNA levels were measured in cells by real-time RT-PCR. Results are an average of three independent experiments. B: NEU activity was measured in live cells 24 h after cells were treated with 22.5 μg/ml of HA-IgG or vehicle only. Substrate 4MU-NANA was added to cells, and fluorescence was measured at the indicated time points. Results are an average of three independent experiments. C and D: cells were treated with increasing concentrations of HA-IgG for 24 h. IL-6 (C) and MCP-1 (D) levels in media were quantified by ELISA and normalized to total protein. Results are representative of at least three independent experiments with similar results. P values were calculated as described in materials and methods. For all graphs, post hoc P values are provided on the graphs: *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Type 3 main effect P values: A, P < 0.0001; C, P < 0.0001; D, P = 0.0008.

Fig. 5.

Inhibiting NEU activity blocks HA-IgG-stimulated IL-6 and MCP-1 production by MRL/lpr lupus prone MCs. A and B: primary MRL/lpr MCs were treated with 22.5 μg/ml of HA-IgG over time. IL-6 (A) and MCP-1 (B) levels were measured in media by ELISA and normalized to total protein. C and D: primary MRL/lpr MCs were cultured with increasing concentrations of the NEU inhibitor oseltamivir phosphate (OP) for 24 h followed by stimulation with 22.5 μg/ml of HA-IgG. IL-6 (C) and MCP-1 (D) levels in the media 6 h (presented) and 3 h (not shown) after stimulation were measured by ELISA and normalized to total protein. E and F: cells were transfected with Neu1 siRNA or nontarget siRNA and stimulated with 45 μg/ml of HA-IgG for 24 h and Neu1 mRNA levels in cells (E) and IL-6 production in media (F) were measured. All results are representative of at least three independent experiments with similar results. For all graphs, post hoc P values are provided on the graphs: *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Type 3 main effect P values: A, P = 0.0026; B, P < 0.0001; C, P < 0.0001; D, P = 0.0018.

Fig. 6.

Inhibiting NEU activity blocks lupus serum-stimulated IL-6 production by MRL/lpr lupus prone MCs. Primary MRL/lpr MCs were treated with increasing concentrations of MRL/lpr serum or B6 serum for 24 h. IL-6 (A) and MCP-1 (B) levels in media were measured by ELISA and normalized to total protein. C: MRL/lpr MCs were cultured with increasing concentrations of OP for 24 h followed by stimulation with 20% MRL/lpr lupus serum. IL-6 levels were quantified 3 h (not shown) and 6 h (presented) after stimulation by ELISA and normalized to total protein. All results are representative of at least three independent experiments with similar results. For all graphs, post hoc P values are provided on the graphs: ***P < 0.001; ****P < 0.0001. Type 3 main effect P values: A, P < 0.0001; C, P < 0.0001.

Fig. 7.

NEU1 and NEU3 expression overlaps with IgG deposits on the surface of MRL/lpr lupus-prone MCs and in the glomeruli of nephritic MRL/lpr mice. MRL/lpr lupus-prone MCs were fixed (but not permeablized) and stained with isotype (A) or HA-IgG for 1 h followed by Alexa Fluor 594 goat anti-mouse IgG secondary antibody (B–D). NEU1 (C) and NEU3 (D) were detected using rabbit anti-NEU1 and anti-NEU3 primary antibodies, respectively followed by a FITC-conjugated anti-rabbit secondary. Staining was not observed with secondary antibodies alone (not shown). HA-IgG, red; NEU1 and NEU3, green; nuclei, blue. Results are representative of two independent experiments. E and F: renal sections from nephritic MRL/lpr mice were stained for IgG deposition with Alexa Fluor 594 goat anti-mouse IgG secondary antibody and for NEU1 (E) or NEU3 (F), as indicated above. Bright-field and fluorescent images for IgG (red) and NEU1 or NEU3 (green) are presented independently, and then a composite image of IgG, NEU1, or NEU3, and nuclei staining (blue) is presented. White arrows indicate areas of overlap, which are largely mesangial, between IgG and NEU1 or NEU3 in the composite images.