Neutrophils Are Essential As A Source Of Il-17 In The Effector Phase Of Arthritis

<div><p>Objective</p><p>Th17 has been shown to have a pivotal role in the development of arthritis. However, the role of IL-17 in the T cell-independent effector phase has not fully been examined. We investigated whether IL-17 is involved in the effector phase of arthritis by using K/BxN serum-induced arthritis model.</p><p>Methods</p><p>K/BxN serum was transferred into IL-17 knockout (KO) mice, SCID mice and their control mice, and arthritis was evaluated over time. In order to clarify the source of IL-17 in the effector phase, neutrophils or CD4+ T cells collected from IL-17 KO or control mice were injected into IL-17 KO recipient mice together with K/BxN serum. To examine if neutrophils secrete IL-17 upon stimulation, neutrophils were stimulated with immune complex in vitro and IL-17 in the supernatant was measured by ELISA.</p><p>Results</p><p>K/BxN serum-induced arthritis was much less severe in IL-17 KO mice than in WT mice. Since K/BxN serum-transferred SCID mice developed severe arthritis with high serum IL-17 concentration, we speculated neutrophils are the responsible player as an IL-17 source. When wild type (WT) but not IL-17 KO neutrophils were co-injected with K/BxN serum into IL-17 KO mice, arthritis was exacerbated, whereas co-injection of WT CD4+ T cells had no effect. In vitro, stimulation of neutrophils with immune complexcaused IL-17 secretion.</p><p>Conclusions</p><p>Neutrophils are essential as a source of IL-17 in the effector phase of arthritis. The trigger of secreting IL-17 from neutrophils may be immune complex.</p></div

A larger number of TAGLN2⁺ B-cells is observed in lymph nodes and kidneys in SLE patients compared to those in controls.

<p>(A-B) Double immunofluorescence analysis of TAGLN2 and the B-cell marker CD20 is shown. Nuclei were stained with DAPI. (A) In control lymph nodes, TAGLN2⁺ B-cells were observed in follicular areas (original magnification, x200). (B) TAGLN2⁺B-cells were distributed from the follicular/germinal center (GC) to perifollicular areas in SLE lymphadenopathy (original magnification, x200). HE staining on the left (original magnification, x40). (C) The ratio of TAGLN2+ cells to CD20+ cells in SLE and controls was calculated. Significantly higher numbers of TAGLN2-positive cells were seen in B-cells in lymph nodes associated with SLE (n = 5) compared to those in controls (n = 5). Data are presented as the mean ± SEM. *p<0.05. (D) Right, a few TAGLN2⁺ CD20⁺ B-cells (arrows) are seen within B-cell aggregates in the interstitium of lupus nephritis, whereas no TAGLN2⁺ B-cells are observed in the control kidney sample (Left). (original magnification, x400). (E) Left, <i>TAGLN2</i> mRNA levels in CD19⁺ B-cells in healthy donors (HC, n = 6) and SLE patients (n = 9) as determined by qRT-PCR (mean ± SEM, 0.98 ± 0.10 in HC and 0.79 ± 0.04 in SLE) (p>0.05). Right, <i>TAGLN2</i> mRNA levels in CD19⁺CD27⁺ memory B-cells in healthy donors (HC, n = 11) and SLE patients (n = 14) (the mean of SEM, 1.0 ± 0.11 in HC and 1.2 ± 0.14 in SLE) (p>0.05.).</p

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

<div><p>Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and <i>TAGLN2</i> mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2⁺B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19⁺ B-cells and CD19⁺CD27⁺ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. <i>TAGLN2</i>-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of <i>TAGLN2</i>-knockdown Raji cells showed decreased expression of the genes associated with immune function including <i>CCR6</i> and as well as of those associated with regulation of the actin cytoskeleton including <i>ABI2</i>, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.</p></div

Down-regulated genes related to the immune system.

<p>Down-regulated genes related to the immune system.</p

Pathway analysis of microarray data. Functional grouping of significant, differentially expressed down regulated genes in <i>TAGLN2</i> siRNA Raji cells versus the control (p<0.01).

<p>Pathway analysis of microarray data. Functional grouping of significant, differentially expressed down regulated genes in <i>TAGLN2</i> siRNA Raji cells versus the control (p<0.01).</p

<i>TAGLN2</i> knockdown results in impaired phosphorylation of PLCγ, which is related to the actin-linked signaling pathway and inhibition of cell migration.

<p>(A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of <i>TAGLN2</i>-knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the <i>TAGLN2</i>-KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p<0.05. (C) Significant inhibition of CXCL13-dependent <i>TAGLN2</i> KD Raji cell chemotaxis as compared with negative controls. *p<0.05. RFU, relative fluorescence units. Migratory cells were lysed and quantified using fluorescent dye. The relative quantification was used to determine the change between various samples. Data were normalized by designating one sample in negative controls as equal to 1. Then, the ratiometric results were used to scale all values relative to that sample. (D) The CXCR5 and IgM expression levels were assessed by flow cytometry in <i>TAGLN2</i> KD and control Raji cells. <i>TAGLN2</i> KD appeared to have no effect on the surface expression of CXCR5 and IgM. Gray shading indicates isotype control.</p

TAGLN2 colocalizes with F-actin and moves together to the periphery after stimulation. TAGLN2-GFP overlaps with LifeAct-RFP in Raji B cells at the outer actin ring.

<p>Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.</p

<i>TAGLN2</i> mRNA is induced upon BCR stimulation and significantly expressed on activated B-cells in the germinal center.

<p>(A) Immunohistochemistry showing TAGLN2 localized in the germinal center in human secondary lymphoid tissues (left) and Bcl-6 expression, a marker of germinal center B-cells (right) (original magnification, x400). GC, germinal center. The figure is representative of 5 tonsils examined. (B) Double immunofluorescence analysis of TAGLN2 (green) and the B-cell marker CD20 (red), showing coexpression of TAGLN2 and CD20 in the germinal center. Nuclei were stained with DAPI (arrows, original magnification, x400). (C) <i>TAGLN2</i> mRNA is induced upon BCR stimulation. <i>TAGLN2</i> mRNA levels were measured in primary human B-cells from six different healthy donors at the indicated time points after IgM+IgG stimulation using qRT-PCR. At each time point, the <i>TAGLN2</i> mRNA levels in anti-IgM+IgG stimulated cells (stim) are expressed fold change relative to the levels in non-stimulated (non-stim) cells. The mean <i>TAGLN2</i> mRNA expression ratio was 0.93-fold at 1 h, 1.09-fold at 6 h, and 2.85-fold at 24 h after stimulation, suggesting that <i>TAGLN2</i> mRNA was induced by 24-h B-cell activation (p<0.05, paired t-test vs. non-stimulated control). Data are presented as the mean ± SEM. *p<0.05.</p

Top 30 genes downregulated in TAGLN2 knockdown versus control cells.

<p>Top 30 genes downregulated in TAGLN2 knockdown versus control cells.</p

TAGLN2 is associated with the functional gene groups of the immune system including <i>CCR6</i> and the regulation of the actin cytoskeleton including <i>ABI2</i>.

<p>(A) Validation of the microarray data regarding the change in expression of specific genes in <i>TAGLN2</i>-knockdown Raji cells compared to control cells. Expression of the indicated genes was assessed using qRT-PCR and is indicated as fold change relative to that in control Raji cells transfected with scrambled siRNA. Results are the mean ± SEM of triplicate experiments. *p<0.05. (B) CCR6 is colocalized with TAGLN2⁺B-cells in GC areas of enlarged follicles in lymph nodes in SLE patients. Double immunofluorescence of CCR6 and TAGLN2 was performed. Nuclei were stained with DAPI. Co-localization of CCR6 and TAGLN2 is indicated in yellow in the merged image (original magnification, x400). GC, germinal center.</p