Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

<div><p>It is well known that B-1 B cells are the main cell type that is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, we unexpectedly found that some epithelial cells also can express rearranged IgM transcript that has natural IgM characteristics, such as germline-encoded and restricted rearrangement patterns. Here we studied IgM expression in human non-B cells and found that IgM was frequently expressed by many human epithelial cancer cells as well as non-cancer epithelial cells. Moreover, CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM. Like the natural IgM, the epithelial cancer cell-derived IgM recognized a series of microbial antigens, such as single-stranded DNA, double-stranded DNA, lipopolysaccharide, and the HEp-2 cell antigen. More important, stimulation of the toll-like receptor 9 (TLR9), which mimics bacterial infection, substantially increased the secretion of IgM in human epithelial cancer cells. These findings indicate that human epithelial cancer cells as well as non-cancer epithelial cells can spontaneously produce IgM with natural antibody activity.</p> </div

HIF-1α activated the signalling pathways controlling IL-33 expression.

<p>A-B, HIF-1α overexpression synergized with TNF-α and IL-1β to induce the activation of ERK and p38 pathways. RASF were transfected with pcDNA3.1 or pcDNA3.1-HIF-1α. 24 h later, the cells were stimulated with 10 ng/ml TNF-α plus 2 ng/ml IL-1β for 5 min, 15 min, and 30 min, respectively. Then the cells were lysised for assessment of the phosphorylation of ERK (A) and p38 (B) by Western blot. C-D, Silencing HIF-1α dampened TNF-α- plus IL-1β-induced ERK and p38 signalling pathway activation. RASF were transfected with HIF-1α siRNA or the control scramble siRNA for 24 h. Then the cells were stimulated with 10 ng/ml TNF-α plus 2 ng/ml IL-1β for 5 min, 15 min, and 30 min, respectively. After that, the cells were harvested for assaying the activation of the ERK (C) and p38 (D) pathways by Western blot. The results were representative of three independent experiments.</p

IL-33 enhanced HIF-1α expression in RASF, forming a self-amplification circuit.

<p>A-D, Knocking-down endogenous IL-33 compromised HIF-1α expression in RASF. RASF were transfected with IL-33 siRNAs or the control scramble siRNA. 24 h later, IL-33 knocking-down efficiency was determined by realtime PCR (A) and cell lysate ELISA (B). Accordingly, HIF-1α expression levels were assessed by realtime PCR (C) and RT-PCR (D) after IL-33 was silenced (Wilcoxon signed-rank test, *<i>p</i><0.05 <i>vs.</i> the control scramble siRNA). E-H, Exogenous IL-33 stimulation promoted HIF-1α expression in RASF. RASF were stimulated with different doses (1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively) of recombinant endotoxin-free human IL-33 for 24 h, then were assayed for HIF-1α expression by RT-PCR (E) and realtime PCR (F) analyses. Similarly, after stimulation with 100 ng/ml recombinant endotoxin-free human IL-33 for different time points (12 h, 24 h, and 48 h, respectively), RASF were harvested for detecting HIF-1α expression by RT-PCR (G) and realtime PCR (H) analyses (Wilcoxon signed-rank test, *<i>p</i><0.05 <i>vs.</i> vehicle control). The results were presented as mean±SEM of four independent experiments.</p

HIF-1α/IL-33 self-amplification circuit exacerbates the inflammation in RA.

<p>Rheumatoid arthritis synovial fibroblast (RASF) hyperplasia leads to tissue hypoxia, which induces upregulated expression of HIF-1α. This upregulated HIF-1α expression provokes IL-33 production by RASF, one of the main sources of IL-33. IL-33 in turn enhances HIF-1α expression in RASF, thus forming a self-amplification circuit that would perpetuate the inflammatory process in RA.</p

Chloroquine abolished effects for TLR9 agonists on IgM secretion in HeLa MR cells.

<p>A, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). The effects of TLR9 agonists were abolished by chloroquine (lower panel). GAPDH, internal control. B, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. *<i>P</i><0.05 <i>vs.</i> vehicle control; **<i>P</i><0.01 <i>vs.</i> vehicle control. C, Western blot analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. D, Western blot analysis showed that IκB was phosphorylated after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine.</p

IgM expression in human non-B cell-derived cancer cell lines.

<p>A, detection of Ig µ and Ig κ transcripts in multiple cancer cell lines by semi-nested RT-PCR. HeLa and HeLa MR, cervical cancer cell lines; SW480, colon cancer cell line; U-2 OS, osteosarcoma cell line; HepG2, hepatic cancer cell line; 293 and 293T, human embryonic kidney cell lines. Raji, human B lymphocytic leukemia cell line, as positive control; GAPDH, internal control. B, flow cytometry study using mouse anti-human Ig µ mAb showed that IgM was localized not only on the plasma membrane but also in the cytoplasm of epithelial cancer cells, especially HeLa MR cells. Red line, isotype control IgG1; Blue line, anti-human IgM. C, Confocal microscopy analysis of HeLa MR cells using mouse anti-human Ig µ mAb showed that IgM was present both on the cell membrane and in the cytoplasm. Mouse IgG, as isotype control. D, whole IgM was detected in HeLa MR cells by non-reducing SDS-PAGE (without β-mercaptoethanol) and Western blot with goat anti-human Ig µ polyclonal antibody. Human IgM, as positive control. E, IgM expression was detected in HeLa MR cells by reducing SDS-PAGE (with β-mercaptoethanol) and Western blot with mouse anti-human Ig µ mAb. Human IgM, as positive control; β-actin, internal control. F, IgM was also detected in the cultural supernatant of HeLa MR cells by reducing SDS-PAGE and Western blot using mouse anti-human Ig µ mAb. Human IgM, as positive control; Medium, as negative control.</p

Expression of BCR-like complex in human epithelial cancer cells.

<p>A, RT-PCR showed that CD79A and CD79B were detected in human cancer cell lines, U-2 OS, HT-29, HeLa MR and HeLa, and that there were two isoforms for both CD79A and CD79B. Raji, as positive control; GAPDH, internal control. The PCR products were separated by gel electrophoresis on 1% agarose and were further confirmed by DNA sequencing. B, Western blot analysis demonstrated the presence of the larger isoform of CD79A and CD79B. β-actin, internal control. C, confocal microscopy analysis demonstrated co-localization (yellow) of CD79A (FITC, green) and Ig M (PerCP/Cy5.5, red) (upper two panels), or CD79B (PE, red) and Ig M (FITC, green) (lower two panels) in HeLa MR cells with or without stimulation with anti-IgM. D, calcium flux analysis by confocal microscopy in HeLa MR cells loaded with Fluo-3/AM and stimulated with goat anti-human IgM (a) or goat IgG as isotype controle (b). Images were captured after stimulation for 0, 1, 15, 30, 60, and 150 seconds. The corresponding time course of the calcium flux is shown. The relative fluorescence from the images was estimated with Leica confocal software. Each line represents the signal derived from a single cell. E, phosphorylation of PKC and Akt downstream of the PLC-γ2 and PI3K signaling pathways, respectively, was detected in HeLa MR cells after stimulation with 20 µg/ml goat anti-human IgM for 5 and 15 minutes, respectively.</p

Soluble CD24 is an inflammatory biomarker in early and seronegative rheumatoid arthritis

Introduction: Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by autoantibody production, joint inflammation and bone destruction. Nearly 1/3 of RA patients with the active disease also exhibit a normal range of ESR and CRP. Here we assessed the performance and clinical significance of soluble CD24 (sCD24) as a biomarker of disease activity in RA. Methods: A total of 269 RA patients, 59 primary Sjogren’s syndrome (SS) patients, 81 systematic lupus erythematosus (SLE) patients, 76 osteoarthritis (OA) patients and 97 healthy individuals (HC) were included in this study. Soluble CD24 in sera were detected by ELISA. Therefore, the concentration of sCD24 was analyzed in RA patients with different disease activity statuses. Results: The sCD24 was significantly increased in RA (2970 pg/mL), compared to other rheumatic diseases (380-520 pg/mL) and healthy individuals (320 pg/mL). Moreover, sCD24 was elevated in 66.67% of early RA and 61.11% of seronegative RA patients. In addition, sCD24 was significantly correlated with the disease duration and inflammatory indicators. Conclusion: The sCD24 could be an inflammatory biomarker in RA patients, especially in early and seronegative patients.</p

Elevated levels of IL-33 in RA patient synovial fluids.

<p>A, levels of IL-33 in the synovial fluids of RA patients (n = 50) and OA patients (n = 30) were detected by ELISA. The differences between the two groups were evaluated by Student’s <i>t</i> test. (*<i>p<</i>0.05). B, IL-33 expression levels were also determined by realtime PCR in the synovial fibroblasts of RA patients (RASF, n = 6) and OA patients (OASF, n = 4), one of the main producers of IL-33 (Wilcoxon signed-rank test, *<i>p</i><0.05). C, Similarly, the expression levels of HIF-1α in RASF (n = 7) and OASF (n = 4) were also determined by realtime PCR (Wilcoxon signed-rank test, *<i>p</i><0.05).</p

Knocking-down HIF-1α compromised IL-33 expression in RASF.

<p>A, RASF were transfected with HIF-1α siRNA or the control scramble siRNA. 24 h later, the cells were stimulated with 10 ng/ml TNF-α plus 2 ng/ml IL-1β. HIF-1α knockdown efficiency was confirmed by realtime PCR (A) and cell lysate ELISA (B) analyses. The IL-33 transcriptional levels were determined by realtime PCR (C) and RT-PCR (D) 4 h after stimulation. And also, the protein levels of IL-33 were assayed by cell lysate ELISA analysis (E) 24 h after stimulation (Wilcoxon signed-rank test, *<i>p</i><0.05). The results were presented as mean±SEM of five independent experiments.</p