Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen : functionality and stability studies

Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab′)(2) fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool

Effect of ADAM17 silencing on cell motility.

<p>(<b>A</b>) Exemplary trajectories of migrating wild type (WT), mock-transfected (M3) and ADAM17-silenced (S35) MC38CEA cells displayed in circular diagrams drawn with the initial point of each trajectory placed at the origin of the plot. Each panel shows the trajectories of 50 cells. The variables of cell movement are presented below diagrams. CME, coefficient of movement efficiency; *<i>P</i><0.05 (<i>vs.</i> wild type and mock-transfected cells). Data are from one representative experiment. Comparable results were obtained in the second experiment with M3 and S35 as well as in two experiments, in which the movements of WT, M2, and S3 were analyzed. (<b>B, C</b>) Wound healing assay (<b>B</b>) Representative wound images. Micrographs taken at 0 and 24 h after scratch wounding of cell monolayers. (<b>C</b>) Quantification of the results of two independent experiments performed in duplicates for each cell line. The lengths of cell displacement are expressed as a difference between average pre- and post-healing wound. * – <i>P</i><0.01 <i>vs.</i> M3. (<b>D</b>) Influence of rmNRG-1β on mock-transfected- and ADAM17-silenced MC38CEA cells – representative wound images. Wounded monolayers were incubated for 22 h in serum-free medium in the absence (control) or in the presence of rmNRG-1β. (<b>E</b>) Quantification of the results from a single experiment representative of three performed. * – <i>P</i><0.01 <i>vs.</i> the same cell line incubated w/o rmNRG-1β.</p

Expression of growth factors and growth factor receptors by MC38CEA colon carcinoma.

<p>(<b>A</b>) RT-PCR analysis of the expression of TGFα, EGFR and ErbB4 in MC38CEA cells in culture, in MC38CEA tumors and in positive controls: murine brain endothelial cells (MBE) and murine breast cancer cell line (4T1) and murine brain. cDNA from: M – mock-transfected cells, TM – mock-transfected tumors, S – ADAM17-silenced cells, TS – ADAM17-silenced tumors. Amplification of cDNA coding for elongation factor 2 (EF2) was performed as a control of samples' quality. (<b>B</b>) Analysis of the effect of exogenous EGF on ³H-thymidine incorporation into MC38CEA or 4T1 cells (positive control). * – <i>P</i><0.01 <i>vs.</i> control. (<b>C</b>) Quantitative RT-PCR analysis of ErbB2 and ErbB3 expression in ADAM17-silenced (S2, S3) and mock-transfected (M1, M2, M3) cell lines and in control cell lines, MBE and 4T1 as well as in murine tissues. (<b>D</b>) The levels of NRG-1 released to the medium from ADAM17-silenced (S2, S3) and mock-transfected (M1, M3) cells measured by ELISA. * – <i>P</i><0.01 <i>vs.</i> M1 and M3. (<b>E</b>) Western blotting analysis of ErbB2 phosphorylation in ADAM17-silenced (S2, S3), mock-transfected (M1, M2, M3) and wild-type (wt) MC38CEA cells incubated in the serum-free medium alone or in the medium enriched in exogenous rmNRG-1β. GAPDH was used as a loading control. Quantification of Western blot signals is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050791#s2" target="_blank">Materials and Methods</a> and is based on at least 3 independent experiments for a given cell line. * – <i>P</i><0.01 <i>vs.</i> the same cell line incubated w/o rmNRG-1β; & – <i>P</i><0.001 <i>vs.</i> M1. All data in panels A, B, C and D come from at least 3 independent experiments performed in duplicates.</p

Effect of ADAM17 silencing on proliferation and viability of MC38CEA cells.

<p>The cells were grown in serum-free or serum-low DMEM and their growth and viability were monitored daily using the MTT assay. Bars represent average values ± SD from 3 independent experiments performed in sextuplicates.</p

The effect of ADAM17-silencing on tumor angiogenesis and lymphangiogenesis.

<p>(<b>A</b>) Representative images of mock-transfected and ADAM17-silenced tumors on the 17^th day after s.c. injection of tumor cells. (<b>B</b>) Representative micrographs of peripheral tumor tissues and measurements of the areas covered by CD31-positive cells. Nuclei stained with DAPI (blue) and blood vessel endothelial cells stained with anti-CD31 (red). Experiment was performed twice with 5 tumors of each type analyzed per experiment. Five random fields were analyzed for each tumor section. * – <i>P</i><0.01 <i>vs</i> M2. (<b>C</b>) Relative levels of VEGF-A mRNA analyzed by qRT-PCR and relative VEGF-A concentration in the culture media of mock-transfected and ADAM17-silenced MC38CEA cell lines. The average level of VEGF-A mRNA in mock-transfected (M2) cells was taken as 1 and the average concentration of VEGF-A in M2 culture medium was taken as 100%. Bars represent average values ± SD from 4 independent experiments performed in duplicates. * – <i>P</i><0.01 <i>vs</i> M2 and M3. (<b>D</b>) Zymographic analysis of gelatinase activity in the culture media of mock-transfected (M1 and M3) and ADAM17-silenced (S2 and S3) MC38CEA cell lines. The result (shown as a photographic negative) is representative of 4 performed analyses. (<b>E</b>) Immunochemical analysis of lymphangiogenesis in peritumoral areas of mock-transfected and ADAM17-silenced MC38CEA tumors on the 17^th day after s.c. injection of tumor cells. The representative sections were stained with DAPI to visualize nuclei and immunostained with anti-LYVE-1, anti-CD45, and anti-CD11b antibodies. Single images and overlays of LYVE-1/CD11b and CD45/CD11b are presented. White lines indicate arbitrarily recognized borders between tumors (denoted as T) and peritumoral areas. Arrows indicate exemplary elongated mock-transfected LYVE-1⁺ cells arranged in a regular pattern.</p

Analysis of ADAM17-silencing in MC38CEA cells.

<p>(<b>A</b>) Quantitative RT-PCR analysis of ADAM17 mRNA levels in the wild-type MC38CEA (WT) and in the MC38CEA cell lines stably transfected with control shRNA (M1, M2, M3), or with ADAM17 shRNAs (S2, S3, S35). * – <i>P</i><0.01 <i>vs.</i> WT and each M cell line. (<b>B</b>) The levels of TNF released to the medium from mock-transfected or ADAM17-silenced MC38CEA cell lines incubated in the absence or presence of PMA, measured by ELISA. Bars represent average values ± SD from 3 independent experiments performed in duplicates. * – <i>P</i><0.01 <i>vs.</i> unstimulated cells.</p

Analysis of the cytokine profiles in MC38CEA tumors and their cytotoxic effects on MC38CEA cells.

<p>(<b>A</b>) Relative protein and transcript levels of TNF, IFNγ and MCP-1 in lysates from wild type (WT), mock-transfected (M2) and ADAM17-silenced (S3) MC38CEA tumors measured by cytometric bead array immunoassay (CBA) or qRT-PCR, respectively. Average levels of each cytokine or its mRNA in M2 and/or WT tumor lysates were taken as 100% (CBA) or 1 (qRT-PCR). Control grups were as follows: M2 (three experiments, 5 mice per each experiment), WT (5 mice in one experiment) and both M2 (4 mice) and WT (5 mice) in one experiment. (<b>B</b>) Relative levels of TNF, IFNγ, and MCP-1 in sera from mice bearing WT, mock-transfected (M2), and ADAM17-silenced (S3) MC38CEA tumors at the end of the experiments. (<b>C, D</b>) The influence of TNF and IFNγ on MC38CEA viability. (<b>C</b>) Mock-transfected and ADAM17-silenced MC38CEA cells were incubated in DMEM/2% FCS with TNF (10 ng/ml) or IFNγ (50 ng/ml) or with TNF+IFNγ for 24, 48 or 72 h and then cell viability was assayed using MTT test. (<b>D</b>) Mock-transfected and ADAM17-silenced MC38CEA cells were incubated for 48 h in DMEM/2% FCS with TNF or IFNγ or with TNF+IFNγ at various concentrations and then cell viability was assayed using MTT test. (<b>E</b>) The influence of TNF and IFNγ on caspase activity in mock-transfected (M1 and M3) and ADAM17-silenced (S2 and S3) MC38CEA cells. Bars represent average values ± SD—for (<b>A</b>) and (<b>B</b>) from 5 independent experiments with at least 5 mice per each experimental group, * – <i>P</i><0.05 <i>vs.</i> M2 and WT controls; for (<b>C</b>) and (<b>D</b>) from 3 independent experiments performed in triplicates, * – <i>P</i><0.05 <i>vs.</i> untreated controls; (<b>E</b>) from 4 independent experiments performed in sextuplicates, * – <i>P</i><0.01 <i>vs.</i> untreated controls.</p