Journal: Molecular Therapy

Article Title: A Novel C5a-neutralizing Mirror-image ( l -)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

doi: 10.1038/mt.2013.178

Figure Lengend Snippet: NOX-D20 binds to C5 but does not inhibit complement-mediated hemolysis. SPR measurement of NOX-D20 binding to human ( a ) C5a, ( b ) C5a(desArg), and ( c ) C5. Kinetic rate constants k a and k d are shown as mean ± SEM. Data are representative for at least three individual measurements. ( d ) Human serum pretreated with NOX-D20 (black squares) or C5-binding aptamer C5C6 (black triangles) was incubated with opsonized sheep erythrocytes. Hemolysis was quantified by photometric measurement of hemoglobin in the supernatant at 405 nm. Normalized data representative for three independent experiments is shown. RU, response units.

Article Snippet: Recombinant human and mouse C5a was from R&D Systems (Wiesbaden, Germany), recombinant human C5a(desArg) from Hycult Biotech (Beutelsbach, Germany), and human C5 purified from serum from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Binding Assay, Incubation

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

doi: 10.1038/srep21391

Figure Lengend Snippet: ( a ) Exhaustive cytokine array for sera from mice fed HFD. Cytokine array showed that C5a upregulates most in various cytokines by HFD. Sera pooled from eight mice were analyzed. (b) Feeding HFD for 4 weeks increased serum C5a levels (left) (n = 4 and 4, respectively). In addition, HFD increased C5 mRNA levels, the precursor of C5a, in the liver (right) (n = 8 and 8, respectively). Data are presented as the mean ± SEM. ** P < 0.01 and * P < 0.05 by unpaired; 2-tailed Student’s t test. (c) Leukocyte recruitment in wt mice fed NC with recombinant mouse C5a after 1.5 h. Leukocyte recruitment was not detected when treated with vehicle (left panel), but observed when treated with C5a (right panel). Bar, 50 μm. C5a significantly increased leukocyte recruitment (n = 5 and 5, respectively). Data are presented as the mean ± SEM. ** P < 0.01 by unpaired; 2-tailed Student’s t test. (d) 2 μg/kg of recombinant murine C5a was administered to wt mice fed NC. C5a induced neutrophilia for 1.5 hours (n = 4 and 4, respectively). Data are presented as the mean ± SEM. * P < 0.05 by unpaired; 2-tailed Student’s t test. (e) Leukocyte recruitment in wt mice fed HFD for 4 weeks with C5aR antagonist. Administration of C5aR antagonist diminished leukocyte recruitment induced by HFD (** P < 0.01) (n = 4 and 4, respectively). Data are presented as the mean ± SEM. ** P < 0.01 by unpaired; 2-tailed Student’s t test. Bar, 50 μm.

Article Snippet: Differentiated cells were treated with 3nM of recombinant human C5a (PeproTech, inc).

Techniques: Recombinant

Journal: Scientific Reports

Article Title: Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

doi: 10.1038/srep21391

Figure Lengend Snippet: ( a) HFD for 4 weeks significantly increased MCP-1 levels in sera of mice (n = 8 and 8, respectively). Data are presented as the mean ± SEM. ** P < 0.01 by unpaired; 2-tailed Student’s t test. (b) MCP-1 mRNA levels in various tissues including bone marrow (n = 8 and 8, respectively), leukocytes (n = 4 and 5, respectively), spleen (n = 7 and 8, respectively), aorta (n = 6 and 8, respectively), visceral fat (n = 8 and 8, respectively) and liver (n = 8 and 7, respectively), of mice fed HFD or NC, involving 4 independent experiments. HFD significantly increased the MCP-1 level in circulating leukocytes. Data are presented as the mean ± SEM. * P < 0.05 by two-way ANOVA with Bonferroni’s post test. (c) HFD increased MCP-1 mRNA levels in neutrophils such as Ly-6G + leukocytes, but not Ly-6G − leukocytes (n = 3/each group), involving 4 independent experiments. Data are presented as the mean ± SEM. * P < 0.05 by two-way ANOVA with Bonferroni’s post test. (d) MCP-1 level in sera was measured by ELISA when C5aR antagonist was administrated in wt mice fed HFD for weeks. C5aR antagonist significantly decreased serum MCP-1 levels increased by HFD (n = 4 and 4, respectively). Data are presented as the mean ± SEM. * P < 0.05 by unpaired; 2-tailed Student’s t test. (e) mRNA level of MCP-1 in neutrophil-like HL-60 treated with 3nM recombinant C5a. C5a significantly increased MCP-1 expression level compared to control (n = 5 and 5, respectively), involving 4 independent experiments. Data are presented as the mean ± SD. * P < 0.05 by unpaired; 2-tailed Student’s t test.

Article Snippet: Differentiated cells were treated with 3nM of recombinant human C5a (PeproTech, inc).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Control

Journal: Scientific Reports

Article Title: Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

doi: 10.1038/srep21391

Figure Lengend Snippet: ( a) Flow cytometry for leukocytes in femoral artery of wt mice or CCR2 −/− mice fed NC or HFD. CD45 + cells were gated by the square (upper). Leukocytes in the intima significantly increased in wt mice fed HFD compared with NC. However, the CD45-positive cells did not change in CCR2 −/− mice between HFD and NC (n = 7/each group). These leukocytes were positive for CD11b, CD11c, and CCR2, involving 8 independent experiments. An isotype control antibody was used as the negative control (gray filled). Data are presented as the mean ± SEM. **** P < 0.0001 by two-way ANOVA with Bonferroni’s post test. (b) Flow cytometric analysis for single-cell suspension of femoral arteries of wt mice fed HFD for 4 weeks when neutrophils in peripheral blood were depleted by specific antibody. Neutrophil depletion significantly decreased the intimal leukocytes in the femoral artery (n = 6 and 6, respectively). Data are presented as the mean ± SEM. * P < 0.05 by unpaired; 2-tailed Student’s t test. (c) Flow cytometric analysis for single-cell suspension of the femoral arteries of wt mice treated with C5a for 4 weeks. C5a significantly increased leukocytes in the femoral artery (n = 8 and 7, respectively). Data are presented as the mean ± SEM. * P < 0.05 by unpaired; 2-tailed Student’s t test.

Article Snippet: Differentiated cells were treated with 3nM of recombinant human C5a (PeproTech, inc).

Techniques: Flow Cytometry, Control, Negative Control, Suspension

Journal: Scientific Reports

Article Title: Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

doi: 10.1038/srep21391

Figure Lengend Snippet: HFD increased C5 expression levels in the liver and serum C5a levels. C5a induced neutrophilia and neutrophil recruitment in the femoral artery. Neutrophil recruitment contributed to the CD11c + intimal leukocytes in the femoral artery.

Article Snippet: Differentiated cells were treated with 3nM of recombinant human C5a (PeproTech, inc).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: The co-expression of C5AR1 and its ligand C5a in intracranial aneurysm lesions. Specimens of intracranial aneurysm (IA) lesions (Aneurysm) or control arterial walls (Superficial temporal artery in ( A ), Anterior cerebral artery-olfactory artery bifurcation in ( B )) from human cases ( A ) or rats ( B ) were harvested and examined in immunohistochemistry. The representative images of immunofluorescent staining of IA lesions for C5AR1 (green), C5a/C5a des-arg (red in ( A )), C5 (red in ( B )), and merged images with nuclear staining by DAPI (blue) are shown. A demagnified image of lower panels in ( B ) is shown on the right. The square in the demagnified image corresponds to the area shown in the magnified images. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: Tissue-type Plasminogen Activator as an up-stream factor to produce C5a from C5. ( A ) The expression of tissue-type Plasminogen Activator (tPA) and Plasminogen in intracranial aneurysm (IA) lesions. IA lesions from human cases or induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for tPA (green), Plasminogen (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( B ) Co-expression of tPA with C5a in IA lesions. Human IA lesions were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5a/C5a des-arg which is the derivative of C5a (green), tPA (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( C ) Enzymatic cleavage of C5 into C5a by Plasmin. Recombinant C5 (100 μg/ml) was co-incubated with each dose of recombinant Plasmin as indicated (0–100 μg/ml) for 1.5 h in a cell-free system. The incubation mixture was subjected to western blot analyses. The representative images from the western blot analyses to detect C5 or C5a are shown. M; size marker. ( D ) The alternation in the coagulation-fibrinolysis system in IA lesions of rats. The heat map of genes related with the coagulation-fibrinolysis system from the comprehensive gene expression profile data set (GEO accession: GSE161044) is shown.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Recombinant, Incubation, Western Blot, Marker, Coagulation

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory factors. ( A – C ) The C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory genes in phorbol myristate acetate-primed cell line from human case with diffuse histiocytic lymphoma, U937 cell line. Cells were treated with recombinant C5a as indicated in the panels (0–10 h in ( A ), 0–100 nM in ( B ), 10 nM and 10 h in ( C ). Some cells were pre-treated with the selective C5AR1 inhibitor, W54011 (300 nM, 30 min) prior to the treatment with recombinant C5a. Total RNA was then purified from treated cells subjected to quantitative RT-PCR analyses to examine the induction of TNF (a gene for TNF-α), IL1B or PTGS2 (a gene for COX-2; cyclooxygenase-2). Data represents as mean ± s.e.m (( A ), n = 4; ( B , C ), n = 6). Statistical analysis was done by the Wilcoxon rank sum test in ( A ), the Kruskal–Wallis test followed by the Steel–Dwass test in ( B , C ). *p < 0.05. ( D ) Co-expression of C5a with pro-inflammatory factors in intracranial aneurysm (IA) lesions. IA lesions induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5ar1 (green), TNF-α (red), IL-1β (red), COX-2 (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Recombinant, Purification, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining