Journal: PLoS ONE

Article Title: Nanoparticulate matter exposure results in neuroinflammatory changes in the corpus callosum

doi: 10.1371/journal.pone.0206934

Figure Lengend Snippet: (A) Filtered air (n = 8) or nPM (n = 8) exposed mice stained for C5 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (B) Low magnification representation of region analyzed. (C) C5 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.001). (D) Filtered air (n = 8) and nPM (n = 8) exposed mice stained for C5α (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (E) Low magnification representation of region analyzed. (F) C5α immunostaining in the corpus callosum of nPM exposed animals was significantly greater than in the filtered air group (p = 0.02). (G) Filtered air (n = 18) and nPM (n = 18) exposed mice stained for CD88 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (200x). (H) Low magnification representation of region analyzed. (I) CD88 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.04). * signifies p< 0.05, ** signifies p ≤ 0.001. Error bars represent standard deviation. Scale bars indicate 50 μm.

Article Snippet: Slides were incubated overnight with anti-C5 (mouse 1:50 Hycult Biotech, Netherlands; clone BB5.1), anti-CD88 (rat 1:200 Hycult Biotech, Netherland; HM1076) or rabbit complement component C5α (125kDa) antibody (1:50 Santa Cruz, SC-21941).

Techniques: Staining, Immunostaining, Standard Deviation

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 1. Immunohistochemistry of C5aR in the RCC specimens. FFPE samples of RCC were stained with the anti-C5aR antibody. Representative examples of C5aR-negative RCC without metastasis (A) and C5aR-expressing mRCC (B) are shown. Original, x200 magnification. C5aR, C5a receptor; RCC, renal cell carcinoma; mRCC, metastatic RCC; FFPE, formalin-fixed paraffin-embedded.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Expressing, Formalin-fixed Paraffin-Embedded

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 3. C5a elicits cytoskeletal rearrangement and changes in cellular mor- phology in the C5aR-expressing Renca cells. Renca/empty cells (A-D) and Renca/C5aR cells (E-G) were incubated with C5a (10 nM) and fixed at the indicated time-points. F-actin was visualized by immunofluorescent staining with Alexa 488-conjugated phalloidin (green), and nuclei with TO-PRO-3 (red). Scale bars, 20 µm. Arrowheads and the arrow indicate membrane ruffing and lamellipodia, respectively. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Incubation, Staining, Membrane

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 4. C5a-C5aR axis triggers ERK and PI3K-dependent cellular inva- sion. (A) Renca-derived cells were stimulated with or without 10 nM C5a for 15 min after serum starvation for 24 h. Samples were harvested and subjected to immunoblotting using antibodies indicated. (B and C) Invasion assays were carried out using (B) Renca/empty and Renca/C5aR cells or (C) Renca/C5aR cells only. (C) Medium in both the upper and lower wells were mixed with the indicated kinase inhibitors or vehicle (DMSO). U0126 was used at 20 µM and PI-103 was used at 0.5 µM. Data are presented as the mean ± SD. *P<0.02 and **P<0.01. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Derivative Assay, Western Blot

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.

Article Snippet: The construction and characterization of Chinese hamster ovary (CHO) cells expressing human C5a receptor 1 (C5aR1) and human C5aR1-green fluorescent protein (GFP) have been described previously [ ]. pCMV6 mouse C5aR1 was purchased from OriGene Technologies (catalog number MC208206; GenBank Accession NM_007577).

Techniques: Expressing, Transfection, Positive Control, Negative Control, Flow Cytometry, Knockdown

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value <0.0001; ***).

Article Snippet: The construction and characterization of Chinese hamster ovary (CHO) cells expressing human C5a receptor 1 (C5aR1) and human C5aR1-green fluorescent protein (GFP) have been described previously [ ]. pCMV6 mouse C5aR1 was purchased from OriGene Technologies (catalog number MC208206; GenBank Accession NM_007577).

Techniques: Expressing, Transfection, Knockdown, Flow Cytometry, Negative Control, Gene Expression, Quantitative RT-PCR, Control

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: Production of C5a and expression of C5aR in the injured spinal cord. A, C5a protein concentration in injured WT spinal cord is significantly increased compared with both naive (time point 0) and sham levels at 2 h, 6 h, 12 h, 1 d, 4 d, and 7 d after SCI (n = 4 or 5 per time point, two-way ANOVA with Newman–Keuls post hoc tests). *p < 0.05. **p < 0.01. ****p < 0.0001. B, WT C5a levels are also increased in the plasma in response to SCI, exceeding levels observed in sham-operated mice at 30 min, 2 h, 6 h, 12 h, and 1 d after injury (n = 4 or 5 per time point, two-way ANOVA with Newman–Keuls post hoc tests). *p < 0.05. **p < 0.01. ***p < 0.001. C–E, Representative confocal images showing C5aR expression in the injured spinal cord at 1 and 7 d after injury. C, At 1 d after injury, C5aR appeared present at the lesion epicenter on nucleated Iba1+ cells (arrow) as well as numerous smaller circular/ovoid cells that did not express Iba1 (arrowheads). Scale bar, 6.0 μm. D, At 7 d after injury, C5aR is present on cells with amoeboid morphology in WT mice, which appear to be clustered Iba1+ macrophages/microglia (arrow). Scale bar, 10 μm. E, C5aR expression was also observed on more elongated GFAP+ astrocytes (arrows), alongside C5aR+GFAP− cells with macrophage-like morphology (arrowhead). Scale bar, 13 μm. F, Only a very low level of nonspecific background fluorescence was observed following C5aR staining of lesioned C5ar−/− spinal cord tissue. Scale bar, 35 μm. G, Representative Western blots demonstrating C5aR expression by astrocytes in vitro. Left and middle lanes contain WT and C5ar−/− whole mouse brain homogenates, respectively. Right lane contains protein sample from cultured WT astrocytes.

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: Expressing, Protein Concentration, Fluorescence, Staining, Western Blot, In Vitro, Cell Culture

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: C5ar−/− mice have a dual phenotype after SCI. A, BMS locomotor scoring revealed that C5ar−/− mice have significantly improved hindlimb motor function at 7 d after injury compared with WT mice. This trend reversed with time such that, at 28 and 35 d after SCI, WT mice had regained significantly more motor function than C5ar−/− mice (two-way ANOVA with Bonferroni post hoc tests, n = 8–12). B, Pooled BMS scores for individual mice from various experiments at 7 d after injury (Student's two-sided t test, n = 18–21). C, Graph showing the BMS scores for individual mice at 35 d after injury from longitudinal scores plotted in A (Student's two-sided t test, n = 8–12). D, Postmortem T2*-weighted MRI images showing lesion sites in WT and C5ar−/− mice. Scale bar (top left image): coronal images, 400 μm; sagittal images, 1 mm. E, Quantitative analysis revealed significantly larger lesion core volumes in C5ar−/− mice. Representative reconstructions of lesion cores for each genotype are shown in gray. F, G, A reduction in myelin content was observed in C5ar−/− mice at 35 d after SCI (Student's two-sided t test, n = 8 per group). Scale bar: F (top left image), 200 μm. H, I, Reductions in the proportional area (H) and intensity (I) of GFAP staining were also observed in C5ar−/− mice (Student's two-sided t tests, n = 8 per group). J, K, A more widespread presence of Ly6b.2+ cells (J) and CD3+ T cells (K) was observed in C5ar−/− mice at 35 d after injury (two-way ANOVA with Newman–Keuls post hoc tests, n = 5 per group), as also confirmed by area under the curve analysis (Student's two-sided t test, n = 5 per group). *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: Acute, but not sustained, antagonism of C5aR improves SCI outcomes in Macgreen mice. A, BMS locomotor scores of mice treated with a C5aR antagonist (C5aR-A) for 7 d after SCI have significantly higher BMS scores than vehicle (Veh)-treated mice at 21, 28, and 35 d after injury. *p < 0.05 (two-way ANOVA with Bonferroni post hoc tests, n = 8–10). B, A scatter plot depicting the BMS scores of individual mice at 35 d after SCI. *p = 0.029 (Student's two-tailed t test, n = 8–10). C, D, Data from the ledged tapered beam walk task also indicated that C5aR blockade during the acute period improved long-term recovery, with C5aR-A-treated mice making significantly fewer stepping mistakes (C), and also traversing the beam faster (D) than vehicle-treated SCI controls. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA with Newman–Keuls post hoc tests, n = 6–10). E, F, (Sub)acute C5aR-A treatment resulted in significantly more myelin being present at the lesion epicenter at 35 d after SCI compared with vehicle-treated mice. **p = 0.0088 (Student's t test, n = 5 per group). Scale bar: E (top left), 200 μm. E, G, The GFP+ infiltrate in the lesion core of Macgreen mice was also significantly reduced following the C5aR-A treatment regimen. **p = 0.0061 (Student's two-sided t test, n = 5 per group). H–J, GFAP immunoreactivity at 35 d after SCI was not significantly different between treatment groups based on analysis of both proportional area (I) and staining intensity (J). Scale bar: H (top left), 200 μm. K, Improved recovery from SCI was not sustained with continued C5aR-A administration. *p < 0.05, C5ar−/− + vehicle versus WT + vehicle (two-way ANOVA with Bonferroni post hoc tests, n = 4 or 5).

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: Two Tailed Test, Staining

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: Inflammation in the (sub)acute period of SCI is reduced with C5aR elimination. A–F, Levels of MCP-1 (A), TNF (B), CXCL1 (C), IL-6 (D), IL-1β (E), and IL-10 (F) were all significantly increased at 12 h after SCI compared with sham-operated controls, regardless of genotype. C5aR deficiency did, however, result in significant reductions in CXCL1, IL-6, and IL-1β in the injured spinal cord compared with WT mice. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-way ANOVA with Newman–Keuls post hoc tests, n = 4 or 5). G, H, No significant differences in the Ly6b.2+ inflammatory cell infiltrate were observed between WT and C5ar−/− mice at 1 d (G) and 7 d (H) after SCI. Scale bar: G (bottom image), 40 μm. I, A significant reduction in the number of inflammatory monocytes/macrophages was observed in injured C5ar−/− spinal cord at 7 d after SCI. **p < 0.01 (Student's two-sided t test). n = 5 per group.

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: C5aR deficiency in the peripheral immune compartment does not alter the outcome from SCI. A, Flow cytometry data showing chimerization efficacy. [C5ar−/− → WT] BM chimeras (i.e., WT mice that received a C5ar−/− bone marrow transplant) only express background levels of C5aR on circulating granulocytes, equivalent to the amount of nonspecific staining observed on C5ar−/− cells. ***p < 0.001 (one-way ANOVA with Newman–Keuls post hoc). n = 3–7. B, BMS locomotor scoring revealed no significant differences in functional recovery as a result of select C5aR deficiency in the peripheral immune compartment compared with [WT → WT] controls (two-way ANOVA with Bonferroni post hoc tests; n = 6 or 7). C, Scatter plot showing main BMS scores for individual mice at 35 d after SCI. D, No difference was observed between the experimental groups in the amount of myelin within the ventrolateral white matter at 35 d after SCI (Student's two-sided t test, p > 0.05; n = 6 or 7). Scale bar: D (top), 200 μm. E–G, Quantification of the inflammatory infiltrate also showed no differences between the experimental groups in the number of Ly6b.2+ cells (E), the proportional area of CD11b+ immunoreactivity (F), and the number of CD3+ lymphocytes (G) present at the lesion epicenter (Student's two-sided t tests, p > 0.05; n = 6 or 7).

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: Flow Cytometry, Staining, Functional Assay

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: Signaling through the C5a-C5aR axis promotes astrocyte proliferation in vivo and in vitro. A, Quantification of BrdU+GFAP+ astrocytes at the lesion site of WT SCI mice that were chronically administered C5aR-A (orange) or vehicle (Veh, blue) solution, and vehicle-treated C5ar−/− mice (red). Note the significantly reduced presence of newly generated astrocytes along the rostral and caudal margins of the lesion in the absence of C5aR signaling (two-way ANOVA with Newman–Keuls post hoc tests; n = 4 or 5 per group). *p < 0.05. **p < 0.01. B, A significant, negative correlation was observed between the total number of BrdU+GFAP+ cells and lesion volumes (Pearson's correlation, p < 0.0001). C, C5a stimulates the proliferation of WT astrocytes in vitro in a dose-dependent manner at concentrations >10 nm (one-way ANOVA with Newman–Keuls post hoc tests). *p < 0.05. ***p < 0.001. Graph is representative of three experimental repeats. D, C5ar−/− astrocytes did not proliferate in response to high-dose C5a (50 nm; Student's two-sided t test, p > 0.05). E, Exposure of cultured astrocytes to C5a (50 nm) resulted in a significant increase in the ratio of P-STAT3 to STAT3. Addition of the STAT3 Inhibitor BP-1-102 (10 μm) just before C5a stimulation blocked this increase in STAT3 phosphorylation (one-way ANOVA with Newman–Keuls post hoc tests). **p < 0.01. F, Stimulation of C5ar−/− astrocytes with 50 nm of C5a did not lead to a significant change in STAT3 phosphorylation (Student's two-sided t test, p > 0.05). G, Addition of BP-1-102 (5 μm) blocked C5a-induced astrocyte proliferation in vitro (one-way ANOVA with Newman–Keuls post hoc tests). **p < 0.01. ***p < 0.001. Dotted line indicates the initial number of cells plated. D–F, Data are mean ± SEM, with n = 6 biological replicates. ns, Not significant.

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: In Vivo, In Vitro, Generated, Cell Culture

Journal: The Journal of Neuroscience

Article Title: The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury

doi: 10.1523/JNEUROSCI.5218-14.2015

Figure Lengend Snippet: Diagram showing the proposed dual and time-dependent role of C5aR in SCI. In the (sub)acute period (0–7 d after SCI), activated astrocytes and microglia proliferate and/or migrate to the site of injury. Activation of these cells occurs in part through increased C5a levels as a result of complement activation, which in turn augments their production and release of inflammatory cytokines at the lesion site. Release of CXCL1 is a key signal for neutrophil recruitment to the site of SCI. IL-1β and IL-6 aid in the recruitment of blood monocytes and macrophages, which promote secondary injury if adopting an M1 phenotype. In the postacute to chronic period of SCI (7 d after SCI onwards), C5aR signaling is critically required for STAT3-mediated astrocyte proliferation and glial scar formation, which seals the injury site and prevents the spread of secondary injury. Through its regulation of IL-6 levels, C5aR may also be involved IL-6R-dependent astrocyte proliferation. 1(Anderson et al., 2004, 2005; Nguyen et al., 2008); 2(Lacy et al., 1995; Griffin et al., 2007; Ager et al., 2010); 3(Gasque et al., 1995; Lacy et al., 1995; Woodruff et al., 2008); 4(Acarin et al., 2000; Pineau and Lacroix, 2007; Pineau et al., 2010); 5(Klusman and Schwab, 1997; Romano et al., 1997; Dinarello, 2009); 6(Baggiolini and Clark-Lewis, 1992; Harada et al., 1994; Taub et al., 1996); 7(Gensel et al., 2009; Kigerl et al., 2009; Blomster et al., 2013a); 8(Gensel et al., 2009; Shechter et al., 2009, 2013); 9(Okada et al., 2006); and 10(Bush et al., 1999; Faulkner et al., 2004; Okada et al., 2006; Herrmann et al., 2008; Wanner et al., 2013).

Article Snippet: Rat anti-C5aR antibody (clone 10/92; 1:1000; Hycult Biotech, #HM1077), in combination with goat anti-rat IgG IRDye-700CW (1:10,000; LI-COR, #926-32219), was used to detect C5aR using similar procedures as detailed above.

Techniques: Activation Assay