Survival of TH-positive neurons and outgrowth from SIRPα mutant cultures.

<p>TH-immunohistochemistry over the tissue slice area demonstrates that the TH-positive neurons appeared healthy in control culture (a), and cultures from CD47^−/− (b), SIRPα mutant (c), and control culture treated with antibodies against TSP-1 (d). Measurements of TH-positive nerve fiber density (e), length (f), and astrocytic migration revealed similar values for SIRPα wildtypes and mutants. Scale bar = 25 µm.</p

TH-positive nerve fiber outgrowth and astrocytic migration in CD47^−/− cultures.

<p>TH-positive nerve fiber outgrowth and vimentin–positive astrocytic migration from CD47^+/+ (a, c) and CD47^−/− (b, d) VM cultures after 7 (a, b) and 14 (c, d) DIV. The astrocytic migration was shorter at 7 DIV as compared to 14 DIV. Short TH-positive nerve fiber outgrowth was observed in CD47^+/+ (a) and CD47^−/− (b) cultures at 7 DIV, while at 14 DIV the TH-positive nerve fibers had reached over longer distances and with higher density in CD47^−/− cultures (d) compared to control cultures (c). The dotted line delineates the border of the tissue slice. Scale bar = 100 µm.</p

Effects of treatment with TSP-1 antibodies.

<p>Non-glial-associated nerve fiber outgrowth in control culture (a), cultures from SIRPα mutant (b, c, d), and control cultures treated with antibodies against TSP-1 (e) as revealed by TH-immunohistochemistry. In all cultures, dotted nerve fibers were present, and in higher magnification it is clear that the TH-immunoreactive dots have diffused from the axon and is also present in the near vicinity of the axon (c), while in non-disrupted axons TH-immunoreactivity stays within the nerve fiber (d). Neither nerve fiber density (f) nor the distance that nerve fibers reached (g) nor astrocytic migration (h) were affected by treatment with antibodies against TSP-1. Scale bar a, b, e = 70 µm, c, d = 25 µm.</p

Collagen I dependent 2D and 3D cell migration is regulated by CD47.

<p>(A) Wound healing assay; Int cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes, thereafter cells were plated onto 10 µg/ml collagen I coated dishes for 2 hours, after which a wound was made in the monolayer, and the cells were allowed to migrate for 18 hours. Pictures of the wound were taken after 0 and 18 hours. The wound closure was measured and is presented as the percentage of wound closure as compared to time zero. (B, C) 3D cell migration assay; (B) Cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or the β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes after which 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. (C) Int 407 cells were transfected with 50 nM siRNA against CD47 (as indicated) or with scrambled control siRNA, for 48 hours. Thereafter 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. Cell migration was examined by staining with crystal violet blue and measuring the absorbance at 590 nm. The data are given as percent of control and represent means ± SE of five separate experiments. The statistical analyses were performed with unpaired Students t-test; *P<0.05, **P<0.01 relative to the control.</p

Differentiation of distinct M1 and M2 macrophage populations.

<p>(A) Morphological evaluation of macrophage phenotypes. (B) Apoptosis and necrosis of macrophage populations was evaluated by staining with Annexin V and PI, respectively, followed by flow cytometry. Shown is percent necrotic/apoptotic cells (mean ± SD) from three independent experiments. (C) Expression of extracellular markers for macrophage populations as evaluated by immunostaining and flow cytometry. Relative mean flourescense intensity (MFI) ± SD of three or more independent experiments is shown, where each sample was normalized against its respective isotype control. Relevant statistically significant differences are indicated by * (p<0.05), ** (p<0.01), or *** (p<0.001), non-significant <i>p</i> values are indicated by n.s.</p

Collagen I induces COX-2 expression through a PTX dependent G-protein.

<p>(A) Int 407 cells were incubated with or without 500 ng/ml PTX for 2 hours, after which they were plated out onto 10 µg/ml collagen I or 6% BSA (control) coated dishes for 1 hour. Adherent cells were then lysed and analysed for COX-2 expression by Western blotting as previously described. (B) Cells were transiently transfected with empty vector or vectors expressing small inhibitor peptides against either Gα_i1-2 or Gα_i3 before plated onto 10 µg/ml collagen I coated dishes for 1 hour. Adherent cells were the lysed and analysed for COX-2 expression by Western blotting. All membranes were re-probed for actin to ensure equal loading. The accumulated data of the densitometric analyses are given as percent of control and represent means ± SE of four separate experiments. The statistical analyses were performed with unpaired Students t-test; *P<0.05, **P<0.01 relative to the control and compared to collagen I treatment.</p

The endocytic ability of the different macrophage populations.

<p>Endocytosis by M1 and M2 macrophages, or macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 CRC cell lines was evaluated by measuring internalization of FITC-dextran by flow cytometry. Shown is percent relative endocytosis (mean ± SD) with endocytosis by IL10 stimulated M2 macrophages set as 100%. Statistically significant differences are indicated by * (p<0.05) or ** (p<0.01). All other differences were tested but found non-significant (not indicated in figure).</p

The migratory ability of M1 and M2 macrophage populations.

<p>Migration of macrophage phenotypes was evaluated by Boyden chamber experiments, where macrophages were allowed to migrate towards conditioned media from RKO or SW480 CRC cell lines, or culture medium control (RPMI+10% FBS). Mean numbers of migrating cells (n) ± SD is shown. Relevant statistically significant differences are indicated by * (p<0.05), non-significant <i>p</i> values are indicated by n.s.</p

The gene expression profiles of the different macrophage populations.

<p>Gene expression was studied in M1 and M2 macrophages, or macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 CRC cell lines using a Cytokine & Chemokine PCR Array. (A) Expression of pro-inflammatory and anti-inflammatory cytokines. (B) Expression of chemotactic factors. Shown is fold gene expression, with IL4 stimulated M2 macrophages set as 1.</p

Effects on the macrophage phenotype by secreted factors from CRC cells.

<p>(A) Morphological evaluation of macrophages stimulated with conditioned media (Cm) from RKO, SW480 or Caco2 cell lines. (B) Expression of extracellular markers was evaluated by immunostaining and flow cytometry in macrophages of M1 and M2 subtype or in macrophages stimulated with conditioned media (Cm) from CRC cell lines. Relative mean flourescense intensity (MFI) ± SD of three or more independent experiments is shown, where each sample was normalized against its respective isotype control and with the M-CSF stimulated control sample set as 1.</p