Table 1.
Clinical characteristic of Crohn's disease patients and ulcerative colitis patients.
Figure 1.
CD163 transcripts are increased in inflamed IBD mucosa.
A. CD163 RNA expression was evaluated in colonic biopsies taken from 22 normal controls (CTR), 25 patients with Ulcerative Colitis (UC) and 19 patients with Crohn's disease (CD) by real-time PCR and levels were normalized to β-actin. Data indicate mean ± SEM of all samples; *p = 0.01; **p<0.0001. B. CD163 RNA expression was evaluated in ileal biopsies taken from 7 ileal CTR and 6 ileal CD patients by real-time PCR. Data indicate mean ± SEM of all samples; *p = 0.03. C. Colonic biopsies taken from 22 CTR, 11 IBD patients (4 CD and 7 UC) receiving no therapy (w/o therapy), 18 IBD patients (9 CD and 9 UC) treated with mesalamine (5-ASA), 10 IBD patients (3 CD and 7 UC) treated with steroids (CS) and 5 IBD patients (3 CD and 2 UC) treated with immunomodulators (ISS) were analyzed for CD163 RNA expression by real-Time PCR. Levels are normalized to β-actin. Data are expressed as mean ± SEM of all samples; *p = 0.01; **p<0.01. D. CD163 RNA expression was evaluated in LPMC from 6 normal controls (CTR), 4 patients with UC and 6 patients with CD by real-time PCR. Levels are normalized to β-actin. Data indicate mean ± SEM of all samples; *p = 0.02.
Figure 2.
CD163 protein expression is increased in IBD.
A. Representative Western blots showing CD163 and β actin in total proteins extracted from mucosal samples of 2 CTR, 2 UC patients and 2 CD patients. Right panel shows the quantitative analysis of CD163/β-actin ratio in mucosal samples taken from 8 CTR, 8 UC patients and 8 CD patients as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of all samples; *p = 0.003; **p = 0.03. B. Representative photomicrographs (100× original magnification) of CD163-stained paraffin-embedded sections of surgical samples taken from 1 CTR, 1 patient with UC and 1 patient with CD. Isotype control antibody-stained section is also shown. Right panel shows the number of CD163-positive cells for high power field (hpf) in colonic sections taken from 3 CTR, 3 UC patients and 4 CD patients. Data are expressed as mean ± SD; *p = 0.03; **p = 0.02. C Representative photomicrographs (200× original magnification) of CD163-stained paraffin-embedded sections of surgical samples taken from 1 patient with UC and 1 patient with CD. CD163-positive cells are evident inside and around vessels. Insets show higher magnification (400×) images.
Figure 3.
CD163 RNA and protein expression is increased in the inflamed areas of IBD.
A. Paired biopsies taken from the involved (Involv) and uninvolved (Uninv) mucosa of 6 UC patients and 3 CD patients were analyzed for CD163 RNA expression by real-time PCR. Levels are normalized to β-actin; horizontal bars indicate the median values; *p = 0.003. B. Representative photomicrographs (original magnification 100×) of CD163-stained sections of colonic mucosal samples taken from involved and uninvolved mucosa of 1 UC patient. Right panel shows the number of CD163-positive cells per high power field (hpf) of colonic sections taken from the involved (Involv) and uninvolved (Uninv) mucosa of 3 UC patients. Data indicate the mean values ± SEM of all samples; **p = 0.01.
Figure 4.
Increased CD163+cells/CD68+ cells ratio in IBD tissue.
A. Representative photomicrographs (100× original magnification) in serial paraffin-embedded sections of surgical samples taken from 1 CTR, 1 patient with UC and 1 patient with CD and stained with CD163 or CD68. Right panel shows the ratio of CD163+ and CD68+ cells counted in colonic sections taken from 3 CTR, 3 UC patients and 3 CD patients. Horizontal bars indicate the median values; *p = 0.03; **p = 0.02.
Figure 5.
CD163-expressing cells are increased in the blood of IBD patients.
A. The Histograms show the percentage of CD163-expressing cells in total PBMC (A), CD14+ cells (B), CD16+ cells (C) and CD14+ CD16+ cells (D) of 23 IBD patients (5 UC patients, 18 CD patients) and 9 CTR. Cells were isolated from the blood of IBD patients and controls and examined by flow-cytometry as indicated in material and methods. Data indicate the mean values ± SEM of all samples; *p<0.001 **p = 0.001.
Figure 6.
IL-6 enhances CD163 expressions in normal colonic explants and LPMC.
A. Representative Western blots showing CD163 and β-actin in total proteins extracted from normal colonic explants treated with or without (unstimulated = UNST) TNF-α (20 ng/ml) or IL-6 (50 ng/ml) for 24 hours. Right panel shows the quantitative analysis of CD163/β-actin ratio in normal colonic explants. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of four separate experiments; *p = 0.04. B. Representative Western blots showing CD163 and β-actin in total proteins extracted from normal LPMC treated as above for 48 hours. Right panel shows the quantitative analysis of CD163/β-actin ratio in LPMC protein extracts. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of three separate experiments; **p = 0.01.
Figure 7.
Cross-linking of CD163 with the EDHU1-Ab enhances TNF-α expression in LPMC, PBMC and in purified HLADR-expressing LPMC of IBD patients.
A–B. Left panels. TNF-α RNA expression was evaluated in LPMC of 4 CD patients and 1 UC patients (A) and in PBMC of 3 CD patients and 3 UC patients (B) treated with EDHU1-Ab or control IgG for 6 hours by real-time PCR. Levels are normalized to β-actin; horizontal bars indicate the median values; *p = 0.03; **p = 0.01. Right panels. TNF-α secretion was measured in supernatants of IBD LPMC and PBMC treated as described above for 48 hours. Horizontal bars indicate the median values; *p = 0.03; **p = 0.01. C. TNF-α RNA expression was evaluated by real-time PCR in HLA-DR-expressing, CD3- and CD19-negative LPMC of 3 patients with CD and treated with EDHU1-Ab or control IgG for 6 hours. Levels are normalized to β-actin.