Monomeric C-Reactive Protein Potential Utilization in the Histological Assessment of Inflammatory Bowel Disease (IBD) Patients

Abstract

Introduction Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), represent chronic progressive inflammatory gastrointestinal disorders, without a single reference standard for their diagnosis. The histological assessment gained an important role in accurately measuring disease activity, and mucosal healing (MH) was recently proposed to be an ideal treatment goal for patients with IBD because of its favorable prognosis, with a lower risk of recurrence or surgical treatment. This paper aims to add to the histological classical findings for IBD patients the identification of the monomeric form of the C-reactive protein (mCRP) as a supplementary marker that could be stained at the level of tissue samples and could be correlated with the pathogenic mechanism. Methods Two groups of 10 patients were each selected for the study, for both UC and CD, together with a control group. All samples collected through digestive endoscopy were analyzed by using H&E-stained slides, followed by immunohistochemical examination with antibodies to mCRP (M8C10), and markers of inflammatory activity through CD3, CD45(leukocyte common antigen (LCA)), CD138/syndecan-1 and CD68. Results For the CD study group, all histological elements identified with H&E and afterward stained with CD138, CD68, CD3, and CD45/LCA were correlated with the standards imposed by the European Crohn's and Colitis Organization (ECCO). For the group of patients with UC, histological images obtained with H&E and IHC stainings also confirmed the recommendation of ECCO. The main cells considered in the literature as histological markers for IBD are neutrophils, lymphocytes, and plasmocytes, stained in our study with CD45/LCA, CD3, and CD138. For all 20 cases of IBD (UC and CD), the staining with anti-Ab8C10 antibodies for mCRP was positive, while negative results were noticed within the control group. An mCRP protein visualized with anti-Ab8C10 antibodies presented an intracytoplasmatic localization in the neutrophils, plasma cells, lymphocytes, and macrophages from the lamina propria and glandular epithelium, without expression in endothelial cells. Conclusions Our study represents one of the first papers that identifies the localization of mCRP molecules within the intestinal mucosa of patients with IBD (both UC and CD) by using immunohistochemistry (IHC) staining. This finding opens a new perspective for considering mCRP as a marker correlated with histological disease activity and/or definition of histological remission in IBD.

Keywords: crohn’s disease (cd); imunohistochemistry; inflammatory bowel disease; monomeric c-reactive protein; ulcerative colitis (uc).

Figure 1. Immunohistochemical staining of individual anti-Ab8C10 antibodies for mCRP in the control group

1A. Colonic mucosa with normal histological aspect (H&E, 10x) 1B. Colonic mucosa with normal histological aspect (H&E, 20x) 1C. Colonic mucosa with normal histological aspect (H&E, 40x) 1D. Normal colonic mucosa with negative staining for anti-Ab8C10 antibodies (10x) 1E. Normal colonic mucosa with negative staining for anti-Ab8C10 antibodies (20x) 1F. Normal colonic mucosa with negative staining for anti-Ab8C10 antibodies (40x) (10x: images are magnified to a size 10 times larger than the original; 20x: images are magnified to a size 20 times larger than the original; 40x: images are magnified to a size 40 times larger than the original)

Figure 2. Immunohistochemical staining of individual anti-Ab8C10 antibodies for mCRP in ulcerative colitis and Crohn’s disease

2A. Histology of ulcerative colitis with activity features in the lamina propria. Note the presence of neutrophils, plasma cells, lymphocytes in the lamina propria and glandular epithelium, highlighted with anti-Ab8C10 antibodies (40x) 2B. Histology of ulcerative colitis with a mild increase in lymphocytes and plasma cells within the lamina propria immunostained with anti-Ab8C10 antibodies (40x) 2C. Histology of ulcerative colitis with an abundant chronic inflammatory infiltrate in the lamina propria. Note the presence of a blood vessel with inflammatory cells in the lumen immunostained with anti-Ab8C10 antibodies, without the staining of endothelial cells (40x) 2D. Histology of Crohn’s disease with neutrophils in the lamina propria and in the surface epithelium highlighted with anti-Ab8C10 antibodies (40x) 2E. Histology of Crohn’s disease with an abundant lympho-plasmocytic infiltrate in the lamina propria highlighted with anti-Ab8C10 antibodies (40x) (mCRP: monomeric C-reactive protein; 40x: images are magnified to a size 40 times larger than the original)

Figure 3. Histological findings and immunohistochemical staining in ulcerative colitis

3A. Colonic mucosa with normal glandular architecture and moderate chronic inflammatory infiltrate in the lamina propria (H&E, 20x) 3B. Positive cytoplasmatic staining of plasma cells, lymphocytes and macrophages with anti-Ab8C10 antibodies for mCRP (20x) 3C. Positive membrane and cytoplasmatic staining of lymphocytes with anti-CD3 antibodies (20x) 3D. Positive cytoplasmatic staining of the macrophages with anti-CD68 antibodies (20X) 4E. Positive cytoplasmatic staining of plasma cells and lymphocytes with anti-LCA antibodies (20x) 3F. Positive cytoplasmatic staining of plasma cells with anti-CD138 antibodies (20x) (mCRP: monomeric C-reactive protein; 20x: images are magnified to a size 20 times larger than the original; LCA: leukocyte common antigen)

Figure 4. Histological findings and immunohistochemical staining in Crohn’s disease

4A. Colonic mucosa with a moderate chronic inflammatory infiltrate in the lamina propria and mild architectural distortions (H&E, 20x) 4B. Positive cytoplasmatic staining of plasma cells, lymphocytes, and macrophages with anti-Ab8C10 antibodies for mCRP (20x) 4C. Positive membrane and cytoplasmatic staining of lymphocytes with anti-CD3 antibodies (20x) 4D. Positive cytoplasmatic staining of the macrophages with anti-CD68 antibodies (20x) 4E. Positive cytoplasmatic staining of plasma cells and lymphocytes with anti-LCA antibodies (20x) 4F. Positive cytoplasmatic staining of plasma cells with anti-CD138 antibodies (20x) (mCRP: monomeric C-reactive protein; 20x: images are magnified to a size 20 times larger than the original; LCA: leukocyte common antigen)