ABSTRACT
BACKGROUND & AIMS: Clinical sepsis seldom accompanies inflammatory bowel disease. The aim of this study was to measure colonic mucosal levels of the neutrophil product bactericidal/permeability-increasing protein (BPI), which kills gram-negative bacteria in addition to inactivating endotoxin. METHODS: Enzyme-linked immunosorbent assay and immunohistochemistry for BPI were performed on homogenates and tissue secretions of biopsy specimens from patients with ulcerative colitis (n=11) and Crohn's disease (n=5) and from normal controls (n=5). RESULTS: Mucosal neutrophil content (144 +/- 23 vs. 35 +/- 9 neutrophils/mg protein; P<0.007) and BPI content (2.07 +/- 0.75 vs. 0.12 +/- 0.02 ng/mg protein; P<0.002) were greater in the colitis groups and correlated closely (r=0.68; P<0.001). This relationship held for both ulcerative colitis (P<0.002) and Crohn's disease (P<0.01) with a trend towards greater levels in Crohn's disease. There was a trend towards higher BPI levels with an increasing endoscopic inflammation score (grade I, 1.32 +/- 0.6 ng/mg protein; grade II, 2.82 +/- 1.4 ng/mg protein). Immunohistochemistry and the biopsy culture showed BPI to be both intracellular and extracellular, to be present in the crypt lumen, and to be released into incubating medium. CONCLUSIONS: Mucosal levels of BPI are increased in colitis. Such localization may ameliorate mucosal responses to gram-negative bacteria and their products.
Subject(s)
Blood Proteins/metabolism , Colon/metabolism , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/metabolism , Membrane Proteins , Adolescent , Adult , Animals , Antimicrobial Cationic Peptides , Biopsy , Blotting, Western , Colitis, Ulcerative/metabolism , Colon/pathology , Crohn Disease/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunohistochemistry , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/pathology , Male , Middle Aged , Neutrophils/pathologyABSTRACT
Mucosal inflammation is associated with altered expression of cell membrane molecules. Disaggregation of tissue for flow cytometry may introduce artefactual changes. In an attempt to prevent the induction of artefacts, cells were fixed prior to isolation. The addition of 0.1% buffered formaldehyde to the collagenase/dispase digestion of mucosal biopsy specimens from patients with inflammatory bowel disease enhances detection of CD3, CD11b, CD16, CD63, and CD14. No significant effect was noted for CD19, CD67 or CD45. The expression of CD3, CD11b and CD45 correlated with the degree of endoscopic inflammation. Dilute buffered formaldehyde may be a useful adjunct to the enzymatic isolation of cells from mucosal specimens, by protecting surface antigens from digestion or alterations in expression.
