Differential activation of cytokine secretion in primary human colonic fibroblast/myofibroblast cultures.
BACKGROUND
Fibroblasts and myofibroblasts are known to secrete a wide spectrum of cytokines, but the individual spectrum is tissue-specific. We investigated the effect of cell activation on cytokine secretion of isolated human colonic fibroblasts/myofibroblasts from control patients and patients with mucosal inflammation.


METHODS
Primary cultures of human colonic submucosal fibroblasts/myofibroblasts were incubated with IL-1alpha (100 U/ml), IL-Ibeta (10 ng/ml), IL-10 (10 ng/ml), TNF (10 ng/ml), PMA (10 ng/ml), LPS (50 ng/ml), IL-4 (10 ng/ml), or a combination of IL-1 and TNF. Secreted cytokines were determined by ELISA. NF-kappaB activation was demonstrated by electrophoretic mobility-shift assays (EMSA).


RESULTS
Incubation of colonic fibroblasts/myofibroblasts with IL-1, LPS, TNF and PMA induced secretion of IL-6, IL-8, M-CSF and GM-CSF. IL-8 and IL-6 secretion could be stimulated by IL-1alpha, IL-1beta, TNF, PMA and LPS within 6 h of incubation. IL-6 secretion was stimulated from 0.5 +/- 0.01 pg/h x microg fibroblast protein to 18.5 +/- 2.6 pg/h x microg fibroblast protein with IL-1beta (P < 0.01). IL-8 secretion was stimulated from 1.0 +/- 0.1 pg/h x microg fibroblast protein to 41.1 +/- 3.6 pg/h x microg (P < 0.005). IL-4 and IL-10 did not change cytokine secretion significantly. No significant differences between cultures from normal and inflamed mucosa were observed. TNF and IL-1 induced NF-kappaB activation. ALLN, a proteasome and NF-kappaB activation inhibitor, reduced TNF-mediated IL-8, GM-CSF and M-CSF induction significantly, whereas induction of IL-6 secretion remained unchanged.


CONCLUSIONS
Human colonic myofibroblasts can secrete large amounts of IL-6, IL-8, M-CSF and GM-CSF upon stimulation. The induction of IL-8, M-CSF and GM-CSF, but not of IL-6 secretion, is mediated mainly by NF-kappaB activation. The cytokine profile and the total amounts of cytokines released suggest that colonic myofibroblasts can play a role in leukocyte recruitment and during mucosal inflammation. They therefore have to be regarded as an important part of the mucosal immune system.
Rogler
G
G
Dept. of Internal Medicine I, University of Regensburg, Germany. grogler@ucsd.edu
Gelbmann
C M
CM
Vogl
D
D
Brunner
M
M
Schölmerich
J
J
Falk
W
W
Andus
T
T
Brand
K
K
D000328:Adult; D000704:Analysis of Variance; D001707:Biopsy, Needle; D002478:Cells, Cultured; D003093:Colitis, Ulcerative / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism*; D003424:Crohn Disease / Q000473:pathology; D016207:Cytokines / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D011336:Probability; D012016:Reference Values; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10