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. 1999 May;44(5):643-52.
doi: 10.1136/gut.44.5.643.

Mast Cells Are an Important Cellular Source of Tumour Necrosis Factor Alpha in Human Intestinal Tissue

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Free PMC article

Mast Cells Are an Important Cellular Source of Tumour Necrosis Factor Alpha in Human Intestinal Tissue

S C Bischoff et al. Gut. .
Free PMC article

Abstract

Background: Several inflammatory disorders of the intestine are characterised by enhanced expression of tumour necrosis factor alpha (TNF-alpha). Monocytes and macrophages have been suggested as a major cellular source of TNF-alpha in human gut, whereas mast cells, although known to be capable of producing TNF-alpha, have been poorly examined in this respect.

Aims: To investigate whether human intestinal mast cells can produce TNF-alpha, and which factors regulate TNF-alpha production in these cells.

Methods: Mast cells were isolated from surgery tissue specimens of patients undergoing bowel resection because of cancer. Immunohistochemical studies were performed in biopsy specimens derived from 13 patients (two healthy controls, four with Crohn's disease, four with ulcerative colitis, three others). TNF-alpha mRNA and protein expression were studied in vitro by polymerase chain reaction, RNAse protection assay, western blot, and enzyme linked immunosorbent assay in isolated purified human intestinal mast cells stimulated by IgE receptor crosslinking, intestinal bacteria, and lipopolysaccharide. Cellular localisation of TNF-alpha was examined by immunohistochemistry.

Results: TNF-alpha mRNA and protein were expressed constitutively in isolated human intestinal mast cells. Expression of TNF-alpha mRNA and release of TNF-alpha protein were substantially enhanced by IgE receptor crosslinking and by coculture of mast cells with intestinal bacteria; lipopolysaccharide had only marginal effects. Immunohistochemical studies revealed that approximately 60% of the lamina propria cells with immunoreactivity for TNF-alpha were mast cells.

Conclusions: The data show that mast cells are an important source of TNF-alpha in the human intestinal mucosa.

Figures

Figure 1
Figure 1
Expression of TNF-α mRNA in purified human intestinal mast cells. (A) RNAse protection assay: 10 µg of total RNA from THP.1 cells stimulated with 300 U/ml IFN γ (a), and 8 µg each of total RNA from unstimulated mast cells (b), and from mast cells stimulated with 100 ng/ml 29C6 (c), respectively (mast cell purity 98%) were hybridised with radiolabelled TNF-α cRNA (a,b,c) and c-kit cRNA (b,c). (B) Primer dropping RT-PCR: total RNA derived from purified mast cells (99% purity) either unstimulated (a), or stimulated with 100 ng/ml 29C6 (b), 4 × 106/ml bacteria (c), or 10 µg/ml LPS (d) was amplified in the presence of primer pairs for TNF-α and (after seven PCR cycles) GAPDH. DNA fragments obtained after 33 cycles are shown.
Figure 2
Figure 2
Immunostaining of human intestinal mast cells with anti-TNF-α antiserum (original magnification × 1000). (A-C) Cytocentrifuge smears of purified mast cells. A, cytocentrifuge of a mast cell preparation containing 96% mast cells, negative control (staining with a non-immune control antiserum); B, same cells as in A, unstimulated mast cells (staining with rabbit antihuman TNF-α antiserum, immunoreactive cells are stained); C, same cells as in A, stimulation of the mast cells by incubation with intestinal bacteria for six hours (staining as in B). (D-F) Immunohistochemistry of intestinal tissue sections derived from a patient with active Crohn's disease using different primary antibodies. D, negative control using non-immune control antiserum; E, staining with antihuman tryptase monoclonal antibody; F, staining with antihuman TNF-α antiserum. Closed arrows indicate TNF-α positive mast cells, open arrow indicates a TNF-α negative mast cell.
Figure 3
Figure 3
TNF-α immunoreactivity in isolated human intestinal mast cells. Cultured mast cells derived from seven cell preparations were challenged with buffer control (O), 29C6 (29C6), bacteria, or LPS. Mean (SD) percentages of mast cells with no (o), weak (+), and strong (++) immunoreactivity are shown. *p<0.05, **p<0.01 versus buffer control.
Figure 4
Figure 4
TNF-α protein in lysates of purified human intestinal mast cells. Recombinant human TNF-α (0.5 µg, lanes a,f), culture medium without cells (lane b), and lysates of 106 mast cells purified to 96% (lanes c-e). Prior to cell lysis, mast cells had been challenged for six hours with buffer control (c), 29C6 (d), or with intestinal bacteria (e). Arrows indicate the position of the molecular weight (MW) markers. The MW of the TNF-α protein bands of mast cell samples corresponds to that of recombinant TNF-α (17.9 kDa).
Figure 5
Figure 5
Production of TNF-α and inflammatory mediators by human intestinal mast cells. (A) Measurement of TNF-α in supernatants and cell lysates of mast cells, incubated for six hours with buffer control (O), mAb 29C6 (29C6), intestinal bacteria, or lipopolysaccharide (LPS). (B) Measurement of TNF-α, histamine, and sLT in supernatants of mast cells. *p<0.05, †p<0.005 versus buffer control.
Figure 6
Figure 6
Expression of tryptase and TNF-α by human intestinal lamina propria cells. Intestinal biopsy specimens were stained with monoclonal antibody directed against tryptase (Tryp+) and TNF-α (TNF+). (A) Tryp+ and TNF+ cells were counted in two adjacent tissue sections and the percentage of Tryp+ and TNF+ lamina propria cells was calculated (n=13; the circled point represents two experiments with identical results). (B) Comparison of percentages of Tryp+ and TNF+ lamina propria cells in patients with Crohn's disease (CD, n=4), ulcerative colitis (UC, n=4), food allergy (FA, n=3), and healthy controls (HC, n= 2). (C) Ratio of TNF+ mast cells and mast cells (mast cells defined as Tryp+ cells), and ratio of TNF+ mast cells and TNF+ cells, respectively, in histologically normal (n=6) and inflamed tissue (n=7).

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