Tumour necrosis factor-alpha production stimulated by heat shock protein 70 and its inhibition in circulating dendritic cells and cells eluted from mucosal tissues in Crohn's disease

Abstract

Summaryand interleukin (IL)-12 by dendritic cells (DC) from patients with Crohn's disease. TNF-alpha concentration was increased significantly when DC from Crohn's disease were stimulated with HSP70 or CD40L and this was associated with signalling by the extracellular signal regulated kinase (ERK) 1/2 and p38 mitogen activated protein (MAP) kinase pathway. IL-12 production was also increased when DC were stimulated with HSP70. Cells eluted from inflamed intestinal mucosa from Crohn's disease, stimulated with HSP70, CD40L or lipopolysaccharide produced significantly greater TNF-alpha and IL-12 concentrations than cells from uninflamed mucosa. Significant inhibition of TNF-alpha production was demonstrated when DC from peripheral blood mononuclear cells or cells eluted from intestinal mucosa of Crohn's disease were treated with either the HSP70 inhibitory peptide (aa 457-496) or peptides derived from CD40 and CD40L. These inhibitory peptides target the CD40-CD40L and the emerging CD40-HSP70 co-stimulatory pathway. Our findings offer a novel strategy to prevent excessive production of TNF-alpha in Crohn's disease.

Fig. 1

Stimulation of monocyte-derived dendritic cells (DC) with heat shock protein (HSP)70, CD40L or lipopolysaccharide (LPS) producing (a) tumour necrosis factor (TNF)-α and (b) interleukin (IL)-12 in Crohn's disease (CD) □ (n = 16 and 12), ulcerative colitis (UC) (n = 9 and 8) and healthy controls ▪ (n = 13 and 14), respectively; statistical analyses were performed between CD and controls and UC and controls for each stimulant (*P < 0·05, **P < 0·02). Stimulation of monocytes in these groups producing (c) TNF-α (n = 10–16) and (d) IL-12 (n = 7–9), respectively.

Fig. 2

Monocyte-derived dendritic cells (DC) from Crohn's disease stimulated with heat shock protein (HSP)70, CD40L or lipopolysaccharide (LPS) to produce tumour necrosis factor (TNF)-α and interleukin (IL)-12p40, and treated with increasing concentrations of p38 inhibitor (SB023580) (a, b, c) or extracellular signal regulated kinase (ERK) inhibitor (PD098059) (d, e, f); presented as mean ± s. e.m. pg/ml.

Fig. 3

Stimulation by heat shock protein (HSP)70, CD40L or lipopolysaccharide (LPS) of tumour necrosis factor (TNF)-α production by cells eluted from mucosal biopsies from patients with (a) Crohn's disease (inflamed n = 13 and uninflamed n = 13) or (b) ulcerative colitis (inflamed n = 8, uninflamed n = 12) and inhibition of HSP70-stimulated cells with p457–496 or pCD40/CD40L (c) in Crohn's disease (inflamed n = 11, uninflamed n = 12) and (d) in ulcerative colitis (inflamed n = 7, uninflamed n = 11).

Fig. 4

A novel strategy for inhibition of CD40–CD40L or CD40–heat shock protein (HSP)70 interaction by peptides derived from HSP70 or CD40 and CD40L, stimulating production of tumour necrosis factor (TNF)-α, interleukin (I)-12 and CCL-5.