Werner Falk

IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells

Lack of the IL-1 receptor accessory protein (IL-1RAcP) abrogates responses to IL-33 and IL-1 in the mouse thymoma clone EL-4 D6/76 cells. Reconstitution with full-length IL-1RAcP is sufficient to restore responsiveness to IL-33 and IL-1. IL-33 activates IL-1 receptor-associated kinase-1, cJun-N-terminal kinase, and the NF-κB pathway in an IL-1RAcP-dependent manner and results in IL-2 release. IL-33 is able to induce the release of proinflammatory cytokines in bone marrow-derived (BMD) mast cells, indicating that IL-33 may have a proinflammatory potential like its relatives IL-1 and IL-18, in addition to its Th2-skewing properties in the adaptive response described previously. Blocking of murine IL-1RAcP with the neutralizing antibody 4C5 inhibits response of mouse thymoma cells and BMD mast cells to IL-33. The interaction of either membrane-bound or soluble forms of IL-1RAcP and IL-33Rα-chain depends on the presence of IL-33, as demonstrated by coimmunoprecipitation assays. These data demonstrate that IL-1RAcP is indispensable for IL-33 signaling. Furthermore, they suggest that IL-1RAcP is used by more than one α-chain of the IL-1 receptor family and thus may resemble a common β-chain of that family.

The Interleukin-1 Receptor Accessory Protein (IL-1RAcP) Is Essential for IL-1-induced Activation of Interleukin-1 Receptor-associated Kinase (IRAK) and Stress-activated Protein Kinases (SAP Kinases)

Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL-1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFκB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression.

Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice.

The cytokines TNF and IL‐1 have been implicated as mediators of the inflammatory processes in patients with inflammatory bowel disease (IBD). To investigate the role of these cytokines in mucosal inflammation we used anti‐cytokine strategies in a mouse model of acute and chronic colitis. Mice which received 5% dextran sulphate sodium (DSS) in their drinking water showed signs of acute colitis on day 4, with severe weight loss and bloody diarrhoea. Chronic colitis was established after four cycles of feeding 5% DSS for 7 days and water for 10 days, with the mice showing diarrhoea but no weight loss. In acute colitis, treatment with anti‐IL‐1 reagents, anti‐TNF MoAb, or dexamethasone (DEX) led to aggravation. By contrast, in chronic colitis, treatment of mice with several IL‐1 activity‐inhibiting reagents failed to show significant effects, whereas anti‐TNF MoAb or DEX significantly reduced the colitis. We conclude that in acute colitis IL‐1 and TNF are beneficial, whereas in chronic colitis, TNF but not IL‐1 seems to play a major role in perpetuation of chronic inflammation.

Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease.

To test whether there is a difference in the expression of interleukin 8 (IL8) between Crohns disease and ulcerative colitis and to determine the main site of its synthesis this study analysed IL8 in mucosal biopsy specimens of patients with Crohns disease and ulcerative colitis by enzyme linked immunosorbent assay (ELISA) and by in situ hybridisation. IL8 was measured by ELISA in 38 normal control patients, eight inflammatory control patients, 55 Crohns disease biopsy specimens (26 patients), and 67 ulcerative colitis biopsy specimens (35 patients). IL8 mRNA was determined in samples by in situ hybridisation using a specific IL8 RNA probe. IL8 protein was significantly increased in macroscopically inflamed specimens of Crohns disease (median 118 pg/specimen, p < 0.0001), ulcerative colitis (median 140 pg/specimen, p < 0.001), and inflammatory controls (median 30 pg/specimen, p = 0.010) compared with normal controls (median 4 pg/specimen). IL8 was also increased in uninflamed specimens of Crohns disease (median 46 pg/specimen, p < 0.001) but not of ulcerative colitis patients (median 9 pg/specimen, p = 0.3). IL8 protein in the mucosa correlated significantly with macroscopic inflammation in Crohns disease (r = 0.47, p < 0.001) and in ulcerative colitis (r = 0.60, p < 0.001). IL8 mRNA was detected by in situ hybridisation in 31 of 55 biopsy specimens (56%) of Crohns disease patients, in 38 of 67 specimens of ulcerative colitis patients (57%), in five of eight inflammatory controls (63%) and in five of 38 normal controls (13%). Mucosal IL8 mRNA expression correlated with mucosal IL8 protein (r = 0.46, p < 0.001). IL8 mRNA was only detected in inflammatory cells of the interstitium but not in mucosal epithelial cells. IL8 is produced mainly in the lamina propria of the colon in inflammatory bowel disease and correlates with mucosal inflammation.

Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-κB pathway in adipocytes links nutritional signalling with innate immunity

To study the effects of fatty acids and the involvement of the Toll‐like receptor‐4/nuclear factor‐κB (TLR‐4/NF‐κB) pathway with respect to the secretion of adipokines from adipocytes 3T3‐L1 adipocytes were stimulated with increasing doses of fatty acids. The secretion of adiponectin, resistin and monocyte chemoattractant protein‐1 (MCP‐1) was measured by enzyme‐linked immunosorbent assay. The NF‐κB p65 nuclear translocation and TLR‐4 expression were investigated by Western blot. The effects mediated by NF‐κB were tested using a specific NF‐κB‐inhibitor and TLR‐4‐induced effects were analysed with a neutralizing TLR‐4 antibody. Binding of 14C‐labelled fatty acids to TLR‐4/MD‐2 was investigated using a FLAG‐tagged extracellular part of TLR‐4 fused to full‐length MD‐2 via a linker (lipopolysaccharide‐Trap). The messenger RNA (mRNA) expression of adipokines in abdominal adipose tissue of rats fed a standard chow or a high‐fat diet was investigated by reverse transcription–polymerase chain reaction. The TLR‐4 is induced during adipocyte differentiation and its expression is enhanced following fatty acid stimulation. The stimulatory effects of stearic and palmitic acids on MCP‐1 secretion and of palmitoleic acid on resistin secretion are mediated via NF‐κB. The stimulatory effects of stearic, palmitic and palmitoleic acids on resistin secretion and the stimulatory effect of stearic acid on MCP‐1 secretion are mediated via TLR‐4. Fatty acid‐mediated effects are caused by an endogenous ligand because fatty acids were shown not to bind directly to TLR‐4/MD‐2. Adipose tissue mRNA expression and serum levels of adipokines did not differ in rats fed a high‐fat diet. These data provide a new molecular mechanism by which fatty acids can link nutrition with innate immunity.

Interferon-gamma (IFN-γ)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice

Excess nitric oxide formation caused by the activity of the inducible nitric oxide synthase has been implicated as a toxic effector molecule in the pathogenesis of experimental colitis and inflammatory bowel disease. It was therefore investigated whether inhibition of this synthase or the cytokines TNF and IFN‐γ, inducers of nitric oxide synthase, had effects on chronic colitis in mice. Chronic colitis was induced in mice by repeated feeding of DSS. Cytokines were neutralized by treatment with MoAbs and nitric oxide synthase was inhibited by aminoguanidine. The degree of colonic inflammation was assessed by a histological score and colon length. Aminoguanidine treatment reduced nitric oxide activity by 60% (P = 0.0004), the histological score by 31% (P = 0.005) and increased colon length by 1.4 cm (P = 0.002). Neutralization of TNF and IFN‐γ resulted in increased colon length (0.7 cm, P = 0.07 and 0.8 cm, P = 0.03), improved histological score (19%, P = 0.045 and 25%, P = 0.013), and reduced nitric oxide activity (31%, P = 0.07 and 54%, P = 0.004) compared with controls. The combination of anti‐cytokine treatments had additive effects. TNF and IFN‐γ are involved in perpetuation of chronic DSS‐induced colitis, and induction of excessive nitric oxide activity could be their common effector mechanism.

Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment. Cell Death and Differentiation (2001) 8, 679–686

Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.

Dialogue between the CNS and the immune system in lymphoid organs

Abstract It is well known that the CNS influences the responses of the immune system via humoral substances such as cortisol. Here, Rainer Straub and colleagues discuss aspects of the local interaction between nerves and immune cells in lymphoid organs. They provide evidence for chemically mediated transmission between nerves and immune cells in the spleen which is modified by the microenvironment.

Strictures in Crohn’s disease are characterised by an accumulation of mast cells colocalised with laminin but not with fibronectin or vitronectin

BACKGROUND/AIMS Intestinal fibrosis and stricture formation is an unresolved problem in Crohn’s disease. The aim of this study was to investigate whether mast cells accumulate in these tissues and whether their localisation is associated with extracellular matrix components. METHODS Mast cells were visualised by immunohistochemical staining of the mast cell specific proteases chymase and tryptase. Their localisation in relation to extracellular matrix components was shown by immunohistochemical double labelling. RESULTS In strictures in Crohn’s disease, a striking accumulation of mast cells was seen particularly in the hypertrophied and fibrotic muscularis propria, with a mean (SEM) mast cell number of 81.3 (14.9)v 1.5 (0.9)/mm2 in normal bowel (p<0.0005). All mast cells in the muscularis propria were colocalised with patches of laminin. In contrast, in the submucosa, laminin was exclusively found in the basal lamina of blood vessels where many adherent mast cells were seen. No colocalisation of mast cells was found with fibronectin or vitronectin. CONCLUSIONS The large accumulation of mast cells in the muscle layer of strictured bowel suggests a functional role for these cells in the hypertrophic and fibrotic response of the smooth muscle cells. The colocalisation with laminin indicates a mechanism of interaction between smooth muscle cells and mast cells that may be important in the role of mast cells in the process of fibrosis.