Maikel P. Peppelenbosch

Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn's disease

Background and aims: Treatment with infliximab induces remission in about 70% of patients with steroid refractory Crohns disease. Because Crohns disease is considered to be mediated by uncontrolled activation of mucosal T lymphocytes, we hypothesised that infliximab could induce apoptosis of T lymphocytes. Methods: Induction of apoptosis in vivo was studied in 10 patients with therapy refractory Crohns disease. In vitro, resting or stimulated Jurkat T cells were incubated with infliximab. Results: Infusion of infliximab (5 mg/kg) in steroid refractory patients with Crohns disease induced a clinical response in 9/10 patients but did not influence expression of activation markers, homing receptors, memory cells, Fas expression, or Bax/Bcl-2 expression on peripheral blood T lymphocytes. In contrast, a significant increase in CD3 and TUNEL positive cells within colonic biopsies was detected 24 hours after infusion of infliximab, suggesting that infliximab stimulates apoptosis of activated T lymphocytes but not of resting T cells. To test this hypothesis, the effects of infliximab on Jurkat T cells were investigated. We observed that infliximab induced apoptosis and an increase in the Bax/Bcl-2 ratio of CD3/CD28 stimulated Jurkat T cells but not of unstimulated Jurkat cells. Conclusions: Our data indicate that infliximab treatment causes a rapid and specific increase in apoptosis of T lymphocytes in the gut mucosa. These findings may explain the rapid and sustained therapeutic effects of infliximab in Crohns disease.

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4–β-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-κB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-κB is seen in stromal macrophages that also express COX-2 and TNF-α, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-κB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Contrasting roles of IL-12p40 and IL-12p35 in the development of hapten-induced colitis

IL‐12(p70), a heterodimer composed of two subunits (p35 and p40), is a key cytokine for Th1 mediated inflammatory responses. We dissected the role of IL‐12 in the development of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis by studying mice deficient in IL‐12p40, IL‐12p35, or IL‐12Rβ1. TNBS‐treated IL‐12Rβ1–/– and IL‐12p35–/– mice developed only a mild disease associated with low level IL‐18 expression in IL‐12p35–/– mice. In contrast, IL‐12p40–/– mice developed more severe colitis than wild‐type mice associated with high level colonic IL‐18 expression. Administration of IL‐12p40 neutralizing mononuclear antibody dramatically increased pathology in IL‐12p35–/– mice similar to disease scored in IL‐12p40–/– mice. Numbers of IFN‐γ‐producing cells infiltrating the lamina propria were comparably augmented in the different groups of IL‐12‐mutant and wild‐type mice. These results demonstrate that IL‐12p40, in contrast to IL‐12p70, inhibits TNBS‐induced colitis and IL‐18 expression independent of IFN‐γ.

Feed a Cold, Starve a Fever?

ABSTRACT An English old wives’ tale advises us to “feed a cold and starve a fever.” Here we report that the nutritional status modulates the T helper 1 (Th1)-Th2 balance of activated T cells in human volunteers. Food intake resulted in increased levels of gamma interferon production, whereas food deprivation stimulated interleukin-4 release.

Human plasma very low density lipoprotein carries Indian hedgehog.

Hedgehog is one of the major morphogens and fulfils critical functions in both the development and maintenance of the vasculature. Hedgehog is highly hydrophobic and its diffusion toward target tissues remains only partly understood. In Drosophila, hedgehog transport via lipophorins is relevant for development, but neither the presence nor a function for a mammalian Hedgehog carried by human plasma lipoproteins has been established. We investigated the presence of Hedgehog on lipoprotein particles and determined its importance for maintaining the endothelium. LTQ-Orbitrap XL analysis of defined plasma lipoproteins revealed that Indian Hedgehog (Ihh) is present in the human very low density lipoprotein (VLDL) fraction but not in other plasma lipoprotein fractions (low density lipoprotein (LDL) and high density lipoprotein (HDL)). Using the same approach, neither Sonic Hedgehog nor Desert Hedgehog could be detected in plasma lipoprotein fractions. Most likely, primary white adipocytes are the source of Ihh loading on VLDL as both transcriptome as well as immunofluorescence analysis showed high expression of Ihh in these cells. Additionally, we show that the endothelial compartment is most likely to be affected by the presence of Ihh on VLDL. Indeed, VLDL increased survival of primary endothelial cells, suggesting that Ihh transport by VLDL is important for maintaining the human endothelium. In conclusion, our study shows that VLDL carries Ihh throughout the body in mammals and Hedgehog signaling by human plasma VLDL particles may affect blood vessel pathophysiology. A combination of three state-of-the-art technologies, proteomics, genomics, and confocal microscopy, appeared to be a powerful tool for analyzing plasma lipoprotein-associated proteins.

Expression of the Chemokine Receptors CXCR1 and CXCR2 on Granulocytes in Human Endotoxemia and Tuberculosis: Involvement of the p38 Mitogen—Activated Protein Kinase Pathway

The chemokine receptors CXCR1 and CXCR2 critically determine the functional properties of granulocytes. To obtain insight in the regulation of these receptors during infection, CXCR expression was determined on blood granulocytes by fluorescence-activated cell sorter analysis in healthy subjects intravenously injected with lipopolysaccharide (LPS) and in patients with active tuberculosis. In healthy subjects, LPS induced a transient decrease in granulocyte CXCR1 and CXCR2 expression, whereas in tuberculosis patients, only CXCR2 showed reduced levels. In whole blood in vitro, LPS, lipoarabinomannan from Mycobacterium tuberculosis, and lipoteichoic acid from Staphylococcus aureus reduced expression of CXCR2 but not of CXCR1. CXCR2 down-regulation induced by LPS or tumor necrosis factor-alpha in vitro was abrogated by a p38 mitogen-activated protein kinase (MAPK) inhibitor. Granulocytes may down-regulate CXCR2 and, to a lesser extent, CXCR1 at their surface upon their first interaction with mycobacterial or bacterial pathogens by a mechanism that involves activation of p38 MAPK.

Invited review: Lipopolysaccharide recognition, internalisation, signalling and other cellular effects

Despite the importance of bacterial lipopolysaccharide (LPS) in infection and inflammation, many aspects of LPS action remain poorly understood. Especially, the mechanisms by which cells recognise and react to endotoxins or endotoxin-containing particles and how cellular responses are translated into systemic effects have long remained obscure. However, the recent identification of Toll-like receptors as essential participants in endotoxin signal transduction has provided the first answers in clarifying cellular LPS responses. In this review, we discuss the consequences of the clarification of the cellular effects of LPS. Furthermore, for LPS to exert its effects, it has to be transported to its target cells and be recognised before signalling may be induced, and we shall review the current state of affairs with regard to these recognition processes. Finally, we shall investigate how current knowledge may explain endotoxin neutralisation and subsequent detoxification, either through LPS internalisation or via LPS immobilisation, or through the actions of LPS-binding molecules.

Dichotomal effect of the coumadin derivative warfarin on inflammatory signal transduction

ABSTRACT Warfarin, a widely prescribed drug for preventing thrombosis, is thought to act solely through inhibition of vitamin K-dependent coagulation factors. Low concentrations of warfarin inhibit interleukin-6 production and phosphorylation of I-κB but not activation of p38 mitogen-activated protein kinase. Thus, warfarin inhibits inflammatory signal transduction, and this may contribute to clinical effects of warfarin.

A dual role for 7-dehydrocholesterol reductase in regulating Hedgehog signalling?

In a recent paper published in Development , Koide et al. provide evidence for a negative regulatory action of 7-dehydrocholesterol reductase (DCHR7) on the Hedgehog pathway ([Koide et al., 2006][1]). In a series of elegant experiments, the authors show that: (1) DHCR7 expression is intimately