Studies on patients establish Crohn's disease as a manifestation of impaired innate immunity.

The fruitless search for the cause of Crohn's disease has been conducted for more than a century. Various theories, including autoimmunity, mycobacterial infection and aberrant response to food and other ingested materials, have been abandoned for lack of robust proof. This review will provide the evidence, obtained from patients with this condition, that the common predisposition to Crohn's is a failure of the acute inflammatory response to tissue damage. This acute inflammation normally attracts large numbers of neutrophil leucocytes which engulf and clear bacteria and autologous debris from the inflamed site. The underlying predisposition in Crohn's disease is unmasked by damage to the bowel mucosa, predominantly through infection, which allows faecal bowel contents access to the vulnerable tissues within. Consequent upon failure of the clearance of these infectious and antigenic intestinal contents, it becomes contained, leading to a chronic granulomatous inflammation, producing cytokine release, local tissue damage and systemic symptoms. Multiple molecular pathologies extending across the whole spectrum of the acute inflammatory and innate immune response lead to the common predisposition in which defective monocyte and macrophage function plays a central role. Family linkage and exome sequencing together with GWAS have identified some of the molecules involved, including receptors, molecules involved in vesicle trafficking, and effector cells. Current therapy is immunosuppressant, which controls the symptoms but accentuates the underlying problem, which can only logically be tackled by correcting the primary lesion/s by gene therapy or genome editing, or through the development of drugs that stimulate innate immunity.

Rare coding variant analysis in a large cohort of Ashkenazi Jewish families with inflammatory bowel disease.

Rare variants are thought to contribute to the genetics of inflammatory bowel disease (IBD), which is more common amongst the Ashkenazi Jewish (AJ) population. A family-based approach using exome sequencing of AJ individuals with IBD was employed with a view to identify novel rare genetic variants for this disease. Exome sequencing was performed on 960 Jewish individuals including 513 from 199 multiplex families with up to eight cases. Rare, damaging variants in loci prioritized by linkage analysis and those shared by multiple affected individuals within the same family were identified. Independent evidence of association of each variant with disease was assessed. A number of candidate variants were identified, including in genes involved in the immune system. The ability to achieve statistical significance in independent case/control replication data was limited by power and was only achieved for variants in the well-established Crohn's disease gene, NOD2. This work demonstrates the challenges of identifying disease-associated rare damaging variants from exome data, even amongst a favorable cohort of familial cases from a genetic isolate. Further research of the prioritized rare candidate variants is required to confirm their association with the disease.

Innate immunity in inflammatory bowel disease: a disease hypothesis.

Crohn's disease arises from a defective interaction between the highly concentrated mass of bacteria in the gastrointestinal tract and the underlying tissues. It has generally been believed to result from an excessively exuberant inflammatory response or from 'autoimmunity'. Recent evidence has emerged that the problem is instead a failure of the way in which the body responds to the penetration of bacteria and other bowel contents through the intestinal mucosal barrier. Rather than Crohn's disease being caused by excessive inflammation, the primary mechanism is actually that of an immunodeficiency. Failure of inflammatory mediator production leads to insufficient recruitment of neutrophils, resulting in inadequate removal of bacteria and other debris. This impairment of acute inflammation can be compensated in some circumstances by signalling through NOD2. If not cleared, the foreign material in the bowel wall is taken up within macrophages, eliciting a granulomatous reaction and the local and systemic sequelae so characteristic of Crohn's disease.

The function of the NADPH oxidase of phagocytes, and its relationship to other NOXs.

The NADPH oxidase of 'professional' phagocytic cells transfers electrons across the wall of the phagocytic vacuole, forming superoxide in the lumen. It is generally accepted that this system promotes microbial killing through the generation of reactive oxygen species and through the activity of myeloperoxidase. An alternative scenario exists in which the passage of electrons across the membrane alters the pH and generates a charge that drives ions into, and out of, the vacuole. It is proposed that the primary function of the oxidase is to produce these pH changes and ion fluxes, and the issues surrounding these processes are considered in this review. The neutrophil oxidase is the prototype of a whole family of NOXs (NAPDH oxidases) that exist throughout biology, from plants to humans, which might function, at least in part, in a similar fashion.

Modified skin window technique for the extended characterisation of acute inflammation in humans.

OBJECTIVE: To modify the skin window technique for extended analysis of acute inflammatory responses in humans, and demonstrate its applicability for investigating disease. SUBJECTS: 15 healthy subjects and 5 Crohn's patients. TREATMENT: Skin windows, created by dermal abrasion, were overlaid for various durations with filter papers saturated in saline, 100 ng/ml muramyl dipeptide (MDP) or 10 microg/ml interleukin-8 (IL-8). METHODS: Exuded leukocytes were analyzed by microscopy, immunoblot, DNA-bound transcription factor arrays and RT-PCR. Inflammatory mediators were quantified by ELISA. RESULTS: Infiltrating leukocytes were predominantly neutrophils. Numerous secreted mediators were detectable. MDP and IL-8 enhanced responses. Many signalling proteins were phosphorylated with differential patterns in Crohn's patients, notably PKC alpha/beta hyperphosphorylation (11.3 +/- 3.1 vs 1.2 +/- 0.9 units, P < 0.02). Activities of 44 transcription factors were detectable, and sufficient RNA isolated for expression analysis of over 400 genes. CONCLUSIONS: The modifications enable broad characterisation of inflammatory responses and administration of exogenous immunomodulators.

Impaired neutrophil chemotaxis in Crohn's disease relates to reduced production of chemokines and can be augmented by granulocyte-colony stimulating factor.

BACKGROUND: Defective neutrophil recruitment has been described as a primary pathogenic abnormality in Crohn's disease. Cantharidin-induced blisters provide a novel investigative tool to assess cellular influx and inflammatory mediator production during acute inflammation and allows the effects of therapy on these parameters to be measured. AIMS: To determine whether reduced neutrophil tissue penetration in Crohn's disease relates to impaired production of inflammatory mediators, and whether it can be reversed by granulocyte-colony stimulating factor (G-CSF). METHODS: Neutrophil and monocyte/macrophage populations and inflammatory mediators were measured in cantharidin blisters at 24 h. Neutrophil chemotaxis was assessed in vitro using blister fluid as the chemoattractant. The effect of s.c. G-CSF on blister phenotype was determined. RESULTS: Significantly fewer neutrophils migrated into blisters in Crohn's patients. The production of neutrophil chemokines, but not other inflammatory mediators, was reduced. This significantly correlated with reduced chemotaxis in vitro. Differences were unrelated to caspase-recruitment domain 15 genotype. G-CSF significantly increased blister neutrophil concentrations in control subjects and Crohn's patients. CONCLUSIONS: Reduced neutrophil migration during acute inflammation in Crohn's disease is associated with impaired production of appropriate chemoattractants. G-CSF therapy increases neutrophil tissue migration, which may partially account for its observed therapeutic effect.

Can unresolved infection precipitate autoimmune disease?

Autoimmune diseases are frequently postulated to arise as post-infectious phenomena. Here we survey the evidence supporting these theories, with particular emphasis on Crohn's disease and ankylosing spondylitis. Direct proof that infection establishes persistent autoimmunity remains lacking, although it may provoke a prolonged inflammatory response when occurring on a susceptible immunological background. The argument of infective causality is by no means trivial, since it carries important consequences for the safety of vaccine development.

Effects of microinjected small GTPases on the actin cytoskeleton of human neutrophils.

This paper describes a method for microinjection of proteins (Rho GTPases) into neutrophils and observations on the responses of the cells to these injections. Neutrophils are extremely difficult to inject because of their small size, complex morphology and fragility. To allow microinjections they must be cultured on a substrate that enables them to settle, adhere and spread. We determined that fibronectin- and/or collagen-coated coverslips are the best substrates and we used very fine needles and short microinjection times to minimize cell damage. These methods permitted us to inject up to 100 cells in a single preparation over a period of 30 min. Effects of microinjection were assessed by using tetramethylrhodamine isothiocyanate (TRITC)-phalloidin to label F-actin filaments, and observation by fluorescence and confocal scanning microscopy. Microinjection alone resulted in cell rounding and some changes in the F-actin cytoskeleton but injected cells remained adherent at the substrate, were able to respond to microinjected GTPases (V12Rac, V14RhoA, V12Cdc42) and continued to be responsive to activation by exposure to fMet-Leu-Phe (fMLP) or O-tetradecanoylphorbal 13-acetate (TPA). V12Rac caused an increase in neutrophil membrane ruffling and short protrusions from the cell membrane, whereas V14RhoA induced a large increase in punctate F-actin structures. V12Cdc42 produced focal condensation of F-actin and induced the formation of small microspikes. The differences between these responses of neutrophils and those of other similarly treated cell types are discussed. Our findings demonstrate that microinjection is a valuable technique for studying the role of individual proteins in neutrophils.

Cantharidin blisters: a technique for investigating leukocyte trafficking and cytokine production at sites of inflammation in humans.

A skin blister technique is described which allows the investigation of acute inflammation in humans in vivo. Filter paper discs are placed on the skin, impregnated with cantharidin and covered with impermeable film held by adhesive tape. The assembly is easily applied, unobtrusive, stable and may be worn during normal activities. The blister formed at 24 h contains approximately 5x10(5)-5x10(6) cells, predominantly neutrophils and macrophages. Inflammatory cytokines and chemotactic factors are detectable in the blister fluid. The technique is useful for characterizing the acute inflammatory response in health and disease.

The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain.

The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071-3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin-radixin-moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.

Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils.

Neutrophils play a key role in host-defence mechanisms against invading pathogens, using their capacity to migrate, engulf micro-organisms and produce toxic radicals. Protein kinase C (PKC) isotypes are important intracellular regulators of these processes in neutrophils. PKC isotypes themselves are controlled by interactions with lipids, Ca(2+) and proteins. The C2-like domain of PKC-delta (deltaC2) has been identified as a protein-interaction domain in this PKC isotype. In the present paper we have investigated the contribution of protein interactions at this domain to the regulation/function of PKC-delta in neutrophils. Using affinity chromatography we identified actin as a deltaC2 binding partner in these cells. Fluorescein-labelled deltaC2, microinjected into immobilized neutrophils, interacts with filamentous actin (F-actin) inside the cell. PKC-delta co-localizes with F-actin in neutrophils, in lamellipodia at the leading edge of the cell. Stimulation with phorbol ester or IgG-opsonized Staphylococcus aureus results in co-ordinated redistribution of PKC-delta and F-actin, and a PKC-delta inhibitor inhibits these changes. Microinjection of deltaC2 also inhibits F-actin redistribution. Thus PKC-delta binds to F-actin through its C2 domain, and these interactions are important in regulating actin redistribution in neutrophils.

Evidence for a pool of coronin in mammalian cells that is sensitive to PI 3-kinase.

Coronin, a 57 kDa actin binding protein elutes with an apparent molecular mass of 400-600 kDa from gel filtration columns. This fraction is not unrelated to the reported 200 kDa complex where coronin is associated with phox proteins of the NADPH-oxidase. Phosphatidylinositol 3-kinase (PI 3-kinase) solubilizes coronin from the 400-600 kDa complex, thus constitutive active PI 3-kinase is sufficient to disrupt the complex, whereas wortmannin stabilizes it. Conversely, the phox protein associated pool of coronin is PI 3-kinase independent. During phagocytosis coronin is recruited together with PI 3-kinase to membranes of nascent and early phagosomes co-localizing with the actin cytoskeleton, confirming that coronin contributes to phagocytosis.

Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.

We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils.

Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G.

While the critical role of reactive oxygen intermediates (ROI) in the microbicidal activity of polymorphonuclear granulocytes is well established, the function of the nonoxidative effector mechanisms in vivo remains unclear. Here we show that mice deficient in the neutrophil granule serine proteases elastase and/or cathepsin G are susceptible to fungal infections, despite normal neutrophil development and recruitment. The protease deficiencies but not the absence of ROI leads to enhanced resistance to the lethal effects of endotoxin LPS, although normal levels of TNFalpha are produced. The data demonstrate a critical role of the nonoxidative effector mechanisms of neutrophils in host immunity and immunopathology and identify elastase and cathepsin G as effectors in the endotoxic shock cascade downstream of TNFalpha.

Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils.

p47(phox) is an essential component of the NADPH oxidase, and phosphorylation of p47(phox) is associated with activation of the enzyme. Here we have used p47(phox) affinity chromatography to extract a p47(phox) kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-beta(I), -beta(II) and -delta. The C-terminus of p47(phox) represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47(phox), we investigated the functional relevance of the interaction between PKC and p47(phox). Subcellular fractionation revealed an abnormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to particulate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47(phox) affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47(phox) can act as a regulator of PKC in neutrophils.

Activation of the neutrophil NADPH oxidase is inhibited by SB 203580, a specific inhibitor of SAPK2/p38.

Activation of the neutrophil NADPH oxidase by either the bacterial peptide fMLP or phorbol myristate acetate (PMA) is partially suppressed by SB 203580, a specific inhibitor of the MAP kinase family member, SAPK2/p38. The concentration of SB 203580 that suppresses activation of NADPH oxidase is similar to that which inhibits SAPK2/p38 in vitro, and both fMLP and PMA induce an extremely rapid and potent activation of SAPK2/p38 in neutrophils. SB 203580 does not exert its effect by preventing the neutrophil priming reaction, by suppressing the phosphorylation of p47phax, or by preventing the translocation of p47phax/p67phax to the plasma membrane.

Phosphorylation of p67phox in the neutrophil occurs in the cytosol and is independent of p47phox.

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine-phosphorylate p67phox in vitro. Further experiments with anti-phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.