Anthony W. Segal

Killing activity of neutrophils is mediated through activation of proteases by K+ flux.

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.

Defective acute inflammation in Crohn's disease : a clinical investigation

BACKGROUND The cause of Crohns disease has not been mechanistically proven. We tested the hypothesis that the disease is a form of immunodeficiency caused by impaired innate immunity. METHODS We investigated inflammatory responses in patients and controls by quantifying neutrophil recruitment and cytokine production after acute trauma, interleukin 8 secretion by cultured monocyte-derived macrophages after exposure to inflammatory mediators, and local inflammatory and vascular changes in response to subcutaneous injection of heat-killed Escherichia coli. FINDINGS In patients with Crohns disease, trauma to rectum, ileum, or skin led to abnormally low neutrophil accumulation (differences from healthy individuals of 79%, n=8, p=0.0003; 57%, n=3, p=0.05; 50%, n=13, p<0.0001, respectively) and lower production of proinflammatory interleukin 8 (63%, n=7, p=0.003; 63%, n=3, p=0.05; 45%, n=8, p<0.0001) and interleukin 1beta (50%, n=8, p=0.0005). Interleukin 8 secretion by cultured macrophages was reduced after exposure to acute wound fluid (38%, n=50, p<0.0001), C5a (48%, n=41, p=0.0005), or tumour necrosis factor alpha (52%, n=27, p<0.0001). Local inflammatory reaction to inoculation with E coli was attenuated, as quantified by changes in bloodflow (ileal disease 50%, n=6, p=0.01; colonic disease 77%, n=6, p=0.0003). This response was mediated by nitric oxide in controls, was increased by sildenafil in patients, and was not related to CARD15 genotype. INTERPRETATION In Crohns disease, a constitutionally weak immune response predisposes to accumulation of intestinal contents that breach the mucosal barrier of the bowel wall, resulting in granuloma formation and chronic inflammation. Polymorphisms in CARD15 do not underlie this phenotype, but incapacitate the NOD2 pathway that can compensate for impairment of innate inflammation. Current treatment of secondary chronic inflammation might exaggerate the underlying lesion and promote chronic disease.

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.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

The cause of Crohns disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Absence of Cytochrome b-245 in Chronic Granulomatous Disease

The heme-containing protein cytochrome b-245 has been proposed as a primary component of the microbicidal oxidase system of phagocytes that normally generates superoxide-free radicals but when defective is associated with chronic granulomatous disease. We measured this cytochrome in granulocytes from 27 patients with chronic granulomatous disease and from 64 members of their families. It was undetectable in all 19 of the men in whom the defect appeared to be located on the X chromosome. Female relatives who were heterozygous carriers had reduced concentrations of the cytochrome and variable proportions of cells that were unable to generate superoxide; these two characteristics were closely related (r = 0.93 in the 16 mothers and 0.85 in all 24 carriers, P less than 0.001). In contrast, in all eight patients (seven women) with a probable autosomal recessive inheritance the cytochrome was present but nonfunctional. The properties tested, including midpoint potential, carbon monoxide binding, and organelle distribution, were normal, but the cytochrome did not undergo reduction on cellular stimulation. Thus, absence or malfunction of the cytochrome b-245 may be the causal molecular defect in chronic granulomatous disease, implicating it in the microbicidal oxidase system.

Chronic granulomatous disease

Chronic granulomatous disease is an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal infections and disordered inflammation. We propose that NADPH oxidase has a key role in regulating acute neutrophilic and T cell responses, which in turn restrains fungal growth and calibrates the inflammatory response to minimize injury and allergy. In this model, superoxide-induced activation of indoleamine 2,3-dioxygenase (IDO) is a central mechanism by which the optimal balance of antifungal host defense and immune tolerance occurs. This model is based on studies in mice and requires correlation in humans.

Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein.

Phagocytosing neutrophils, monocytes, macrophages and eosino-phils produce a burst of non-mitochondrial respiration that is important for the killing and digestion of microbes. Much of the information about the oxidase system involved comes from studies on patients with chronic granulomatous disease (CGD), a syndrome in which an undue predisposition to infection results from complete absence of this burst of stimulated respiratory activity1. The basis of the oxidase activity is an electron transport chain, the only established component of which is a very unusual b-type cyto-chrome (b−245) (ref. 2). The molecular defect in the X-linked subgroup of CGD is the absence of this cytochrome b−245, which, however, appears to be normal in those subjects with the autosomal recessive mode of inheritance3. In an attempt to identify an abnormality of activation, or an absence or malfunction of a proximal component of the electron transport chain in this latter group, we examined protein phosphorylation in neutrophils after activation of the oxidase with phorbol myristate acetate. All four of the patients studied demonstrated a selective lack of the enhanced phosphorylation of a protein of relative molecular mass (Mr) 44,000 (44K) that was observed in normal subjects and in two CGD patients with an X-linked inheritance. This molecule, therefore, could be an important functional component of the oxidase.

The large-conductance Ca2+-activated K+ channel is essential for innate immunity

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BKCa) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BKCa channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the α-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BKCa channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Intramembrane Bis-Heme Motif for Transmembrane Electron Transport Conserved in a Yeast Iron Reductase and the Human NADPH Oxidase

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91phox protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Inflammatory Bowel Disease in CGD Reproduces the Clinicopathological Features of Crohn's Disease

OBJECTIVES:Patients with chronic granulomatous disease (CGD), a rare congenital disorder characterized by defective neutrophil function, frequently develop an inflammatory bowel disease similar to Crohns disease. The clinical presentations and concordance between the features of the bowel disease in these two conditions have never been formally evaluated.METHODS:Retrospective case note analysis of all adult patients with CGD treated at a tertiary care hospital.RESULTS:A total of 25 eligible patients were identified. Of these, 14 (56%) had experienced gastrointestinal symptoms in the preceding 3 years; and 11 (44%) had documented gastrointestinal inflammation not secondary to infection, manifesting throughout the alimentary canal including the upper gastrointestinal tract (45%), small intestine (27%), colon (73%), and rectum (73%). All had discontinuous inflammation and perianal involvement, and approximately half (55%) demonstrated epithelioid granulomata on histology. All patients fulfilled the Lennard–Jones criteria for the diagnosis of Crohns disease. Therapeutic responses were observed in five patients to 5-aminosalicylates, and in individual patients to thalidomide, interferon-γ, azathioprine, infliximab, and intestinal resection.CONCLUSIONS:There are striking clinical and pathological resemblances between the bowel diseases observed in CGD and Crohns disease, supporting the possibility of mechanistic similarities in their pathogenesis. Patients with CGD appear particularly prone to developing perianal disease.