Dysregulation of Complement System and CD4+ T Cell Activation Pathways Implicated in Allergic Response

Allergy is a complex disease that is likely to involve dysregulated CD4+ T cell activation. Here we propose a novel methodology to gain insight into how coordinated behaviour emerges between disease-dysregulated pathways in response to pathophysiological stimuli. Using peripheral blood mononuclear cells of allergic rhinitis patients and controls cultured with and without pollen allergens, we integrate CD4+ T cell gene expression from microarray data and genetic markers of allergic sensitisation from GWAS data at the pathway level using enrichment analysis; implicating the complement system in both cellular and systemic response to pollen allergens. We delineate a novel disease network linking T cell activation to the complement system that is significantly enriched for genes exhibiting correlated gene expression and protein-protein interactions, suggesting a tight biological coordination that is dysregulated in the disease state in response to pollen allergen but not to diluent. This novel disease network has high predictive power for the gene and protein expression of the Th2 cytokine profile (IL-4, IL-5, IL-10, IL-13) and of the Th2 master regulator (GATA3), suggesting its involvement in the early stages of CD4+ T cell differentiation. Dissection of the complement system gene expression identifies 7 genes specifically associated with atopic response to pollen, including C1QR1, CFD, CFP, ITGB2, ITGAX and confirms the role of C3AR1 and C5AR1. Two of these genes (ITGB2 and C3AR1) are also implicated in the network linking complement system to T cell activation, which comprises 6 differentially expressed genes. C3AR1 is also significantly associated with allergic sensitisation in GWAS data.     

The Role of Heparanase in Pulmonary Cell Recruitment in Response to an Allergic but Not Non-Allergic Stimulus

Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa^-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa^-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa^-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.     

Induction of Allergic Responses to Peanut Allergen in Sheep

Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.     

A Role for Complement in Phagocytosis of Myelin

The mechanisms for phagocytosis of myelin in cell-mediated demyelinating diseases have not been clarified. We have previously shown with cultured phagocytic cells that myelin opsonized with antiserum to myelin constituents is phagocytized in much higher amounts than untreated myelin, indicating that Fc receptors may be involved in the demyelinating process. Using various treatments of antisera, such as heating to destroy complement, and purification of IgG, we show here that complement is a necessary factor for maximal myelin phagocytosis by cultured macrophages. If myelin is sonicated to decrease its particle size, however, complement is not an active factor. Cultured microglia, on the other hand, required complement for maximal phagocytosis of both unsonicated and sonicated myelin. Addition of serum complement greatly increased phagocytosis of untreated CNS and PNS myelin, both unsonicated and sonicated, by macrophages and microglia. From these results it appears that the most important effect of complement is to fragment the myelin, making it more easily phagocytized. Prefragmentation of myelin by sonication can substitute for complement. Complement receptors may, in addition, be important for maximal myelin phagocytosis by microglia.This work was done at the VA Medical Center in fulfillment of the research requirement at the University of Amsterdam     

Complement activation in heart diseases. Role of oxidants

Increasing evidence demonstrated that atherosclerosis is an immunologically mediated disease. Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Three major components are recognized as the major contributing factors in reperfusion injury. These are: (1) molecular oxygen; (2) cellular blood elements (especially the neutrophils); and (3) components of the activated complement system. The latter two often act in concert. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue damage mediated by neutrophils can be initiated by complement fragments, notably C5a, which are potent stimulators of neutrophil superoxide production and adherence to coronary artery endothelium. The complement cascade, particularly the alternative pathway, is activated during myocardial ischemia/reperfusion. Complement fragments such as the anaphylatoxins C3a and C5a, are produced both locally and systematically, and the membrane attack complex is deposited on cell membranes and subsequent release of mediators such as histamine and platelet activating factor (PAF), thereby causing an increase in vascular permeability with concomitant manifestation of cellular edema. Complement increases the expression of CD18 on the neutrophils and increases P-selectin expression on the surface of the endothelium. Mitochondria may be a source of molecules that activate complements during ischemia/reperfusion injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexities of the complement cascade provide numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vitro models of ischemia/reperfusion injury. The mechanism(s) that initiates complement activation is not clearly known, although loss of CD59 (protectin) from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex. Therapeutic approaches to ischemia/reperfusion injury in general, and especially those involving complements, are at the very beginning and their potential benefits have still to be adequately evaluated. It may be noted that complement activation has both positive and negative effects and, therefore, might be modulated rather than abruptly blunted.     

线粒体神经酰胺对细胞凋亡的诱导作用

凋亡诱导期,线粒体内神经酰胺水平升高,当每纳摩尔线粒体膜磷脂内含4～6皮摩尔神经酰胺时,神经酰胺即在线粒体外膜形成稳定的跨膜通道,从而使外膜通透性增加,线粒体膜间蛋白释放,启动细胞凋亡.神经酰胺通道只能在线粒体外膜形成,它是由神经酰胺柱组成的桶装结构,神经酰胺的反式双键具有增加通道的稳定性的作用.     

Local Inflammation Induces Complement Crosstalk Which Amplifies the Antimicrobial Response

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection.     

Microwaves in the Induction of Immune Response to VI-Antigen

In this work the possibility of using microwaves (MW) for immuno-modulation in the immunization of animals with thymus-independent antigen was studied. The projection zones of the thyroid and adrenal glands of the test animals were subjected to the action of decimeter MW, while the corresponding zones of control animals were subjected to imitation MW. The endocrine activity of rabbits was estimated by radioim-mune methods. Vi-antigen was shown to be a thymus-independent antigen for rabbits, according to the results of fluorescent probes to study the structural rearrangements in surfaces of thymocyte membranes and their nuclei, which reflect early changes during the physiological activation of cells. The irradiation by MW on the projection zone of the thyroid was accompanied by a decrease in the glucocorticoid activity of the adrenal cortex and a simultaneous pronounced immunostimulating effect. MW irradiation of the zone of the adrenal glands was accompanied by immunosuppression in combination with enhanced glucocorticoid activity of the adrenal cortex.     

Role for Complement in the Development of Seizures following Acute Viral Infection

Complement, part of the innate immune system, acts to remove pathogens and unwanted host material. Complement is known to function in all tissues, including the central nervous system (CNS). In this study, we demonstrated the importance of the complement system within the CNS in the development of behavioral seizures following Theiler''s murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice, deficient in complement component C3, developed significantly fewer behavioral seizures following TMEV infection, whereas mice depleted of complement component C3 in the periphery through treatment with cobra venom factor had a seizure rate comparable to that of control mice. These studies indicate that C3 participates in the induction of acute seizures during viral encephalitis.The complement system, a component of the innate immune system, functions to recognize and eliminate pathogens and unwanted host material (1). Activation of complement can occur by the classical, alternative, lectin, and terminal pathways (1). The classical pathway is activated by antigen-antibody complexes, some viruses, Gram-negative bacteria, or C-reactive protein complexes (4). The alternative pathway is activated by lipopolysaccharides and polysaccharides on the surfaces of viruses, bacteria, fungi, and parasites. The lectin pathway is activated by mannose or N-acetylglucosamine on the surfaces of bacteria and other pathogens (4). The complement system, consisting of >40 proteins, is highly regulated by the expression of complement inhibitors and complement receptors, as the complement system can have deleterious effects when unregulated (1). Two important steps in the complement cascade are the cleavage of the multifunctional complement proteins C3 and C5 into C3a and C3b proteins and C5a and C5b proteins, respectively. The anaphylatoxins C3a and C5a function to recruit leukocytes and induce inflammation. C3b functions in opsonization, the process of coating pathogens or particulate material with opsonin and thus making it more susceptible to phagocytosis. C5b functions to initiate the assembly of the C5b-C9 complex, the membrane attack complex (MAC), leading to pathogen lysis. Proteins of the complement system are found throughout all tissues and bodily fluids. Although primarily produced by hepatocytes in the liver, other cell types constitutively express low levels of complement proteins (1).Complement proteins are constitutively produced by neurons, microglia, astrocytes, and oligodendrocytes in the central nervous system (CNS) (14, 16, 44; reviewed in references 1 and 13). Astrocytes, the predominant glial cell type in the brain, are comparable to hepatocytes in terms of the number of complement components they produce (4). The levels of various complement mRNAs and proteins are markedly increased in the CNS following viral infection; for example, C1q and C3 proteins and mRNAs are increased in the rat brain following infection with Borna disease virus (11), and C1q protein and mRNA are increased in the rhesus macaque brain following infection with simian immunodeficiency virus (10). In both cases, the increase in the production of C1q in the brain was localized to microglia/macrophages (10, 11). Complement activation has also been shown to be involved in the control of other viral infections, such as the spread of West Nile virus to the CNS (24).We have been studying the role of the innate immune system in the development of acute behavioral seizures following CNS infection of C57BL/6 mice with a neurotropic virus (19, 23). Infection with Theiler''s murine encephalomyelitis virus (TMEV) results in acute seizures developing in more than 50% of C57BL/6 mice (both male and female) generally between days 3 and 10 postinfection (p.i.) (23). Two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and concomitant inflammatory changes in the brain (perivascular cuffing comprised of infiltrating mononuclear cells, infiltration of macrophages, and/or activation of microglial cells and gliosis) were implicated as contributors to the development of acute seizures (19). In contrast, the proinflammatory cytokine IL-1, TMEV-specific CD8⁺ T cells, and viral persistence were discounted as playing a role in seizures (19). It was found that both the pattern of days on which the mice were observed to have seizures and the seizure score (Racine scale) for any given day varied from mouse to mouse (23). Typically, day 3 p.i. was the first day on which a few mice were observed to have seizures, day 6 p.i. was the peak of behavioral seizure activity, and the majority of seizures had a seizure score of 3 (forelimb clonus) and above (score of 4, rearing; score of 5, rearing and falling). The seizures were afebrile and appeared limbic in nature (23). The seizure frequency was observed to be one per mouse per 2-h observation period, and the duration of the seizures was typically 1 to 2 min (23, 31). Mice experiencing seizures were impaired in both coordination and motor function (23).The role of complement in the development of seizures has been studied in humans with Rasmussen''s encephalitis (RE) (41). Although several different viruses have been detected in the tissues of humans with RE by PCR and in situ hybridization, these results are still controversial. Instead it is thought that an autoimmune process underlies RE. Notwithstanding the unknown etiology of RE, the activation of the complement cascade is thought to be a critical component of disease pathogenesis. Several activated components of the complement system (C4, C8, and MAC) were shown to be present in discrete patches of neurons in the cortex of three out of five patients with active RE by immunohistochemistry (41). As a means of demonstrating in vivo that MAC deposition on neurons could trigger seizures, the individual components of the MAC (C5b6, C7, C8, and C9) were sequentially infused into the rat hippocampus, and assembly of the MAC triggered both behavioral and electrographic seizures as well as cytotoxicity (42).In our current study, we examined the role that complement may play in the development of behavioral seizures in the TMEV-induced seizure model. Through the use of mice deficient in complement component C3 and through depletion of complement component C3 in the periphery, we were able to demonstrate the importance of the complement system within the CNS in the development of seizures in the TMEV-induced seizure model.     

Alternative Complement Activity in the Egg Cytosol of Amphioxus Branchiostoma belcheri: Evidence for the Defense Role of Maternal Complement Components

Background

The eggs in most invertebrates are fertilized externally, and therefore their resulting embryos are exposed to an environment full of microbes, many of which are pathogens capable of killing other organisms. How the developing embryos of invertebrates defend themselves against pathogenic attacks is an intriguing question to biologists, and remains largely unknown.

Methodology/Principal Findings

Here we clearly demonstrated that the egg cytosol prepared from the newly fertilized eggs of amphioxus Branchiostoma belcheri, an invertebrate chordate, was able to inhibit the growth of both the Gram-negative bacterium Vibrio anguillarum and the Gram-positive bacterium Staphylococcus aureus. All findings point to that it is the complement system operating via the alternative pathway that is attributable to the bacteriostatic activity.

Conclusions/Significance

This appears to be the first report providing the evidence for the functional role of the maternal complement components in the eggs of invertebrate species, paving the way for the study of maternal immunity in other invertebrate organisms whose eggs are fertilized in vitro. It also supports the notion that the early developing embryos share some defense mechanisms common with the adult species.     

Minor Role of Plasminogen in Complement Activation on Cell Surfaces

Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

Complement as an Endogenous Adjuvant for Dendritic Cell-Mediated Induction of Retrovirus-Specific CTLs

Previous studies have demonstrated the involvement of complement (C) in induction of efficient CTL responses against different viral infections, but the exact role of complement in this process has not been determined. We now show that C opsonization of retroviral particles enhances the ability of dendritic cells (DCs) to induce CTL responses both in vitro and in vivo. DCs exposed to C-opsonized HIV in vitro were able to stimulate CTLs to elicit antiviral activity significantly better than non-opsonized HIV. Furthermore, experiments using the Friend virus (FV) mouse model illustrated that the enhancing role of complement on DC-mediated CTL induction also occurred in vivo. Our results indicate that complement serves as natural adjuvant for DC-induced expansion and differentiation of specific CTLs against retroviruses.     

The Role of the Root in the Induction of Xylem Differentiation in Peas

It is known that growing parts of the shoot induce the differentiationof vascular tissues below them and that this induction is dueto the production of auxin. The problem dealt with here is whythe formation of xylem proceeds in the growing roots. The redifferentiationof parenchyma to tracheary elements in grafts of pea plantswas used in this study. It is proved that this is not due tostimuli coming from the root tip but rather to the movementof a stimulus coming from the shoot into the root. The polarityof movement is maintained even in thin sections, but it canbe reversed by a strong shoot influence.