Anaphylatoxin C5a potentiates allergic inflammation in human lungs

Anaphylatoxin C5a potentiates allergic inflammation in human lungs

Molecular Immunology 44 (2007) 147–266 Abstracts from the XXIst International Complement Workshop 1 Modelling the complex between CD55 and Factor B ...

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Molecular Immunology 44 (2007) 147–266

Abstracts from the XXIst International Complement Workshop

1 Modelling the complex between CD55 and Factor B using electron paramagnetic resonance

an accurate model of the complex between CD55 and Factor B vWF-A.

Rachel J.M. Abbott a , Janet E. Banham b , Christiane R. Timmel b , Gunnar Jeschke c , Susan M. Lea a

doi:10.1016/j.molimm.2006.07.285

a

Sir William Dunn School of Pathology, South Parks Road, Oxford, UK; b Chemistry Department, South Parks Road, Oxford, UK; c Max Planck Institut F¨ur Polymerforschung, Mainz, Germany The human complement regulator CD55 (decay accelerating factor, DAF) is an intrinsic membrane glycoprotein that protects self-cells from complement-mediated lysis. It inactivates the C3 convertases by dissociating them into their constituent proteins. To fully understand how CD55 regulates the convertases more information is needed about the interaction between the components involved. A structure for the complex formed by the interaction of CD55 and human Factor B, a major component of the alternative pathway convertase, would prove particularly informative. Unfortunately, obtaining a structure of the proteins in complex with one another by X-ray and NMR methods is very difficult due to the transient nature of the interaction and the size of the components involved. In an effort to overcome these difficulties, we have applied the four-pulsed EPR method of double electron electron resonance (DEER) to provide experimentally-derived restraints that have allowed us to produce a model for the complex. We have expressed and purified a range of CD55 and Factor B vWF-A domain mutants. The single free cysteine residue on each protein has been reacted with a methane thiosulfonate nitroxide spin-label (MTSSL). DEER has been performed with frozen solutions of CD55 and Factor B vWF-A to measure the distance between the spin-labels within the protein complex. The various mutants produced have enabled us to alter the position of the spin-label within the proteins and this has allowed us to obtain a range of different measurements. Using the distances measured in this way, together with the crystal structures of CD55 and the vWF-A domain of Factor B, we have produced 0161-5890/$ – see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.molimm.2006.07.005

2 Anaphylatoxin C5a potentiates allergic inflammation in human lungs Masayoshi Abe a , Noriko Satoh a , Takehiro Umemura a , Akinori Iwasaki b , Takayuki Shirakusa b , Takeshi Katsuragi a a

Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan; b Department of Surgery, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan Bronchial asthma is a complex inflammatory disorder of the airways. Complement activation produces anaphylatoxic polypeptides, and the anaphylatoxins C3a, C4a, and C5a, are considered to contribute to the pathophysiology of asthma. Among various mediators and cytokines involved in asthma inflammation, cysteinyl-leukotrienes (cysLTs) are one of the most important mediators. Consequently, we studied whether or not C3a and C5a were able to stimulate generation of cysLTs from human lung tissues. The lung tissues that appeared macroscopically normal were obtained from patients with lung cancer. The extraction of RNA from the tissues and then polymerase chain reaction showed C5aR-, C3aR-, and C5L2-mRNA expression in the lungs. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). Incubation with C5a or C3a alone did not stimulate cysLT production from lung tissues. However, the co-addition of C5a or C3a with anti-IgE antibody potentiated cysLT production in comparison with antibody stimulation alone. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1000 ng/ml tended to increase cysLT production, but not to any significant extent. A novel

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C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production from the lung fragments following anaphylactic reaction in the presence of 1 ng/ml C5a. A non-peptide C5aR antagonist (W-54011) similarly suppressed the production of cysLTs by the same stimulation from human lung tissues but not from rat lung tissues. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lungs and contributes to allergic inflammation in disorders such as asthma. Pharmacological manipulation of C5a/C5a receptor signaling may be a promising target for the development of novel anti-asthmatic therapies. doi:10.1016/j.molimm.2006.07.007 3 Role of factor H and complement receptor 3 in mediating attachment of Neisseria gonorrhoeae to eukaryotic cells Sarika Agarwal, Sanjay Ram, Sunita Gulati, Peter A. Rice University of Massachusetts Medical School, Division of Infectious Diseases and Immunology, Worcester, MA 01602, USA Human factor H, an alternative complement pathway regulatory protein, is instrumental in converting complement component C3b to the inactivate form, iC3b, enabling gonococci to evade killing by human complement. In addition to iC3b, factor H, itself, binds to specific receptors on different cell types and in association with complement receptor 3 (CR3, also known as CD11b/CD18) plays a role in adherence to PMNs. Recently it has been reported that CR3 is also present on primary cervical epithelial cells. We hypothesized a potentially cooperative role between factor H and CR3 in gonococcal adherence to epithelial cells that may facilitate their entry into cells. In a model system, we examined association of a factor H binding strain of Neisseria gonorrhoeae, called 252, and the non-factor H binding strain, F62, to Chinese hamster ovary (CHO) cells, transfected with and without the CR3 receptor. In addition to unsialylated gonococcal strains used in the study, cultures were also sialylated with the addition of 5 -cytidinemonophospho-N-acetyl neuraminic acid (CMP-NANA). A fraction of sialylated and unsialylated cultures were each pre-incubated with factor H, then added to CR3 transfected and non-transfected CHO cells. Using either FACS or fluorometer analysis, we measured a significant increase in adherence to CHO cells by GFP expressing gonococci with bound factor H in CR3 transfected CHO cells compared to diminished adherence to non-transfected CHO cells using gonococci with or without bound factor H (ratios range between 2.8 and 5 in gonococci that bind factor H compared to a ratio of ∼1 for gonococci that do not bind factor H). Specificity of the factor H/CR3 interaction in CHO cells was corroborated by increased binding of free factor H and resultant decreased exposure of CD18 on CR3 transfected cells. These results indicate that binding to CR3 bearing cells is enhanced in N. gonorrhoeae with affixed factor H, thereby providing an additional mechanism of adherence of gonococci to host cells. doi:10.1016/j.molimm.2006.07.008

4 Insights into complement amplification and regulation and quenching from the crystal structure of C3b A.A. Ajees a , K. Gunasekaran a , G.J. Kotwal b,c , H.M. Krishna Murthy a a

Center for Biophysical Sciences and Engineering, 1530, 3rd Avenue South, Birmingham, AL 35294, United States; b Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202, United States; c Division of Medical Virology, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, HSC, Cape Town 7925, South Africa Component 3b (C3b) occupies a point in the complement cascade at which classical, alternative and lectin pathways of activation converge through formation of C5 convertases. C3, which in its mature form is a 187 kDa, glycosylated, two-chain, protein, is activated by proteolytic excision of the anaphylatoxic C3a, to its active form, C3b. This proteolysis triggers major conformational changes and results in exposure of a cryptic thioester site for attachment of C3b to cell-surface hydroxyl groups. C3b provides a scaffold for assembly of both C3 and C5 convertases which modulate amplification, regulation and quenching of the complement response against pathogens. C3b thus possesses binding sites for a second molecule of C3b, other complement factors such as factor B and regulatory proteins such as factor H, properdin, decay accelerating factor, membrane cofactor protein, complement receptor 1 as well as for viral regulatory molecules such as vaccinia-virus complement-control protein. Recent determination of the structures of C3 and C3c have contributed greatly to our understanding of complement function. However, because neither C3 nor C3c is active in complement amplification and regulation, many pertinent questions can be answered only through direct observations made on the struc˚ we ture of C3b. Indeed, the crystal structure of C3b at 2.3 A will present, reveal major rearrangement of several domains, from their locations in C3, to form C3b, and suggests potential mechanisms for the pivotal role of C3b in complement amplification and regulation. Much of the versatile activity of C3b in complement function appears to arise from exposure of an array of binding sites for interaction with regulatory and enzymatic factors from their buried environments in C3. The structure of this central component of both convertases, in its active form, is also likely to provide a much anticipated platform of further structural and functional studies of complement amplification and regulation. doi:10.1016/j.molimm.2006.07.009