Jörg Köhl

Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma

The prevalence and severity of allergic asthma continue to rise, lending urgency to the search for environmental triggers and genetic substrates. Using microarray analysis of pulmonary gene expression and single nucleotide polymorphism–based genotyping, combined with quantitative trait locus analysis, we identified the gene encoding complement factor 5 (C5) as a susceptibility locus for allergen-induced airway hyperresponsiveness in a murine model of asthma. A deletion in the coding sequence of C5 leads to C5-deficiency and susceptibility. Interleukin 12 (IL-12) is able to prevent or reverse experimental allergic asthma. Blockade of the C5a receptor rendered human monocytes unable to produce IL-12, mimicking blunted IL-12 production by macrophages from C5-deficient mice and providing a mechanism for the regulation of susceptibility to asthma by C5. The role of complement in modulating susceptibility to asthma highlights the importance of immunoregulatory events at the interface of innate and adaptive immunity in disease pathogenesis.

Anaphylatoxins and infectious and non-infectious inflammatory diseases.

In recent years a plethora of data has accumulated directing toward an important role of polypeptides C3a and C5a and its degradation product C5adesArg, summarized as anaphylatoxins (ATs), in microbial host defense and immune regulation. The ATs exert their various biologic functions by interacting with specific C3a- and C5a-receptors present on cells of myeloid origin, epithelial cells, smooth muscle cells as well as on activated B- and T-cells. Activation of AT receptors mediates signal transduction pathways triggering a variety of proinflammatory events. However, by interacting with the cytokine- and chemokine network C3a and C5a exhibit also anti-inflammatory properties. In this review the focus is on the pathogenetic role of the ATs in sepsis, immune complex disease, delayed type hypersensitivity and asthma. Discussed are data from animal models in which the ATs are blocked by specific C3a or C5a inhibitors or from mice with genetic deletions of the specific receptors of either C3a or C5a/C5adesArg.

Cutting Edge: Guinea Pigs with a Natural C3a-Receptor Defect Exhibit Decreased Bronchoconstriction in Allergic Airway Disease: Evidence for an Involvement of the C3a Anaphylatoxin in the Pathogenesis of Asthma

Asthma is a major cause of morbidity worldwide with prevalence and severity still increasing at an alarming pace. Hallmarks of this disease include early-phase bronchoconstriction with subsequent eosinophil infiltration, symptoms that may be mimicked in vivo by the complement-derived C3a anaphylatoxin, following its interaction with the single-copy C3aR. We analyzed the pathophysiological role of the C3a anaphylatoxin in a model of experimental OVA-induced allergic asthma, using an inbred guinea pig strain phenotypically unresponsive to C3a. Molecular analysis of this defect revealed a point mutation within the coding region of the C3aR that creates a stop codon, thereby effectively inactivating gene function. When challenged by OVA inhalation, sensitized animals of this strain exhibited a bronchoconstriction decreased by ∼30% in comparison to the corresponding wild-type strain. These data suggest an important role of C3a in the pathogenesis of asthma and define a novel target for drug intervention strategies.

Distinct Tissue Site-Specific Requirements of Mast Cells and Complement Components C3/C5a Receptor in IgG Immune Complex-Induced Injury of Skin and Lung

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (KitW/KitW-v mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcγRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3−/− mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3−/− or KitW/KitW-v mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-α, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3−/− mice and KitW/KitW-v mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.

On the role of complement and Fc γ-receptors in the Arthus reaction

Abstract The contribution of either the complement system or the activation of Fc receptors for IgG (FcγRs) to the inflammatory response in immune complex (IC) disease is puzzling. A series of studies has been performed in mice with engineered deficiencies of either FcγRs, the complement components C3, C4 or the C5a receptor. In addition, different C5-deficient mice strains have been evaluated. Mice with gene targeted disruption of the γ-subunit, which mediates surface expression and signal transduction of the high affinity Fc receptor type I for IgG (FcγRI), the low affinity receptor Fc receptor type III for IgG (FcγRIII) and the high affinity receptor type I for IgE (IgϵRI), showed an impaired inflammatory response in the reverse passive Arthus reaction in skin, peritoneum and lung. These data suggest, that the activation of FγRs is the initial event triggering the inflammatory cascade in IC disease. On the other hand, C5aR deficient mice are either protected from tissue injury induced by ICs, as in the lung, or the degree of the inflammatory response is markedly attenuated, as in peritoneum and skin. A detailed analysis of data obtained with the different knock-out strains revealed that both the activation of the complement system as well as the activation of different effector cells via FcγRs contribute to the inflammatory sequelae leading to tissue destruction in IC disease. The relative contributions of FcγRI or FcγRIII and the main effector cells through which these receptors mediate their effector functions are tissue dependent. The activation of the C5a receptor pathway appears to be the prominent contribution of the complement system.

Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures

Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.

Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis.

BACKGROUND The anaphylatoxin C5a is a powerful proinflammatory protein generated on activation of the complement system. Recently, we described an anti-hC5a neoepitope specific mAb, mAb 2925, which was raised against the nonapeptide ISHKDMQLG (C5a-(65-73). This mAb is unique in that it recognizes both hC5a and hC5adesArg, even when it is denatured. It inhibits binding of [125I]C5a to its receptor on Bt2-cAMP differentiated U937 cells. OBJECTIVES To define the epitope of mAb 2925, we used a combined approach of a bacteriophage random octapeptide library, synthetic peptides and site-directed mutagenesis. STUDY DESIGN First a phage peptide library was screened with the anti C5a mAb 2925. Then synthetic peptides were synthesized with respect to the sequence information yielded from the phage approach, and used for binding studies. Site-directed mutagenesis was performed to confirm the results from the mapping experiments. RESULTS AND CONCLUSION Most phages selected by biotinylated Fab 2925 displayed sequences on the minor coat protein which correspond to residues within the C-terminus of human C5a. A first consensus motif comprised amino acids His-Lys or His-Arg, which allowed us to define position 67 and 68 as part of the epitope. A second consensus motif was selected, comprising Arg/Lys-Trp-Trp. This motif did not match any residues within the C5a C-terminus. However, when expressed together with the consensus motif His-Arg, as in HRWWXXXX or in HRXKWWXX, binding of these peptides to Fab 2925 increased as compared to peptides expressing the His-Arg motif only. Thus, the Arg/Lys-Trp-Trp motif serves to stabilize the binding of His-Arg to mAb 2925. Synthetic peptide studies revealed further N-terminal residues Ile65 and Ser66 as part of the epitope. A C5a mutant with an exchange Lys68Glu (C5aGlu68) confirmed the participation of Lys68 as a contact residue within the epitope of mAb 2925. Hence, the epitope recognized by mAb 2925 is linear and comprises residues Ile65, Ser66, His67, and Lys68. Thus, we could demonstrate for the first time that a mAb inhibits C5a receptor binding through specific interaction with receptor binding residues of the ligand.

Chimeric Receptors of the Human C3a Receptor and C5a Receptor (CD88)

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Gα-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.

Characterization of Synthetic C3a Analog Peptides on Human Eosinophils in Comparison to the Native Complement Component C3a

The C3a anaphylatoxin is a potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like Ca2+ transients and the activation of the respiratory burst. These findings support an important role for C3a in diseases typically associated with a peripheral blood or tissue eosinophilia. Synthetic human C3a analogue peptides with variations at the C-terminal effector domain have been evaluated with respect to their binding affinity and signaling potency on human eosinophils. Flow cytometrical analysis and RT-PCR revealed that the C3a receptor is constitutively expressed on human eosinophils. Peptides bearing an N-terminal 9-fluorenylmethoxycarbonyl and the 6-aminohexanoyl motif were the most powerful peptides tested. Amino acid replacements in the conserved C-terminal pentapeptide decreased binding affinity and functional potency substantially. In addition, synthetic C3a analogue peptides induced C3aR internalization, led to transient changes of intracellular Ca2+ concentration, and did release reactive oxygen species in human eosinophils indicating the in vivo relevance of C3a-related sequences. The tripeptide LAR was found to be essential for C3a receptor binding on human eosinophils. Moreover, the putative binding motif of C3a anaphylatoxin is also crucial for the induction of biologic effects in the human system such as changes of intracellular Ca2+ concentration and the release of reactive oxygen species. This study demonstrates that the carboxyl terminus is important for the interaction with the C3aR and the biologic potency of C3a anaphylatoxin in the human system and plays a key role in the activation process of human eosinophils.

Human C5a anaphylatoxin : gene cloning and expression in Escherichia coli

A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.