W Bautsch

Publications of W Bautsch

• Site-specific anti-C3a receptor single-chain antibodies selected by differential panning on cellulose sheets.

  Authors: H Hawlisch, M Müller, R Frank, W Bautsch, A Klos, J Köhl

  Analytical biochemistry. 07/2001; 293(1):142-5.

• Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

  Authors: R S Ames, D Lee, J J Foley, A J Jurewicz, M A Tornetta, W Bautsch, B Settmacher, A Klos, K F Erhard, R D Cousins, A C Sulpizio, J P Hieble, G McCafferty, K W Ward, J L Adams, W E Bondinell, D C Underwood, R R Osborn, A M Badger, H M Sarau

  Journal of immunology (Baltimore, Md. : 1950). 06/2001; 166(10):6341-8.

  The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning ofThe anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

• 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.

  Authors: W Bautsch, H G Hoymann, Q Zhang, I Meier-Wiedenbach, U Raschke, R S Ames, B Sohns, N Flemme, A Meyer zu Vilsendorf, M Grove, A Klos, J Köhl

  Journal of immunology (Baltimore, Md. : 1950). 12/2000; 165(10):5401-5.

  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 subsequentAsthma 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 approximately 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.

• Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin, protein complement 3a, and interleukin-6.

  Authors: O Selberg, H Hecker, M Martin, A Klos, W Bautsch, J Köhl

  Critical care medicine. 08/2000; 28(8):2793-8.

  OBJECTIVE: To evaluate whether plasma concentrations of procalcitonin (PCT), interleukin-6 (IL-6), protein complement 3a (C3a), leukocyte elastase (elastase), and the C-reactive protein (CRP)OBJECTIVE: To evaluate whether plasma concentrations of procalcitonin (PCT), interleukin-6 (IL-6), protein complement 3a (C3a), leukocyte elastase (elastase), and the C-reactive protein (CRP) determined directly after the clinical onset of sepsis or systemic inflammatory response syndrome (SIRS) discriminate between patients suffering from sepsis or SIRS and predict the outcome of these patients. DESIGN: Prospective study. SETTING: Medical intensive care unit at a university hospital. PATIENTS: Twenty-two patients with sepsis and 11 patients with SIRS. MEASUREMENTS AND MAIN RESULTS: The plasma concentrations of PCT, C3a, and IL-6 obtained < or =8 hrs after clinical onset of sepsis or SIRS but not those of elastase or CRP were significantly higher in septic patients (PCT: median, 16.8 ng/mL, range, 0.9-351.2 ng/mL, p = .003; C3a: median, 807 ng/mL, range, 422-4788 ng/mL, p < .001; IL-6: median, 382 pg/mL, range, 5-1004 pg/mL, p = .009, all Mann-Whitney rank sum test) compared with patients suffering from SIRS (PCT: median, 3.0 ng/mL, range, 0.7-29.5 ng/mL; C3a: median, 409 ng/mL, range, 279566 ng/mL; IL-6: median, 98 pg/mL, range, 23-586 pg/mL). The power of PCT, C3a, and IL-6 to discriminate between septic and SIRS patients was determined in a receiver operating characteristic analysis. C3a was the best variable to differentiate between both populations with a maximal sensitivity of 86% and a specificity of 80%. An even better discrimination (i.e., a maximal sensitivity of 91% and a specificity of 80%) was achieved when PCT and C3a were combined in a "sepsis score." C3a concentrations also helped to predict the outcome of patients. Based on the sepsis score, a logistic regression model was developed that allows a convenient and reliable determination of the probability of an individual patient to suffer from sepsis or SIRS. CONCLUSIONS: Our data show that the determination of PCT, IL-6, and C3a is more reliable to differentiate between septic and SIRS patients than the variables CRP and elastase, routinely used at the intensive care unit. The determination of PCT and C3a plasma concentrations appears to be helpful for an early assessment of septic and SIRS patients in intensive care.

• Guinea pig C3 specific rabbit single chain Fv antibodies from bone marrow, spleen and blood derived phage libraries.

  Authors: H Hawlisch, A Meyer zu Vilsendorf, W Bautsch, A Klos, J Köhl

  Journal of immunological methods. 04/2000; 236(1-2):117-31.

  We constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbit immunized with guinea pig complement proteinWe constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbit immunized with guinea pig complement protein C3. By means of the phage display technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross reacted with guinea pig C3a, human C3 or rat C3. The frequency of bone marrow derived C3 positive clones was much higher as compared to blood or spleen derived clones. Additionally bone marrow and spleen derived clones show higher diversity than clones, obtained from blood, as determined by fingerprint analysis with the restriction enzyme AluI. Dissociation rate constants for all scFvs were similar, indicating that the source of the scFvs had no influence on affinities. The antibody fragments were used to analyze complement activation during xenotransplantation. Several blood or bone marrow derived scFvs bound to C3 located on rat liver endothelium after hyperacute rejection of a heterotopically transplanted rat liver into guinea pig. These data demonstrate that monoclonal rabbit scFvs can be easily generated from recombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to detect complement activation during xenotransplantation in guinea pigs.

• Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand.

  Authors: T Crass, W Bautsch, S A Cain, J E Pease, P N Monk

  Biochemistry. 08/1999; 38(30):9712-7.

  Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. TheDespite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 --> Lys199) and the corresponding reciprocal mutants (Lys68 --> Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu(68) has little effect on the ability of either C5a or C5a des Arg(74) to displace [(125)I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.

• Selection of a C5a receptor antagonist from phage libraries attenuating the inflammatory response in immune complex disease and ischemia/reperfusion injury.

  Authors: T Heller, M Hennecke, U Baumann, J E Gessner, A M zu Vilsendorf, M Baensch, F Boulay, A Kola, A Klos, W Bautsch, J Köhl

  Journal of immunology (Baltimore, Md. : 1950). 08/1999; 163(2):985-94.

  A C5a-receptor antagonist was selected from human C5a phage display libraries in which the C terminus of des-Arg74-hC5a was mutated. The selected molecule is a competitive C5a receptor antagonist inA C5a-receptor antagonist was selected from human C5a phage display libraries in which the C terminus of des-Arg74-hC5a was mutated. The selected molecule is a competitive C5a receptor antagonist in vitro and in vivo. Signal transduction is interrupted at the level of G-protein activation. In addition, the antagonist does not cause any C5a receptor phosphorylation. Proinflammatory properties such as chemotaxis or lysosomal enzyme release of differentiated U937 cells, as well as C5a-induced changes in intracellular Ca2+ concentration of murine peritoneal macrophages, are inhibited. The in vivo efficacy was evaluated in three different animal models of immune complex diseases in mice, i.e., the reverse passive Arthus reaction in the peritoneum, skin, and lung. The i.v. application of the C5a receptor antagonist abrogated polymorphonuclear neutrophil accumulation in peritoneum and markedly attenuated polymorphonuclear neutrophil migration into the skin and the lung. In a model of intestinal ischemia/reperfusion injury, i.v. administration of the C5a receptor antagonist decreased local and remote tissue injury: bowel wall edema and hemorrhage as well as pulmonary microvascular dysfunction. These data give evidence that C5a is an important mediator triggering the inflammatory sequelae seen in immune complex diseases and ischemia/reperfusion injury. The selected C5a receptor antagonist may prove useful to attenuate the inflammatory response in these disorders.

• Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

  Authors: B Settmacher, D Bock, H Saad, S Gärtner, C Rheinheimer, J Köhl, W Bautsch, A Klos

  Journal of immunology (Baltimore, Md. : 1950). 07/1999; 162(12):7409-16.

  The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3aThe C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.

• Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis.

  Authors: T Heller, J E Gessner, R E Schmidt, A Klos, W Bautsch, J Köhl

  Journal of immunology (Baltimore, Md. : 1950). 06/1999; 162(10):5657-61.

  The contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgGThe contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgG (FcgammaRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRgamma chain, neutrophil migration was abolished, whereas it was unaffected in FcgammaRIII-deficient mice. Huge amounts of TNF-alpha (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRgamma chain or FcgammaRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcgammaRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.

• Chimeric receptors of the human C3a receptor and C5a receptor (CD88).

  Authors: T Crass, R S Ames, H M Sarau, M A Tornetta, J J Foley, J Köhl, A Klos, W Bautsch

  The Journal of biological chemistry. 04/1999; 274(13):8367-70.

  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. ChimericChimeras 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 Galpha-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.

• Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

  Authors: A Kola, M Baensch, W Bautsch, A Klos, J Köhl

  Molecular immunology. 03/1999; 36(2):145-52.

  The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and theThe human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

• Detection and typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks attached to human skin by PCR.

  Authors: G Liebisch, B Sohns, W Bautsch

  Journal of clinical microbiology. 12/1998; 36(11):3355-8.

  Live Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, whichLive Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.

• Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor.

  Authors: S Lienenklaus, R S Ames, M A Tornetta, H M Sarau, J J Foley, T Crass, B Sohns, U Raffetseder, M Grove, A Hölzer, A Klos, J Köhl, W Bautsch

  Journal of immunology (Baltimore, Md. : 1950). 10/1998; 161(5):2089-93.

  The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. AThe interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.6+/-0.7 nM) and responded functionally to C3a with an intracellular calcium mobilization (ED50 = 0.18+/-0.02 nM). Human rC4a weakly bound to both the hu- and gpC3aR (IC50 > 1 microM). However, only HEK-293 cells expressing the gpC3aR responded functionally to rC4a (ED50 = 8.7+/-0.52 nM), while cells expressing the huC3aR did not (c < or = 1 microM). Thus, through an interaction with the C3aR, huC4a may elicit anaphylatoxic effects in guinea pigs but not in man.

• Genomic organization of the human C3a receptor.

  Authors: D Paral, B Sohns, T Crass, M Grove, J Köhl, A Klos, W Bautsch

  European journal of immunology. 09/1998; 28(8):2417-23.

  The human C3a receptor (C3aR) mediates the activation of cells by the potent proinflammatory chemoattractant C3a, an anaphylatoxin, generated in the early phase of an inflammatory reaction byThe human C3a receptor (C3aR) mediates the activation of cells by the potent proinflammatory chemoattractant C3a, an anaphylatoxin, generated in the early phase of an inflammatory reaction by proteolytic cleavage of the complement component C3. To understand the molecular mechanisms that regulate C3aR gene expression, we initiated studies to determine its genomic and mRNA organization. We now report the following novel findings: (1) The C3aR is a single-copy gene as shown by Southern hybridization of human genomic DNA. (2) Using PCR amplification of DNA from monochromosomal somatic cell hybrid and radiation hybrid panels, the C3aR locus was mapped to chromosome 12p13. (3) Genomic DNA clones encompassing the C3aR locus were isolated from a human genomic DNA library and characterized by restriction mapping, Southern blotting, PCR analysis and DNA sequencing. Comparison of the genomic with the known cDNA sequences revealed a single 6-kb intron sequence located 1 1 bp upstream of the ATG initiation codon. The open reading frame and the complete 3' untranslated region are encoded on a single exon.

• Comparison of the genome organization of pathogenic neisseriae.

  Authors: W Bautsch

  Electrophoresis. 05/1998; 19(4):577-81.

  Current efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other membersCurrent efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other members of these species, or whether, in view of the fact that Neisseriae are highly recombinogenic and exhibit a high degree of horizontal intra- and interspecies genetic transfer, only clone-specific conclusions are valid. From the known physical and genetic maps of each of two gonococcal and meningococcal strains, it would appear that both species exhibit a species-specific conservation in their genetic organization while the interspecies comparison revealed several rearrangements, although still with a high overall similarity. However, these data contrast with other evidence suggesting intra-species rearrangements, such as the nonconserved I-CeuI macrorestriction patterns of different meningococcal and other neisserial strains. Since I-CeuI cuts within the 23S-rRNA sequence, the restriction pattern should give reliable information on the distribution of rrn loci in the neisserial genomes. Further studies are warranted to answer these questions.

• Site-directed C3a receptor antibodies from phage display libraries.

  Authors: H Hawlisch, R Frank, M Hennecke, M Baensch, B Sohns, L Arseniev, W Bautsch, A Kola, A Klos, J Köhl

  Journal of immunology (Baltimore, Md. : 1950). 03/1998; 160(6):2947-58.

  Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellularRecent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.

• A detailed analysis of the C5a anaphylatoxin effector domain: selection of C5a phage libraries on differentiated U937 cells.

  Authors: M Hennecke, A Otto, M Baensch, A Kola, W Bautsch, A Klos, J Köhl

  European journal of biochemistry / FEBS. 03/1998; 252(1):36-44.

  We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known bindingWe have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69-73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70-72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4-10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage-displayed C5a was proven to be a powerful strategy to define receptor-binding points and to select C5aR antagonists based on the structure of the natural ligand.

• Circulating complement proteins in multiple trauma patients--correlation with injury severity, development of sepsis, and outcome.

  Authors: F Hecke, U Schmidt, A Kola, W Bautsch, A Klos, J Köhl

  Critical care medicine. 01/1998; 25(12):2015-24.

  OBJECTIVE: To investigate protein complement 3a (C3a) and protein complement 3 (C3) plasma levels in trauma patients directly after the injury, in relation to the patients' outcome, the developmentOBJECTIVE: To investigate protein complement 3a (C3a) and protein complement 3 (C3) plasma levels in trauma patients directly after the injury, in relation to the patients' outcome, the development of sepsis, or the injury severity, as determined by either the Polytrauma Score (PTS), the Injury Severity Score (ISS), or the Trauma and Injury Severity Score (TRISS). DESIGN: Prospective study. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Thirty-four patients with multiple trauma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: C3a and C3 concentrations, as well as the C3a/C3 ratio, were determined at the time of the accident (T0), at the emergency admission (T1), 8 hrs after the accident (T2), and every 8 hrs until day 3, every 12 hrs until day 6, and once daily on days 7 and 8. The C3a plasma concentrations and the C3a/C3 ratios of nonsurvivors were significantly greater at T0 or T1 as compared with those survivors (p = .008 or .033). Patients who developed sepsis had higher C3a plasma levels at the scene of accident than patients without complications. However, these differences did not reach statistical significance (p = .051), although a clear trend was apparent. Patients were grouped according to the severity of injury, as determined by either the PTS, ISS, or TRISS. We found significant differences in the both the mean C3a values and the C3a/C3 ratio among the different groups, during the first 8 hrs after the injury. In addition, a significant correlation was observed between the C3a concentration or the C3a/C3 ratio at T0 to T2 and either the ISS (r2 = .49), PTS (r2 = .22) or the TRISS (r2 = .45), which was similar to correlations between injury severity scores themselves (r2 = .36 to .58). CONCLUSIONS: Complement activation occurs immediately after the injury. The degree of activation is a hallmark for the outcome of a patient. Determination of C3a concentrations, at the scene of the accident, may prove helpful to assess the severity of the injury and to determine the prognosis. The amount of C3a and the C3a/C3 ratio may be useful as additional parameters to the existing trauma scoring systems, such as, PTS, ISS, and TRISS.

• The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

  Authors: U Martin, D Bock, L Arseniev, M A Tornetta, R S Ames, W Bautsch, J Köhl, A Ganser, A Klos

  The Journal of experimental medicine. 07/1997; 186(2):199-207.

  The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide importantThe pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

• The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization.

  Authors: D Bock, U Martin, S Gärtner, C Rheinheimer, U Raffetseder, L Arseniev, M D Barker, P N Monk, W Bautsch, J Köhl, A Klos

  European journal of immunology. 07/1997; 27(6):1522-9.

  The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR,The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, G alpha 16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.