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Biomedical subjects

J Köhl

Publications and source records attributed to J Köhl.

At least 19 recordsLinked to original sources
PubMed2000

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.

Calcium↗
PubMed2000

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

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 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.

Amino Acid Sequence↗
PubMed2000

A codominant role of Fc gamma RI/III and C5aR in the reverse Arthus reaction.

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRgamma-/- mice, manifestation of disease is almost exclusively determined by FcgammaR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR-/- mice suggested that, dependent on the tissue site, complement is more important than FcgammaR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcgammaRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcgammaR and C5aR. Therefore, FcgammaR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.

Animals↗
PubMed2000

Activation of the acute phase response and complement C3 in patients with IgA nephropathy.

UNLABELLED: Recently we showed systemic complement activation in patients with immunoglobulin A (IgA) nephropathy (measured by "activated C3" [actC3], in other words, neoantigens developing on breakdown products after C3 activation) and reported that plasma levels of actC3 can indicate disease activity and renal outcome. In this study we investigated whether plasma C3a and C-reactive protein (CRP), which require tests that are more readily available, have a similar diagnostic and predictive value. CRP was measured using a highly sensitive enzyme-linked immunosorbent assay and C3a using a specific immunoassay. CRP and C3a levels were significantly higher in 56 patients with IgA nephropathy as compared with 55 healthy controls. C3a levels in IgA nephropathy patients were also significantly increased in comparison with 42 patients with hypertension or nonimmune renal diseases. Neither C3a nor CRP levels correlated with those of actC3 in IgA nephropathy patients. We also compared 10 IgA nephropathy patients with stable, normal renal function with eight IgA nephropathy patients progressing from normal to impaired renal function during mean follow-ups of 7.1 and 5.1 years, respectively. Mean CRP but not C3a levels during the observation period were significantly higher in IgA nephropathy patients with disease progression than in those with stable renal function. CONCLUSION: Systemic complement activation can be detected by measurement of plasma C3a in IgA nephropathy, but C3a levels cannot substitute for actC3 in predicting renal prognosis. Subclinical induction of the acute phase response is also present in patients with progressive IgA nephropathy, but again its prognostic value is limited. Repeated determinations performed over prolonged time courses may possibly improve the prognostic value of CRP levels.

Acute-Phase Reaction↗
PubMed2000

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.

Animals↗
PubMed2000

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

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.

Adult↗
PubMed1999

Acylation-stimulating protein (ASP): structure-function determinants of cell surface binding and triacylglycerol synthetic activity.

Acylation-stimulating protein (ASP or C3adesArg) is a potent lipogenic factor in human and murine adipocytes and fibroblasts. The arginated form of ASP, i.e. complement C3a (C3a), stimulates immunological responses in human granulocytes, mast cells, guinea pig platelets and guinea pig macrophages; however, ASP is inactive in stimulating these responses. Thus both ASP and C3a are bioactive across species but are not functionally interchangeable. Tertiary structure of both proteins by X-ray crystallography and NMR spectroscopy predicts a tightly linked core region consisting of three alpha-helices linked via three disulphide bonds, with one of the alpha-helices extending out from the core and terminating in a flexible conformationally irregular carboxy-tail region. The present studies were undertaken in order to define the functionally active domains of ASP, distinctive from those of C3a, using chemical modifications, enzymic cleavage and synthetic peptide fragments. The results indicate that: (i) the N-terminal region (<10 amino acids) plays little role in ASP receptor binding and triacylglycerol synthesis stimulation; (ii) the native C-terminal region had no activity, but modifications which increased hydrophobicity increased receptor binding, and led to some activation of triacylglycerol synthesis stimulation; (iii) an intact disulphide-linked core region is essential for triacylglycerol synthesis stimulation activity but not for receptor interaction. Finally, basic charges in the carboxy region (His) are essential for ASP triacylglycerol synthesis stimulation but not for receptor binding, whereas both functions are eliminated by the modification of Lys in the disulphide-linked core region. The present results suggest that there are two functional domains in ASP, one that is responsible for the initial binding to the cell surface receptor, and a second domain that activates and increases triacylglycerol synthesis stimulation. This contrasts markedly with the structure-function studies of C3a where both binding competency and function were dependent on the C-terminal Arg. Thus ASP demonstrates distinct bioactivity.

Binding, Competitive↗
PubMed1999

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

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 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.

Amino Acid Substitution↗
PubMed1999

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

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 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.

Acids↗
PubMed1999

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

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 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.

Animals↗
PubMed1999

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 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.

Anaphylatoxins↗
PubMed1999

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

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 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.

Antigens, CD↗
PubMed1999

Selection of phage-displayed anti-guinea pig C5 or C5a antibodies and their application in xenotransplantation.

Xenogeneic liver transplantation in the discordant guinea pig (gp) to rat model results in hyperacute rejection within a few minutes, which is due to activation of the complement system. Currently no antibodies against gp complement factors are available, which allow activation of the gp complement system in serum or complement deposition in tissue to be detected. To close this gap, we started developing single chain Fvs (scFvs) against gpC5 and gpC5a. We generated a combinatorial library of scFv antibodies comprising the variable heavy and light chain repertoire from mice immunized with gpC5. Out of this library we selected several antibodies against gpC5 and C5a after four and six rounds of biopanning, respectively. Selected gpC5-specific scFvs were purified by metal affinity chromatography followed by size exclusion chromatography or by affinity chromatography using Protein L. Purified scFvs were able to inhibit gp complement system in a hemolytic assay and to detect gpC5 deposition in tissue. A surface plasmon resonance based assay on BIAcore was established, with which the C5 concentration in gp serum was determined to 240 microg/ml. As at least 0.04% of the normal gpC5 concentration can be detected, the test provides a powerful tool to investigate the development and the consequence of a hybrid complement system after liver xenotransplantation from gp to rat.

Acute Disease↗
PubMed1998

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

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. 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.

Amino Acid Sequence↗
PubMed1998

Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625-positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS.

Animals↗