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Selection of a C5a Receptor Antagonist from Phage Libraries Attenuating the Inflammatory Response in Immune Complex Disease and Ischemia/Reperfusion Injury¹

Tanja Heller^*, Meike Hennecke^*, Ulrich Baumann, J. Engelbert Gessner, Andreas Meyer zu Vilsendorf, Melanie Baensch^*, Francois Boulay^§, Axel Kola^*, Andreas Klos^*, Wilfried Bautsch^* and Jörg Köhl^2,*

^* Institute of Medical Microbiology, Department of Clinical Immunology, Department of Transplantation Surgery, Medical School Hannover, Hannover, Germany; and ^§ Laboratoire de Biochemie, Centre National de la Recherche Scientifique, Grenoble, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the last few years, complement inhibitors have been developed, which block the complement cascade at different points. The soluble version of the human complement receptor 1, i.e., sCR1³ (1), inhibits classical and alternative pathway activation at the level of C3 and C4 and has been extensively analyzed in vitro and in vivo (for review, see Refs. 2 and 3). The C1 inhibitor (C1-INH), belonging to the family of serine-proteinase inhibitors, blocks C1r and C1s of the classical pathway. In addition, it is the major inhibitor of factor XII and prekallikrein of the contact system. The efficacy of both molecules has been demonstrated in animal studies and they are now assessed in clinical trials (for review, see Ref. 2). However, little is currently known of whether the inhibition of whole complement pathways is really needed to attenuate complement-mediated inflammation, in particular in immune complex (IC) diseases and ischemia/reperfusion (I/R) injury.

Recently, a mAb has been described (4) that inhibits cleavage of C5 and the subsequent generation of C5a and the membrane attack complex (MAC). Surprisingly, this mAb prevented the onset of arthritis and ameliorated established disease in a murine model of collagen-induced arthritis (5). In addition, this mAb attenuated glomerulonephritis and increased survival in a systemic lupus erythematosus-prone mouse strain (6). These data address the question of how much tissue damage in IC disease is produced by C3b and C4b deposition and subsequent phagocytosis and how much is due to generation of C5a and MAC. A critical role for C5a in IC diseases has recently been demonstrated in the reverse passive Arthus reaction using C5a receptor (C5aR)-deficient mice (7, 8).

C5a is a small polypeptide released from the -chain of C5 during complement activation. It mediates a variety of proinflammatory effector functions such as recruitment of polymorphonuclear neutrophils (PMN) and macrophages to inflammatory sites and the activation of these cells to release increased levels of cytokines, chemokines, lysosomal enzymes, products of the arachidonic acid metabolism and histamine (for review, see Refs. 9 and 10). In addition, endothelial cells are stimulated to increase P-selectin (11).

The fact, that C5a appears very early in the inflammatory cascade makes this molecule a promising target for therapy. In addition, a specific inhibition of C5a does not impair C3b-mediated opsonization. Up to now, C5a and C5aR Abs, nonpeptidic and peptidic C5aR antagonists (C5aRA) have been developed (for review, see Ref. 2). Although many of these compounds have been proven to be effective in vitro, only rare examples exist for their potential use in vivo (12, 13, 14, 15).

Recently, we described a method to display C5a on the tip of a filamentous phage using the pJuFo vector (16). C5a is fused to the leucine zipper part of Fos and is expressed with a free C terminus previously demonstrated to be essential for both receptor binding and functional activity (2). Panning C5a C-terminal libraries directly on a C5aR-expressing cell line, we now isolated a potent and specific C5aRA. Using this C5aRA, we were able to address an important role to C5a in IC disease and in intestinal I/R injury.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of C5a C-terminal libraries

All of the constructed libraries are based on pJuFo plus [Ala²⁷]C5a vector including Cys²⁷Ala replacement in the C5a molecule (16). Altogether, five different libraries with modifications at the C5a C-terminus were constructed (Table I). For all libraries, primer MH40 (5' end of C5a) was combined with five different mutagenesis primers (3' end of C5a). Sequences of all primers are given below. All mutagenesis primers introduced a His⁶⁷Phe replacement (Table I). C5a mutants from libraries I–III had 73 aa. C5a mutants from libraries IVa and IVb had 75 aa. For library I, mutagenesis primer MH44 was used, resulting in Asp⁶⁹Phe, Met⁷⁰Lys, and Gln⁷¹Pro replacements and random mutagenesis of positions 72 and 73. For library II, primer MH45 was used resulting in His⁶⁷Phe and Asp⁶⁹Phe replacements and random mutagenesis of positions 70–73. In library III, primer MH46 created random mutagenesis at positions 69–73. In library IVa, Asp⁶⁹Phe, Met⁷⁰Lys, Gln⁷¹Pro replacements were introduced by primer MH47. In addition, positions 72, 73, and 75 were randomly mutated. At position 74, the VDN codon was used encoding all of the 20 naturally occurring amino acids except Arg, Gly, or Cys. Primer MH48, which was used for the construction of library IVb, introduced the same mutations as MH47, with the exception that at position 74 the RGM codon was used, which encodes for Gly and Cys. Thus, by combining libraries IVa and IVb, 19 of the 20 naturally occurring amino acids are encoded at position 74 except the Arg residue, which is conserved in all C5a sequences described (Table I).

Affinity enrichment of C5a mutants

Selection of C5a receptor binding clones was performed on U937 cells, which had been differentiated with 1 mM N⁶,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (Bt₂cAMP; Boehringer Mannheim, Mannheim, Germany) (dU937 cells) for 3 days as described (16). For the first round of panning, 2.5x10⁷ cells grown in RPMI 1640 medium (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated FCS and penicillin (50 IU/ml)/streptomycin (50 µg/ml) at 37°C in a humidified atmosphere with 5% CO₂ were harvested and resuspended in 500 µl medium containing 100 µl phage ( 10¹³ pfu). This mixture was incubated at room temperature for 30 min with gentle agitation. Cells were pelleted (3 min, 800 x g) and washed five times with 1 ml medium. Specifically bound phage were eluted with 200 µl 0.1 M HCl/glycine, pH 2.2, supplemented with 0.1% BSA at room temperature for 10 min. Cells were pelleted by centrifugation and the supernatant was immediately neutralized with 37.5 µl 1 M Tris/HCl, pH 9.1. Then, 100 µl of neutralized eluate were used to infect Escherichia coli TG1 cells. Numbers of phage before and after panning were determined by plating infected E. coli TG1 cells onto TYE+amp+gluc (18).

Altogether, three panning cycles were performed. The washing steps were increased from five times to 10 times in rounds two and three. In panning round three, 2.5 x 10⁶ cells were used. All other steps were performed as described above.

Generation of the pIII-A8 mutant

To obtain the pIII-A8 mutant, gene III, encoding the viral coat protein pIII, was removed by digestion of pJuFo plus [Ala²⁷]C5a-[A8] with SpeI and NheI. SpeI and NheI produce compatible cohesive ends. The digested vector was religated yielding pIII-A8, in which the pIII moiety of the Jun-pIII fusion protein was deleted (see Fig. 1). The PCR reactions, DNA fragment digestion, ligation, transformation, growth of the libraries, helper phage infection, phage preparation, and DNA sequencing of the mutants was performed as described (18).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Cartoon representation of a filamentous phage expressing the selected C5aRA A8. The leucine zipper of transcription factor Jun is fused to the minor coat protein (pIII) of the filamentous M13 phage. The C5a mutant A8 is fused to the leucine zipper of Fos. Jun and Fos form a heterodimer that is stabilized by two disulfide bonds at the N and C terminus of Jun and Fos as described (53 ).

For all in vitro and in vivo experiments, purified soluble C5a mutants were used. For protein expression TG1 bacteria were grown in 2x TY+amp medium containing 100 µg/ml ampicillin and 0.1% glucose. At an OD₆₀₀ of 0.9, isopropyl b-D-thiogalactoside was added to give a final concentration of 0.5 mM. Bacteria were grown overnight with shaking at room temperature. The next day, the periplasmic fraction was prepared by freezing and thawing. Periplasmic fractions from the selected C5a mutants selected were applied to Sepharose columns coupled with C5a-specific mAb 561 as described (16). Highly purified C5a mutants were obtained as determined by SDS-PAGE and subsequent silver staining.

Competitive ¹²⁵I-labeled recombinant human C5a (¹²⁵I-rhC5a) binding studies

¹²⁵I-rhC5a binding assays were conducted on dU937 cells and naive murine peritoneal cells obtained by peritoneal lavage essentially as described (19). Briefly, increasing concentrations of unlabeled C5a or a C5a mutant were tested against a constant amount of ¹²⁵I-rhC5a as tracer (17,000 cpm). After separation of the free ¹²⁵I-rhC5a by filtration, the cell bound ¹²⁵I-rhC5a was determined in a -counter (Packard, Canberra, Australia). The plot of cell-bound ¹²⁵I-rhC5a vs the concentration of unlabeled competitor yielded the half-maximal inhibitory concentrations (ID₅₀).

Degranulation assay

As an example for the C5a-induced degranulation of phagocytic cells, the N-acetyl-ß-D-glucosaminidase release from dU937 cells was determined. The assay was performed exactly as described (20). The ED₅₀ of C5a or C5a mutants was determined as the concentration leading to half-maximal enzyme release.

Chemotaxis assay

Chemotactic activity of dU937 cells was assessed as described by Schwenk et al. (21). Modified Boyden chambers (Nucleopore, Tübingen, Germany) were filled with increasing concentrations of rhC5a (Sigma, Diesenhofen, Germany) or, in case of inhibition experiments, with a mixture of increasing concentrations of rhC5a and pIII-A8 at a constant concentration of 10^-6M. Subsequently, chambers were covered with polycarbonate filters (pore size, 3 µm; Nucleopore). Then, 100 µl of dU937 cells at a concentration of 5 x 10⁵/ml were applied to each chamber. Cells were allowed to migrate for 3 h at 37°C. Migrated cells were lysed by adding 0.1% Triton X-100 (Boehringer Mannheim), and ß-glucuronidase activity in the lysates was determined photometrically using p-nitrophenol-ß-D-glucuronide as substrate (Sigma). Chemotactic activity was expressed as chemotactic index, which is defined as the ratio of the number of migrating cells in presence of stimulus vs the number of migrating cells in presence of medium.

GTPase assay

To determine the GTP-ase activity of C5a or C5a mutants, the method of Gierschik (22) was used. Different concentrations of C5a or the C5a mutants were suspended in 50 µl of 50 mM triethanolamine-HCl, 5 mM MES pH 7.3 containing 5 mM MgCl₂, 143 mM NaCl, 1 mM EDTA, 0.16% BSA, 0.8 mM 5'-adenylylimidodiphosphate (App(NH)p), 0.1 mM ATP, 5 mM creatine phosphate, 0.4 mg/ml creatine phosphokinase, 0.1 µM GTP, and 0.2 µl [-³²P]dGTP (10.8 µCi/µl; 3000 Ci/mmol). The reaction was started by adding 50 µl of dU937 membranes at a concentration of 3 µg dissolved in the same buffer. Cell membranes were prepared as described by Laugwitz et al. (23). After 15 min incubation at 30°C, 700 µl of an ice-cold 5% (w/v) charcoal suspension in 10 mM H₃PO₄, pH 2.0, were added and the samples were centrifuged at 12,000 x g for 30 min at 4°C. Then, 500 µl of the supernatant were counted in a Beckmann LS 6000 SE counter (Beckman Coulter, Fullerton, CA).

Metabolic labeling and immunoprecipitation of C5aR

A stable cell line of C5aR-transfected RINm5F cells was used for the phosphorylation experiments (24). Cells were metabolically labeled with [³²P]orthophosporic acid (0.3–0.5 mCi/ml) for 3 h at 37°C, as described (25). Briefly, phosphorylation of C5aR was initiated by adding a saturating concentration of C5a (5 x 10^-8 M) or pIII-A8 (8 x 10^-7 M). After 10 min incubation, the monolayers were lysed and C5aR was immunoprecipitated as described (26). Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions (27) and autoradiography.

Detection of C5aR by imunoblotting

Immunoblotting of C5aR-transfected RINm5F cells was performed as described (24). In brief, cells were incubated with either rhC5a (5 x 10^-8 M) or pIII-A8 (8 x 10^-7 M) and subsequently lysed in Laemmli buffer (27) supplemented with DTT, separated by SDS-PAGE, and transferred to a 0.22-µM nitrocellulose filter (Schleicher & Schuell, Keene, NH). Filters were blocked with PBS, 0.1% Tween 10, 3% BSA for 1 h at room temperature. Subsequently, filters were incubated with affinity-purified anti-C5aR rabbit IgGs diluted 1:400 in blocking buffer. The filter was washed with PBS, 0.1% Tween 20 and incubated with ¹²⁵I-labeled protein A (1 µCi/ml of blocking buffer) for 1 h at room temperature. Bound radioactivity was detected by autoradiography using Fuji RX film (Tokyo, Japan) at -80°C.

Measurement of intracellular Ca²⁺ concentration ([Ca²⁺]_i) of murine peritoneal cells

The peritoneal cavity of BALB/c mice was lavaged with 2 ml of ice-cold PBS. Cell staining using Diff-Quick (Baxter Merz & Dade, Dudingen, Switzerland) revealed 95–98% macrophages and 2–5% mast cells. The cells were centrifuged, resuspended in HBSS buffer without Ca²⁺, and subsequently loaded with fura-2/AM (Calbiochem, Bad Soden, Germany) as described (20). After reconstitution of the HBSS buffer with Ca²⁺, the C5a-induced rise in [Ca²⁺]_i was determined using the Luminescence Spectrometer LS 50 B (Perkin-Elmer, Beaconsfield, U.K.).

Animals

Female BALB/c mice (8- to 10-wk old) were purchased from Charles River Laboratory (Wilmington, MA) and were maintained under pathogen-free conditions.

Reverse passive Arthus reaction in the peritoneum

To induce the reverse passive Arthus reaction in the peritoneum, chicken egg albumin (20 mg/kg body weight; Sigma) was injected i.v., subsequently followed by an i.p. injection of IgG rich in Ab to chicken egg albumin (800 µg/mouse; ICN, Eschwege, Germany) exactly as described (7). Mice were killed 6 h after injury, and the peritoneal cavity was lavaged with 2 ml PBS, 0.1% ice-cold BSA. Peritoneal cells were stained using Diff-Quick (Baxter Merz & Dade) and subsequently assessed for differential cell count. Where indicated, mice were injected i.v. with 8 µg cobra venom factor (CVF) the day before IC challenge and again with 8 µg CVF 4 h before IC application. CVF was purified from Naja naja venom (Miami Serpentarium Laboratories, Miami, FL) according to a previously described procedure (28). In inhibition experiments, 200 µl (10^-5 M) of the C5aRA pIII-A8 were applied i.p. either at the time of IC challenge (T₀) or 1 h and 2 h later. In a further set of experiments, 100 µl of pIII-A8 at a concentration of 10^-5 M were given i.v. at T₀ or 1 h and 2 h after IC application. In a third set of experiments, 200 µl pIII-A8 were injected i.p. and 100 µl i.v., both at T₀ and 1 h and 2 h later. Serum complement levels were determined as described (29).

Reverse passive Arthus reaction in skin and lung

Mice were anesthetized with ketamine and xylazine and shaved at their basolateral sides. The trachea was cannulated and 150 µg rabbit anti chicken egg albumin Ab (Sigma) was applied. In addition, 30 µg Ab were injected intradermally. Ag (chicken egg albumin, 20 mg/kg) was subsequently given i.v. Where indicated, 200 µl pIII-A8 at a concentration of 7.3 x 10^-6 M were given i.v. before the Arthus reaction, or, additionally, 100 µl pIII-A8 (7.3 x 10^-6 M) were applied 60 and 120 min after IC challenge. After 4 h, mice were killed and PMN accumulation in the skin was assessed by quantitation of myeloperoxidase (MPO). In the lung, the number of PMN was determined by bronchoalveolar lavage.

Bronchoalveolar lavage

The lungs were lavaged with 5 x 1 ml PBS following cannulation of the trachea. Lavaged cells were stained using May-Grünwald and Giemsa stain. The differential cell count of the bronchoalveolar lavage fluid was assessed with a hemocytometer (Neubauer Zählkammer Jürgens, Germany).

MPO assay

Skin punches (1 cm²) of the injection sites were assayed for MPO activity. MPO was extracted and determined as described (30). Briefly, homogenized tissue was suspended in 50 mM potassium phosphate buffer, pH 6.0, 0.5% hexadecyltrimethylammoniumbromide. Cells were broken by three cycles of freezing and thawing and subsequent sonification. The supernatant was mixed with 0.167 mg/ml O-dianisidine dihydrochloride (Sigma) and 0.0005% hydrogen peroxide. The MPO release was calculated by assessing the absorbance at 450 nm. A serial dilution of MPO from human PMN (Calbiochem-Novabiochem, Bad Soden, Germany) served as standard. Samples were run in duplicate.

Model of intestinal I/R injury

Mice were anesthetized with ether and were kept under a heating lamp. A laparatomy was performed, and I/R was induced by occluding the superior mesenteric artery with a microbulldog clamp. The abdomen was covered with warm, moist gauze during this period. Weight-matched control mice underwent sham operation in which the superior mesenteric artery was exposed but not occluded. After 60 min ischemia, the clamp was removed, the incision was closed with a silk suture, and the mice were returned to their cages. The bowel was reperfused for 60 min or until the animals died (50–55 min after starting the reperfusion). Bowel injury (hemorrhage, bowel wall edema, and purple black discoloration) was assessed macroscopically at the end of the reperfusion period or when animals died. Where indicated, mice were treated with CVF before I/R as described for the reverse passive Arthus reaction. The C5aRA was given twice: 200 µl (7.3 x 10^-6 M) were applied i.v. before starting ischemia; another 100 µl were given i.v. directly before removing the clamp.

Quantitation of I/R-induced microvascular dysfunction

Intestinal reperfusion-induced dysfunction was quantitated by measuring the extravasation of plasma proteins into the lung as described (31). Evans blue dye binds avidly to albumin and was used as a marker of protein extravasation. This technology compares favorably with the methodology involving radiolabeled albumin (31). After 60 min of reperfusion, animals were killed and lung tissue was harvested. Pulmonary vessels were emptied by flushing saline into the right ventricle. Subsequently, the lungs were excised, weighed, and the right lung lobe was placed in 1 ml of formamide and homogenized. After incubation in formamide at 37°C for 16 h, the concentration of dye within the eluate was measured spectrophotometrically at 620 nm and expressed as ng dye/mg wet lung weight.

Statistical analysis

Statistical analysis was performed using the SigmaStat version 2.0 statistical package (Jandel, Erkrath, Germany). All data are given as mean ± SEM. First, we tested for a normal distributed population using the Kolomogoroff-Smirnov test. To analyze differences between two normally distributed groups, an unpaired t test was used. Comparison of the means of more than two groups were done by one-way ANOVA. When the differences in the mean values of the groups showed a significant difference, pairwise comparison was performed using the Tukey test. A p value <0.05 was considered to be significant, and a p value <0.001 was considered to be highly significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Panning of the C5a phage libraries on differentiated U937 cells

We constructed 5 C5a-C-terminal libraries, i.e., libraries I, II, III, IVa, and IVb. In libraries I, II, and III, the functionally important Arg⁷⁴ residue was deleted. In libraries IVa and IVb, C5a mutants with a length of 75 aa were constructed, in which no Arg⁷⁴ residue was allowed. In library III, positions 69–73 were randomly mutated (Table I).

For the first panning cycle, 3.0 x 10¹² phage of a mixture of all five libraries were used, which is 6 x 10¹¹ phage from each library. Panning was performed in solution using C5aR-expressing dU937 cells. Next, 2.1 x 10⁸ phage were eluted from dU937 cells giving an output/input ratio of 7.2 x 10^-3%. During the panning cycles, the number of eluted phage increased by a factor of 200, giving a final output/input ratio of 1.1% after three panning rounds. After the third pan, 50 clones were randomly picked and screened for consensus motifs. All clones selected belonged to library III, in which positions 69–73 were randomly mutated. The Phe⁶⁹Lys⁷⁰Pro⁷¹ motif present in library I, II, IVa, and IVb was not found. The motif most often selected included a Leu residue at positions 70, 71, or 72 and a Tyr or an Arg residue at position 73, while no particular amino acid was enriched at position 69, as we have described recently (19).

Inhibition of C5a binding to dU937 cells by the selected C5a mutants

Selected C5a mutants with C-terminal consensus motifs Ser⁷⁰Leu⁷¹Leu⁷²Arg⁷³, i.e., A10, B4, and B8, were expressed as soluble proteins, purified by affinity chromatography, and tested in competitive binding studies using dU937 cells (Table II). All clones showed ID₅₀ values in the range of rhC5a, while the ID₅₀ of one mutant, A8, the structure of which is Jun-pIII/Fos-C5a-[1–66]-Phe⁶⁷Lys⁶⁸Arg⁶⁹Ser⁷⁰Leu⁷¹Leu⁷²Arg⁷³, was about 15-fold higher as compared with rhC5a (Table II). Mutants A10, B4, B8, and A8 share the same C-terminal sequence except position 69, which is Arg in A8 and Ala, Glu, or His in A10, B4, and B8, respectively. In clone A8, a cartoon representation of which is depicted in Fig. 1, the minor coat protein pIII was deleted, resulting in pIII-A8. This deletion did not change binding affinity (Table II). Mutant pIII-A8 was used throughout additional experiments.

The pIII-A8 mutant does not induce degranulation of dU937 cells

Mutants A10, B4, and B8 induced degranulation of dU937 cells and had ED₅₀ values quite similar to C5a (Table II), whereas pIII-A8 did not induce degranulation up to the highest concentration tested, which was 3 x 10^-6 M. At that concentration, rhC5a was completely displaced from the receptor. pIII-A8 was also devoid of any chemotactic activity (data not shown). To check whether signal transduction was blocked at the level of G-protein coupling, hydrolysis of [-³²P]GTP was determined. A dose-dependent increase in GTPase activity was observed for C5a and B4 but not for pIII-A8, even at 2 x 10^-6 M (Fig. 2). Next, we tested the ability of pIII-A8 to induce the phosphorylation of the C5aR. As shown in Fig. 3, C5a caused a strong phosphorylation of the C5aR, whereas no ligand specific phosphorylation occurred for pIII-A8.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. pIII-A8 does not induce GTP hydrolysis in membranes of dU937 cells. Recombinant hC5a or mutant B4 increased GTPase activity from 11 (rhC5a) or 13 pmol/min/mg protein to 35 (rhC5a) and 28 pmol/min/mg protein, respectively. Values of one typical experiment of three are given.

View larger version (61K): [in this window] [in a new window]   FIGURE 3. pIII-A8 does not cause any phosphorylation of RINm5F cells of the human C5aR. A, The C5aR was immunoprecipitated from 32P metabolically labeled cells before (-) and after (+) stimulation with C5a (5 x 10-8M) or pIII-A8 (8 x 10-7M). B, Immunoblot of nonlabeled cell extracts before (-) and after (+) C5a (5 x 10-8 M) or pIII-A8 (8 x 10-7 M) addition revealed with an anti-C5aR serum. Phosphorylation of the C5aR induces a mobility shift from 40 to 43 kDa.

To assess the potency of pIII-A8 to inhibit C5a-induced degranulation of dU937 cells, increasing concentrations of pIII-A8 were incubated with rhC5a at a concentration of 8.6 x 10^-10 M (ED₈₀). The C5a mutant inhibited cell degranulation with an ID₅₀ of 7.85 ± 1.57 x 10^-8 M (Fig. 4A).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. pIII-A8 inhibits C5a-induced cell degranulation and chemotaxis. A, Differentiated U937 cells were challenged with a constant concentration of rhC5a, i.e., 8.6 x 10-10 (ED80) after a 10-min preincubation with increasing concentrations of pIII-A8. pIII-A8 inhibits cell degranulation with an ID50 of 7.85 ± 1.57 x 10-8 M. B, Increasing concentrations of C5a were applied to dU937 cells in the absence or the presence of pIII-A8 (10-6 M). Maximal cell migration induced by C5a at a concentration of 5 x 10-9 M was inhibited by 77%. Lower concentrations of C5a, i.e., 10-9 M (ED90) or 5 x 10-10 M (ED80) were inhibited by 92% and 100%, respectively. Data from one typical experiment of five are given. **, p < 0.001 (t test).

pIII-A8 is a C5aRA on murine peritoneal macrophages

The A8 mutant was selected using the human histiocytic cell line U937. To test whether this mutant also binds to the murine C5aR and, moreover, whether it is able to displace C5a from the murine C5aR, we performed competitive binding studies with naive murine peritoneal macrophages. Indeed, ¹²⁵I-rhC5a was displaced from the murine C5aR with an ID₅₀ of 1.68 ± 0.81 x 10^-8 M and thus did not differ from the data obtained with dU937 cells (Table II). To analyze, whether pIII-A8 also inhibits C5a-mediated effects in mice, the antagonist was tested for its potency to block rhC5a-induced rise in [Ca²⁺]_i of naive peritoneal macrophage. As shown in Fig. 5, pIII-A8 completely inhibited Ca-mobilization mediated by 10^-9 M (ED₅₀) and 2 x 10^-9 M (ED₇₅) C5a, when applied at a constant concentration of 10^-6 M. Higher concentrations of C5a, i.e., 10^-8 M or 2 x 10^-8 M, were inhibited by 50–75%.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. pIII-A8 inhibits C5a-induced [Ca2+]i increase in murine peritoneal macrophage. Naive peritoneal macrophage were challenged with increasing concentrations of rhC5a in the absence or the presence of pIII-A8 at a concentration of 10-6 M. pIII-A8 was applied 10 min before the C5a stimulus. The C5a-induced rise in [Ca2+]i was completely inhibited when C5a was applied at a concentration of 10-9 M or 2 x 10-9 M. At higher concentrations, the inhibition was between 50 and 95%. Data from one typical experiment of three are depicted. **, p < 0.001; *, p < 0.05 (t test).

To analyze the in vivo potency of pIII-A8, it was tested in a murine model of IC peritonitis, i.e., the reverse passive Arthus reaction. BALB/c mice were challenged i.v. with chicken egg albumin and i.p. with anti-chicken egg albumin Ab. This treatment resulted in the influx of 3.8 ± 0.8 neutrophils x 10⁶/mouse, corresponding to 46.5 ± 7.3% of cells in the peritoneum 6 h after IC challenge (Fig. 6). Administration of 200 µl (10^-5 M) of pIII-A8 i.p. at the time of IC challenge or 1 h and 2 h after IC challenge did not alter the PMN influx 6 h after initiation of IC peritonitis. A significant reduction in PMN recruitment was found when 100 µl (10^-5M) of pIII-A8 were applied i.v., independent of whether the molecule was given at the time of IC challenge or 1 h and 2 h after the initiation of IC peritonitis (PMN, 20.1 ± 5.9% or 20.1 ± 7.4%; p < 0.05). When 200 µl pIII-A8 were injected i.p. and 100 µl i.v. (both 10^-5 M) at the time of Ag/Ab application, and again 1 h and 2 h after IC application, PMN influx was abolished (9.5 ± 3.1%) (p < 0.001; ANOVA). An identical reduction in neutrophil recruitment was observed after complement depletion with CVF (10.1 ± 4.8%). Neutrophil accumulation in Ag and Ab controls was 2.1 ± 0.8 or 13.6 ± 6.4% of peritoneal cells, respectively.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. pIII-A8 impairs PMN recruitment in IC peritonitis. Intraperitoneal challenge of BALB/c mice with IC resulted in an increase in the portion of PMN in the peritoneal cavity from 1 to 2% in untreated animals to 46.5 ± 7.3% 6 h after IC application. A combination of i.p. and i.v. application of pIII-A8 1 h and 2 h after IC challenge abrogated PMN migration (46.5% ± 7.3% (n = 26) vs 9.5% + 3.1% (n = 6); p < 0.001, ANOVA and Tukey test). An identical reduction was found for CVF-treated animals (10.1% + 4.8% (n = 8); p < 0.001, ANOVA and Tukey test). PMN recruitment was also significantly blocked when pIII-A8 was injected solely i.v. 1 h and 2 h after IC challenge (PMN, 20.1% + 5.9% (n = 12); p < 0.05, ANOVA and Tukey test) or at the time of IC application (T0) (PMN, 20.1 + 7.4% (n = 8); p < 0.05, ANOVA and Tukey test). Intraperitoneal application of pIII-A8 did not influence PMN migration. **, p < 0.001 .

The properties of pIII-A8 to serve as a C5aRA were further evaluated in two other models of IC diseases, i.e., the reverse passive Arthus reaction in the skin and in the lung. After i.v. application of OVA and a combination of intratracheal and subcutaneous application of anti-OVA Abs, a tremendous accumulation of neutrophils was observed, both in the skin and in the lung (Fig. 7). The MPO activity in the skin, 4 h after challenging mice with IC, was 241.6 ± 51.6 µg/cm² as compared with 44.1 ± 7.1 µg/cm² or 57.3 ± 8.7 µg/cm² in mice receiving only the Ag or the Ab. When mice were treated with a single dose of 200 µl i.v. pIII-A8 at a concentration of 7.3 x 10^-6 M or, additionally, with 100 µl C5aRA 1 h and 2 h after initiation of the Arthus reaction, the MPO activity decreased to 101.2 ± 33.4 or 80.5 ± 23.7 µg/cm² (Fig. 7A).

View larger version (20K): [in this window] [in a new window]   FIGURE 7. The PMN migration into the skin (A) and the lung (B) is attenuated by the C5aRA pIII-A8. A, The i.v. application of a single dose pIII-A8 (n = 5) just before initiation of the Arthus reaction (T0) reduced the skin content of MPO to 26.8%; however, this was statistically not significant (p = 0.052, ANOVA and Tukey test). The additional i.v. application of pIII-A8 (n = 7) 60 min and 120 min after IC challenge reduced the skin content of MPO significantly to 15.8% as compared with untreated animals (n = 6) (p = 0.023, ANOVA and Tukey test). B, The number of neutrophils determined in the BAL was significantly reduced from 3.2 ± 0.8 neutrophils x 105/mouse to 0.7 ± 0.2 neutrophils x 105/mouse in the group that received three doses of the C5aRA i.v. (p = 0.003, ANOVA and Tukey test).

Effects of the C5aRA on I/R-induced local and remote organ injury

Occluding of the superior mesenteric artery for 60 min and subsequent reperfusion for 1 h resulted in severe local and remote tissue injury, i.e., in the bowel and the lung. In the group of untreated animals, 5 of 10 animals died 50–55 min after reperfusion. All animals suffered from heavy bowel injury (hemorrhage, bowel wall edema, and purple black discoloration located at different sites) (Table III). In contrast, all animals receiving CVF survived and showed no signs of bowel injury at all. When mice were treated with 200 µl pIII-A8 i.v. at a concentration of 7.3 x 10^-6 M before arterial occlusion and another 100 µl of the C5aRA just before reperfusion, four of six animals survived. None of the surviving animals showed intestinal injury, while the two nonsurviving animals exhibited intestinal hemorrhage and bowel edema. Sham-operated controls had completely normal bowel appearance and survived.

View this table: [in this window] [in a new window]   Table III. Effects of pIII-A8 on pulmonary microvascular dysfunction, bowel injury, and survival

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction of C5a and its receptor is complex. A minimum of two binding domains has been described, the first of which is located in the core region while the second one is located at the C terminus of C5a. Using C-terminal-derived peptides and site-directed mutagenesis, this domain and in particular residues Lys⁶⁸, Leu⁷², and Arg⁷⁴ have been demonstrated to be crucial for all effector functions of C5a (18, 32, 33, 34, 35, 36). In addition, synthetic C-terminal peptide analogues have been reported, which are C5aRA. The peptide NMePhe-Lys-Pro-dCha-Trp-dArg was described as the most potent antagonist (32). Thus, targeting the C5a C-terminus seemed a reasonable approach to modulate C5a agonist properties.

We constructed C5a libraries, in which either the whole C-terminal domain from positions 69–73 or only particular residues were randomly mutated. These libraries were cloned into the pJuFo vector and were expressed on the tip of a filamentous phage. After a three-round pan on a C5aR-expressing cell line, all of the selected clones were from library III, in which positions 69–73 were randomly mutated. None of the selected clones had the Phe-Lys-Pro motif present in three of the five libraries, although this motif was demonstrated to confer binding affinity in the C5a analogue peptide NMePhe-Lys-Pro-dCha-Trp-dArg (32). In contrast to the native C5a sequence or the sequences selected from the libraries, D-amino acids have been introduced in this hexapaptide resulting in changes in chirality. This in turn may heavily affect the binding of the Phe-Lys-Pro motif to the C5aR.

All of the selected clones were pure C5a agonists except clone A8 (see Fig. 1), which bound to the C5aR with an ID₅₀ in the low nanomolar range, i.e., 1.6 x 10^-8 M, and, however, which was devoid of any signaling activity as determined in different in vitro assays such as cell degranulation, chemotaxis, and increase in [Ca²⁺]_i. With an ID₅₀ only 16-fold lower as compared with the natural ligand C5a, we tested this molecule for its ability to antagonize C5a effects in vitro. We found, that C5a effector functions could be blocked to nearly 100% when pIII-A8 was used at a concentration of 10^-6 M. This concentration was chosen, because it results in a complete displacement of C5a from its receptor, which is a "conditio sine qua non" for a complete and successful inhibition of C5a-mediated effects. In fact, from all C5aRA reported, pIII-A8 is the one with the highest in vitro potency (2).

Common to all effector functions of C5a is the activation of a heterotrimeric G-protein as the first step in signal transduction. C5aR have previously been shown to couple to either PTx-sensitive G-proteins or to the PTx-insensitive G subunit subsequently activating phospholipase C (37, 38). The selected antagonist did not induce any measurable GTPase activity, demonstrating that signal transduction is interrupted at the level of G-protein activation (Fig. 2).

After C5aR binding, cells become rapidly refractory to further stimulation, a phenomenon termed homologous desensitization. The receptors are uncoupled from the G-protein, internalized in endosomes, and subsequently recycled to the cell-surface membrane. Recent data suggest that receptor phosphorylation is the key mechanism for C5aR desensitization (24, 39). In contrast to C5a, pIII-A8 did not induce any phosphorylation of the C5aR (Fig. 3), suggesting that pIII-A8 inhibits C5a effector functions by displacement of C5a from its receptor but not by receptor desensitization.

To check whether pIII-A8 might prove useful to analyze C5a effects in murine animal models, we first tested its ability to antagonize C5a-induced increases in [Ca²⁺]_i in murine peritoneal macrophages. In fact, binding and antagonistic properties of pIII-A8 on murine macrophages were indistinguishable from dU937 cells. Then, pIII-A8 was analyzed in a murine model of IC peritonitis. PMN migration into the peritoneal cavity was significantly impaired 6 h after initiation of the IC disease, when pIII-A8 was applied i.v. Moreover, it was completely abrogated when the C5aRA was injected i.v. and i.p. at the time of IC challenge and 1 h and 2 h after initiation of the Arthus reaction. Because a mere intraperitoneal application of pIII-A8 did not affect the inflammatory response, we draw the conclusion that C5aR on PMN must be already blocked in the circulation to prevent neutrophil migration into the inflammatory focus.

As the antagonist is based on the natural ligand, pharmakokinetics are quite similar (T. Heller et al., manuscript in preparation). This is an important point, because the low molecular mass of pIII-A8, i.e., 18 kDa, allows easy tissue penetration, as is the case for the natural ligand (12 kDa).

At the moment, the most potent and most commonly used inhibitor of complement activation is the soluble complement receptor 1 (sCR1). It has been demonstrated by Weismann et al. (1) that sCR1 blocks complement activation in vitro with an ID₅₀ of 1.5 x 10^-10 M. However, the effective concentration of sCR1 to inhibit complement activation in vivo is 1.3 x 10^-6 M, which is nearly 10,000 times the concentration required for blocking 50% of complement in vitro. In fact, doses of 15–20 mg/kg body weight are commonly used in animal models resulting in micromolar concentrations of sCR1 within the circulation. In accordance to the in vitro data, the C5aRA was applied at concentrations from 3.65 x 10^-7 M to 10^-6 M, given that the injected 200 or 100 µl of a 7.3 x 10^-6 M to 10^-5 concentration were 10-fold diluted in the 2 ml blood volume of the mouse. These concentrations are similar to or even below the molar concentrations commonly used for the sCR1.

Recently, we described that the PMN recruitment into the peritoneal cavity in IC peritonitis is predominantly mediated by complement or the high-affinity Fc receptor type I for IgG (FcRI) (CD64) on macrophages, depending on the mouse strain used (40). In BALB/c mice, complement mainly contributes to PMN migration, whereas in C57BL/6 mice it is the interaction of IC with FcRI. The results obtained in the current manuscript give evidence that the complement-mediated effect in BALB/c mice can be mainly attributed to C5a. In addition, the data give evidence that the antagonist has the potential to protect mice from C5a-induced PMN accumulation in IC peritonitis.

In two further models of IC disease, i.e., the reverse passive Arthus reaction in the skin and in the lung, we found a substantial reduction of the neutrophil influx of about 70–80% when the antagonist was applied i.v. These data demonstrate that the C5a-inhibitory effect is not only restricted to the peritoneum but is also valid for the protection of other tissues, such as the lung or the skin. In addition, these data imply an important general role for C5a in the initiation of the inflammatory sequelae seen in IC disease.

An important role for C5a in IC-triggered inflammation in peritoneum, skin, and lung has recently also been addressed by the use of C5aR-deficient mice (7, 8). These authors found that PMN migration was substantially impaired in C5aR-deficient mice as compared with their normal littermates. They observed a clear tissue dependency with a key role of C5a in the lung and a synergistic role in peritoneum or skin. While we also found a clear contribution of C5a to the IC-mediated inflammation, the most prominent effect of C5a inhibition by pIII-A8 occurred in IC-mediated peritonitis. A possible explanation for the observed differences is that different mouse strains were used for the experiments. The C5aR-deficient mice have a mixed genetic background comprising two strains, 129 and C57BL/6, whereas we used BALB/c mice throughout the experiments.

The overall contribution of complement proteins and FcRs in the initiation of the inflammatory response in IC disease is puzzling. Data obtained with mice lacking the Fc--chain or the FcRII suggest that the inflammatory sequelae in IC-induced skin injury (41, 42), alveolitis (43), and autoimmune glomerulonephritis (44) are solely FcR dependent. This view is supported by the missing attenuation of skin injury in C3-, C4- and C5-deficient mice (45). In addition, the same authors reported that mice lacking the C5aR had only minimally reduced inflammatory responses in IC-triggered alveolitis (43), which is in contrast to the results originally described by Bozic et al. (8). Our data obtained with the C5aRA strongly support the data by Bozic et al. and identify C5a as an important mediator in IC alveolitis. In addition, our results argue against a concept in which only FcRs play a role in the pathogenesis of IC disease but favor the view that both complement, in particular C5a, and FcRs have to be considered as mediators in the inflammatory concert of IC disease.

Several experimental reports suggest that complement activation is critically involved in I/R injury in the heart (1, 46), lung (47), skeletal muscle (48), and intestine (49, 50). Complement activation occurs by either interstitial or intravascular activation of the classical (48) or the alternative pathway (46), leading to the release of the anaphylatoxins C3a and C5a and the deposition of the MAC on injured endothelial cells. Evidence for the contribution of both C5a and the MAC to the inflammatory sequelae, such as neutrophil accumulation, increased vascular permeability, or cellular apoptosis, has been provided by specific inhibition of C5 cleavage with a mAb (51) or by means of the sCR1 during myocardial infarction in rats (1) or pigs (46).

In the model of intestinal I/R, local and remote injury occurs. Local injury is characterized by gross bowel injury including bowel hemorrhage, bowel wall edema, and purple black discoloration. Remote injury is manifested by pulmonary accumulation of neutrophils as well as microvascular dysfunction. The application of pIII-A8 improved both local and remote inflammation. All untreated animals suffered from heavy local bowel injury, while only 33% of the C5aRA-treated group showed signs of bowel damage, demonstrating a bowel protective effect of pIII-A8. The fact that not all animals were protected from local injury, as in the CVF-treated group, suggests that additional mediators from the complement system may be involved. The most likely candidate is the C5b-9 complex, because pIII-A8 has no effect on the formation or the deposition of the MAC on injured endothelial cells. Similar to C5a, the MAC up-regulates CD62P on endothelial cells, resulting in the adherence of neutrophils (52). In addition, it is able to induce apoptosis by Ras activation in ischemic tissue, at least in myocardial tissue (51).

Pulmonary vascular permeability was significantly decreased in the C5aRA-treated group as compared with untreated animals. In fact, the pulmonary microvascular dysfunction was as low as in sham controls or CVF-treated mice. These data provide evidence that C5a is crucial for the formation of the pulmonary edema following intestinal ischemia. C5a may affect the integrity of the endothelial cell layer by a direct activation of endothelial cells or alveolar macrophage. In addition, C5a can initiate and augment PMN adherence, transmigration, and activation (10).

Whatever the most important mechanism is, intestinal ischemia is a common clinical problem, either as a primary event, e.g. after superior mesenteric artery embolism, or associated with the systemic inflammatory response syndrome. Targeting the C5a-C5aR interaction by pIII-A8 has major potential as a clinical tool to reduce the extent and severity of intestinal I/R injury.

In summary, we have selected a potent C5aRA from phage display libraries, which allowed us to identify C5a as an important mediator in IC disease and I/R injury. This novel C5aRA may prove useful as a specific therapeutic agent targeting selectively a particular effector function of the complement system, the C5a-C5aR interaction. The beneficial and necessary functions, such as bacterial opsonization by C3 cleavage products or the clearance of ICs, are not affected, as is the case when "broad spectrum" complement inhibitors such as sCR1 or C1-INH are used.

	Acknowledgments

 
We thank D. Bitter-Suerman for continuous support and R. E. Schmidt for helpful discussions.

	Footnotes

 
¹ This work was supported by a grant of the Bundesministerium für Bildung, Wissenschaft, Forschung and Technologie (FKZ: 01 VM 9305) to J.K., a grant of the Sonderforschungsbereich 265 project B1 to U.B., and a grant of the Deutsche Forschungsgemeinschaft (892/5/1) to J.E.G.

² Address correspondence and reprint requests to Dr. Jörg Köhl, Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany. E-mail address:

³ Abbreviations used in this paper: sCR1, soluble complement receptor 1; IC, immune complex; I/R, ischemia/reperfusion; hC5a, human complement 5a; des-Arg⁷⁴-C5a, human complement 5a without C-terminal Arg residue at position 74; [Ala²⁷]-C5a, hC5a with a Cys²⁷Ala replacement; C5aR, human complement 5a receptor (CD 88); C5aRA, C5aR antagonist; A8, a fusion protein comprising the C-terminal domain of the minor coat protein (pIII) of the filamentous M13 phage and the leucine zipper of Jun, which forms a heterodimer with the leucine zipper of Fos and [Ala²⁷]-C5a-(1–66)-FKRSLLR; pIII-A8, a heterodimer comprising the leucine zipper of Jun, the leucine zipper of Fos and [Ala²⁷]-hC5a-(1–66)-FKRSLLR; ¹²⁵I-rhC5a, ¹²⁵I-labeled recombinant human C5a; dU937 cells, U937 cells differentiated with 1 mM N⁶,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate for 3 days; MAC, membrane attack complex; PMN, polymorphonuclear neutrophil; [Ca²⁺]_i, intracellular Ca²⁺; CVF, cobra venom factor; MPO, myeloperoxidase; FcRI, Fc receptor type I for IgG.

Received for publication March 18, 1999. Accepted for publication May 6, 1999.

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